Nucleic acid‐based therapeutics that regulate gene expression have been developed towards clinical use at a steady pace for several decades, but in recent years the field has been accelerating. To date, there are 11 marketed products based on antisense oligonucleotides, aptamers and small interfering RNAs, and many others are in the pipeline for both academia and industry. A major technology trigger for this development has been progress in oligonucleotide chemistry to improve the drug properties and reduce cost of goods, but the main hurdle for the application to a wider range of disorders is delivery to target tissues. The adoption of delivery technologies, such as conjugates or nanoparticles, has been a game changer for many therapeutic indications, but many others are still awaiting their eureka moment. Here, we cover the variety of methods developed to deliver nucleic acid‐based therapeutics across biological barriers and the model systems used to test them. We discuss important safety considerations and regulatory requirements for synthetic oligonucleotide chemistries and the hurdles for translating laboratory breakthroughs to the clinic. Recent advances in the delivery of nucleic acid‐based therapeutics and in the development of model systems, as well as safety considerations and regulatory requirements for synthetic oligonucleotide chemistries are discussed in this review on oligonucleotide‐based therapeutics.

Porcine parvovirus (PPV) is a major causative agent in reproductive failure, but in the last two decades many novel porcine parvoviruses were described and designated as porcine parvovirus 2 through 6 (PPV2–PPV6). However, their role for pig health is largely unknown. The aim of this study was to better understand the on-farm prevalence of PPVs in different age groups of pigs, and to assess the diagnostic applicability of testing different diagnostic materials. In total, 271 oral fluids, 1244 serum samples, and 1238 fecal samples were collected from 3–21-week-old pigs from 19 farms, and after pooling by 4–6, tested by real-time PCR. The results showed that PPVs are widely spread in Poland and that the highest detection rates were obtained for oral fluids (ranging from 10.7% (PPV1) to 48.7% (PPV2)). Fattening pigs were the age group with the most frequent detection of PPVs (ranging from 8.6% (PPV1) to 49.1% (PPV2)). Porcine parvoviruses were detected mostly in growing-finishing pigs and the infection persisted until the late fattening period, which may suggest the chronic character of the infection (especially for PPV2, which was found to commonly infect animals of all ages). Particularly low Ct values detected for PPV2, PPV3, PPV5, and PPV6 in serum pools from some farms suggested that these viruses may cause high levels of viremia in one or more individuals included in these pools. Further studies are needed to quantify the levels of PPVs viremia and to assess the impact in co-infections with other, often endemic pig viruses, such as porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV).

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Plastids are small organelles equipped with their own genomes (plastomes). Although these organelles are involved in numerous plant metabolic pathways, current knowledge about the transcriptional activity of plastomes is limited. To solve this problem, we constructed a plastid tiling microarray (PlasTi-microarray) consisting of 1629 oligonucleotide probes. The oligonucleotides were designed based on the cucumber chloroplast genomic sequence and targeted both strands of the plastome in a non-contiguous arrangement. Up to 4 specific probes were designed for each gene/exon, and the intergenic regions were covered regularly, with 70-nt intervals. We also developed a protocol for direct chemical labeling and hybridization of as little as 2 micrograms of chloroplast RNA. We used this protocol for profiling the expression of the cucumber chloroplast plastome on the PlasTi-microarray. Owing to the high sequence similarity of plant plastomes, the newly constructed microarray can be used to study plants other than cucumber. Comparative hybridization of chloroplast transcriptomes from cucumber, Arabidopsis, tomato and spinach showed that the PlasTi-microarray is highly versatile.

BackgroundThe gene that encodes tumor protein p53, Tp53, is mutated or silenced in most human cancers and is recognized as one of the most important cancer drivers. Homozygotic Tp53 knockout mice, which develop lethal cancers early in their lives, are already used in cancer prevention studies, and now Tp53 knockout rats have also been generated. This study assessed feasibility of using homozygous Tp53 knockout rats to evaluate the possible outcome of cancer chemoprevention.MethodsA small colony of Tp53 knockout rats with a Wistar strain genetic background was initiated and maintained in the animal house at our institution. Tp53 heterozygotic females were bred with Tp53 homozygous knockout males to obtain a surplus of knockout homozygotes. To evaluate the reproducibility of their lifespan, 4 groups of Tp53 homozygous knockout male rats born during consecutive quarters of the year were kept behind a sanitary barrier in a controlled environment until they reached a moribund state. Their individual lifespan data were used to construct quarterly survival curves.ResultsThe four consecutive quarterly survival curves were highly reproducible. They were combined into a single “master” curve for use as a reference in intervention studies. The average lifespan of untreated male Tp53 homozygous knockout rats was normally distributed, with a median of 133 days. Sample size vs. effect calculations revealed that confirming a 20% and 30% increase in the lifespan would respectively require a sample size of 18 and 9 animals (when assessed using the t-test with a power of 80% and alpha set at 0.05). As an example, the Tp53 homozygous knockout rat model was used to test the chemopreventive properties of carnosine, a dipeptide with suspected anticancer properties possibly involving modulation of the mTOR pathway. The result was negative.ConclusionFurther evaluation of the Tp53 homozygous knockout male rat colony is required before it can be confirmed as a viable tool for assessing new methods of cancer prevention or treatment.

The constant search for successful cancer therapies lasts for decades. Apart from the huge scientific effort and enormous sum of spent money, only a small amount of newly developed medicines move into clinical use (only 94 registered anticancer drugs in the last 12 years). Anticancer regimes are still overcome by drugs invented over 50 years ago such as cisplatin and doxorubicin. Significant progress in the development of improved anticancer drugs was made due to multiple studies on the relationship between the molecular structure of chemical compounds and their cytostatic activity. A number of ligands (mainly organic) with quite effective anticancer properties are known, but they show insufficient activity, selectivity and multidrug resistance. Formation of transition metal - ligand complexes (with proven anticancer effect) changes the properties of the latter. The factors that affect the cytotoxic properties of metal complexes are: the type of ligand and metal, the nature of the connection between metal and ligand, and the distribution of electronic charge density in the formed complexes. Here, we report the recent efforts to improve existing compounds with confirmed anticancer activity. They seem to be unappreciated as their effects appear to be less spectacular than that of targeted anticancer drugs (i.e. based on antibodies or small RNAs).

Sarcomas are a heterogeneous group of malignant tumors, that develop from mesenchymal cells. Sarcomas are tumors associated with poor prognosis and expected short overall survival. Efforts to improve treatment efficacy and treatment outcomes of advanced and metastatic sarcoma patients have not led to significant improvements in the last decades. In the Tp53C273X/C273X rat model we therefore aimed to characterize specific gene expression pattern of angiosarcomas with a loss of TP53 function. The presence of metabolically active tumors in several locations including the brain, head and neck, extremities and abdomen was confirmed by magnetic resonance imaging (MRI) and positron emission tomography (PET) examinations. Limb angiosarcoma tumors were selected for microarray expression analysis. The most upregulated pathways in angiosarcoma vs all other tissues were related to cell cycle with mitosis and meiosis, chromosome, nucleosome and telomere maintenance as well as DNA replication and recombination. The downregulated genes were responsible for metabolism, including respiratory chain electron transport, tricarboxylic acid (TCA) cycle, fatty acid metabolism and amino-acid catabolism. Our findings demonstrated that the type of developing sarcoma depends on genetic background, underscoring the importance of developing more malignancy susceptibility models in various strains and species to simulate the study of the diverse genetics of human sarcomas.

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