Chemically defined polyethylene glycol siRNA conjugates with enhanced gene silencing effect.

The therapeutic application of siRNA suffers from poor bioavailability caused by rapid degradation and elimination. The covalent attachment of PEG is a universal concept to increase molecular size and enhance the pharmacokinetic properties of biomacromolecules. We devised a facile approach for attachment of PEG molecules with a defined molecular weight, and successful purification of the resulting conjugates. We directly conjugated structurally defined PEG chains with twelve ethylene glycol units to the 3'-terminal hydroxyl group of both sense and antisense strands via an aminoalkyl linker. The conjugates were easily purified by HPLC and successful PEGylation and molecule integrity were confirmed by ESI-MS. The evaluation of in vitro gene knockdown of two different targets in MCF-7 breast cancer cells showed stable pharmacologic activity when combined with a standard transfection reagent. Sense strand PEGylation even increased the silencing potency of a CRCX4-siRNA which had modest activity in its wild-type form. The results indicate that PEG chains at the 3'-terminus of both strands of siRNA are well tolerated by the RNAi effector. The attachment of short, chemically defined PEG chains is a feasible approach to improve the pharmacokinetic properties of siRNA, and can be combined with other targeted and untargeted delivery vehicles.

Corrigendum: Identification of extremely GC-rich micro RNAs for RT-qPCR data normalization in human plasma.

[This corrects the article DOI: 10.3389/fgene.2022.1058668.].

Identification of extremely GC-rich micro RNAs for RT-qPCR data normalization in human plasma.

We aimed at extending the repertoire of high-quality miRNA normalizers for reverse transcription-quantitative PCR (RT-qPCR) of human plasma with special emphasis on the extremely guanine-cytosine-rich portion of the miRNome. For high-throughput selection of stable candidates, microarray technology was preferred over small-RNA sequencing (sRNA-seq) since the latter underrepresented miRNAs with a guanine-cytosine (GC) content of at least 75% (p = 0.0002, n = 2). miRNA abundances measured on the microarray were ranked for consistency and uniformity using nine normalization approaches. The eleven most stable sequences included miRNAs of moderate, but also extreme GC content (45%-65%: miR-320d, miR-425-5p, miR-185-5p, miR-486-5p; 80%-95%: miR-1915-3p, miR-3656-5p, miR-3665-5p, miR-3960-5p, miR-4488-5p, miR-4497 and miR-4787-5p). In contrast, the seven extremely GC-rich miRNAs were not found in the two plasma miRNomes screened by sRNA-seq. Stem-loop RT-qPCR was employed for stability verification in 32 plasma samples of healthy male Caucasians (age range: 18-55 years). In general, inter-individual variance of miRNA abundance was low or very low as indicated by coefficient of variation (CV) values of 0.6%-8.2%. miR-3665 and miR-1915-3p outperformed in this analysis (CVs: 0.6 and 2.4%, respectively). The eight most stable sequences included four extremely GC-rich miRNAs (miR-1915-3p, miR-3665, miR-4787-5p and miR-4497). The best-performing duo normalization factor (NF) for the condition of human plasma, miR-320d and miR-4787-5p, also included a GC-extreme miRNA. In summary, the identification of extremely guanine-cytosine-rich plasma normalizers will help to increase accuracy of PCR-based miRNA quantification, thus raise the potential that miRNAs become markers for psychological stress reactions or early and precise diagnosis of clinical phenotypes. The novel miRNAs might also be useful for orthologous contexts considering their conservation in related animal genomes.

Critical evaluation of quantification methods for oligonucleotides formulated in lipid nanoparticles.

There is a very large variety in the types of nanoparticulate lipid formulations for oligonucleotides, and remarkably, also a very large heterogeneity in the methods that are used for analyzing oligonucleotide load, encapsulation efficiency and oligonucleotide release. Furthermore, a literature survey showed that the extent to which these procedures are reported in scientific literature varies greatly, with some of them not even reporting any quantification at all. This greatly hampers the reproducibility of nanoparticle preparation between different researchers and between different laboratories, which slows down the clinical translation of such nanomedicines. In this work, a standardized extraction method from liposomes is proposed, in which potential contaminants from the carrier are removed by a simple extraction of the oligonucleotides. These extracts were then analyzed with seven commonly used methods for oligonucleotide quantification, including several absorbance based methods and the most commonly applied dye binding assay. Strikingly, differences in absolute values up to fourfold were found when the same sample was analyzed using different methods which should be taken into consideration when reports using different methods are compared. Furthermore, these results indicate that the most commonly applied method, the dye binding assay, may -without adaptations- not be suitable for short oligonucleotides like siRNAs. The found differences in quantification methods as described here underscore the need for proper documentation of methods to correctly interpret published results, which -with regard to oligonucleotide analysis- is currently lacking in many reports.

RNAi-Mediated Knockdown of Protein Expression.

RNA interference is an essential method for studying genomic functions of single genes by loss-of-function experiments. Short interfering siRNAs are efficiently transfected into cultured cells to enable RISC-mediated mRNA cleavage and inhibition of translation in a sequence-specific manner. RNAi enables knockdown of single genes and screening for specific cellular processes or outcomes. In this chapter, we describe a detailed universal protocol for lipoplex-mediated siRNA transfection for cell cultures and cell lysis for subsequent RNA or protein analysis. The experimental procedure is described for verification of knockdown and includes cell lysis for mRNA or protein quantification. Important aspects for specific gene silencing and potential pitfalls are discussed.

Going beyond the liver: progress and challenges of targeted delivery of siRNA therapeutics.

Therapeutic gene silencing promises significant progress in pharmacotherapy, including considerable expansion of the druggable target space and the possibility for treating orphan diseases. Technological hurdles have complicated the efficient use of therapeutic oligonucleotides, and siRNA agents suffer particularly from insufficient pharmacokinetic properties and poor cellular uptake. Intense development and evolution of delivery systems have resulted in efficient uptake predominantly in liver tissue, in which practically all nanoparticulate and liposomal delivery systems show the highest accumulation. The most efficacious strategies include liposomes and bioconjugations with N-acetylgalactosamine. Both are in early clinical evaluation stages for treatment of liver-associated diseases. Approaches for achieving knockdown in other tissues and tumors have been proven to be more complicated. Selective targeting to tumors may be enabled through careful modulation of physical properties, such as particle size, or by taking advantage of specific targeting ligands. Significant barriers stand between sufficient accumulation in other organs, including endothelial barriers, cellular membranes, and the endosome. The brain, which is shielded by the blood-brain barrier, is of particular interest to facilitate efficient oligonucleotide therapy of neurological diseases. Transcytosis of the blood-brain barrier through receptor-specific docking is investigated to increase accumulation in the central nervous system. In this review, the current clinical status of siRNA therapeutics is summarized, as well as innovative and promising preclinical concepts employing tissue- and tumor-targeted ligands. The requirements and the respective advantages and drawbacks of bioconjugates and ligand-decorated lipid or polymeric particles are discussed.

miRNA-based therapies: strategies and delivery platforms for oligonucleotide and non-oligonucleotide agents.

The discovery of miRNAs as important regulatory agents for gene expression has expanded the therapeutic opportunities for oligonucleotides. In contrast to siRNA, miRNA-targeted therapy is able to influence not only a single gene, but entire cellular pathways or processes. It is possible to supplement downregulated or non-functional miRNAs by synthetic oligonucleotides, as well as alleviating effects caused by overexpression of malignant miRNAs through artificial antagonists, either oligonucleotides or small molecules. Chemical oligonucleotide modifications together with an efficient delivery system seem to be mandatory for successful therapeutic application. While miRNA-based therapy benefits from the decades of research spent on other therapeutic oligonucleotides, there are some specific challenges associated with miRNA therapy, mainly caused by the short target sequence. The current status and recent progress of miRNA-targeted therapeutics is described and future challenges and potential applications in treatment of cancer and viral infections are discussed.