RNase H-sensitive multifunctional ASO-based constructs as promising tools for the treatment of multifactorial complex pathologies.

Combined therapies play a key role in the fight against complex pathologies, such as cancer and related drug-resistance issues. This is particularly relevant in targeted therapies where inhibition of the drug target can be overcome by cross-activating complementary pathways. Unfortunately, the drug combinations approved to date -mostly based on small molecules- face several problems such as toxicity effects, which limit their clinical use. To address these issues, we have designed a new class of RNase H-sensitive construct (3ASO) that can be disassembled intracellularly upon cell entry, leading to the simultaneous release of three different therapeutic oligonucleotides (ONs), tackling each of them the mRNA of a different protein. Here, we used Escherichia coli RNase H1 as a model to study an unprecedented mode of recognition and cleavage, that is mainly dictated by the topology of our RNA·DNA-based hybrid construct. As a model system for our technology we have created 3ASO constructs designed to specifically inhibit the expression of HER2, Akt and Hsp27 in HER2+ breast cancer cells. These trifunctional ON tools displayed very low toxicity and good levels of antiproliferative activity in HER2+ breast cancer cells. The present study will be of great potential in the fight against complex pathologies involving multiple mRNA targets, as the proposed cleavable designs will allow the efficient single-dose administration of different ON drugs simultaneously.

Inborn Errors of Nucleoside Transporter (NT)-Encoding Genes (SLC28 and SLC29).

The proper regulation of nucleotide pools is essential for all types of cellular functions and depends on de novo nucleotide biosynthesis, salvage, and degradation pathways. Despite the apparent essentiality of these processes, a significant number of rare diseases associated with mutations in genes encoding various enzymes of these pathways have been already identified, and others are likely yet to come. However, knowledge on genetic alterations impacting on nucleoside and nucleobase transporters is still limited. At this moment three gene-encoding nucleoside and nucleobase transporter proteins have been reported to be mutated in humans, SLC29A1, SLC29A3, and SLC28A1, impacting on the expression and function of ENT1, ENT3, and CNT1, respectively. ENT1 alterations determine Augustine-null blood type and cause ectopic calcification during aging. ENT3 deficiency translates into various clinical manifestations and syndromes, altogether listed in the OMIM catalog as histiocytosis-lymphoadenopathy plus syndrome (OMIM#602782). CNT1 deficiency causes uridine-cytidineuria (URCTU) (OMIM#618477), a unique type of pyrimidineuria with an as yet not well-known clinical impact. Increasing knowledge on the physiological, molecular and structural features of these transporter proteins is helping us to better understand the biological basis behind the biochemical and clinical manifestations caused by these deficiencies. Moreover, they also support the view that some metabolic compensation might occur in these disturbances, because they do not seem to significantly impact nucleotide homeostasis, but rather other biological events associated with particular subtypes of transporter proteins.

OncomiRs miR-106a and miR-17 negatively regulate the nucleoside-derived drug transporter hCNT1.

High-affinity uptake of natural nucleosides as well as nucleoside derivatives used in anticancer therapies is mediated by human concentrative nucleoside transporters (hCNTs). hCNT1, the hCNT family member that specifically transports pyrimidines, is also a transceptor involved in tumor progression. In particular, oncogenesis appears to be associated with hCNT1 downregulation in some cancers, although the underlying mechanisms are largely unknown. Here, we sought to address changes in colorectal and pancreatic ductal adenocarcinoma-both of which are important digestive cancers-in the context of treatment with fluoropyrimidine derivatives. An analysis of cancer samples and matching non-tumoral adjacent tissues revealed downregulation of hCNT1 protein in both types of tumor. Further exploration of the putative regulation of hCNT1 by microRNAs (miRNAs), which are highly deregulated in these cancers, revealed a direct relationship between the oncomiRs miR-106a and miR-17 and the loss of hCNT1. Collectively, our findings provide the first demonstration that hCNT1 inhibition by these oncomiRs could contribute to chemoresistance to fluoropyrimidine-based treatments in colorectal and pancreatic cancer.

The Physicochemical, Biopharmaceutical, and In Vitro Efficacy Properties of Freeze-Dried Dexamethasone-Loaded Lipomers.

Dexamethasone-loaded polymer hybrid nanoparticles were developed as a potential tool to treat alopecia areata due to their follicular targeting ability. Freeze drying (FD) is a common technique used to improve nanoparticle stability; however, there are few studies focused on its effect on ethyl cellulose lipid-core nanoparticles. Nanoparticles were lyophilized with different cryoprotectants. Sucrose was selected because it allowed for a good resuspension and provided acceptable physicochemical parameters (374.33 nm, +34.7 mV, polydispersion 0.229%, and 98.87% encapsulation efficiency). The nanoparticles obtained were loaded into a pleasant xanthan gum hydrogel, and the rheological, release, and skin permeation profiles of different formulations were studied. The FD formulation significantly modified the particle size, and the drug release and permeation properties were also altered. In addition, analyses of the cytotoxicity and anti-inflammatory efficacy of FD and non-FD particles on human keratinocytes indicated no differences.

Dexamethasone-Loaded Lipomers: Development, Characterization, and Skin Biodistribution Studies.

Follicular targeting has gained more attention in recent decades, due to the possibility of obtaining a depot effect in topical administration and its potential as a tool to treat hair follicle-related diseases. Lipid core ethyl cellulose lipomers were developed and optimized, following which characterization of their physicochemical properties was carried out. Dexamethasone was encapsulated in the lipomers (size, 115 nm; polydispersity, 0.24; zeta-potential (Z-potential), +30 mV) and their in vitro release profiles against dexamethasone in solution were investigated by vertical diffusion Franz cells. The skin biodistribution of the fluorescent-loaded lipomers was observed using confocal microscopy, demonstrating the accumulation of both lipomers and fluorochromes in the hair follicles of pig skin. To confirm this fact, immunofluorescence of the dexamethasone-loaded lipomers was carried out in pig hair follicles. The anti-inflammatory (via TNFα) efficacy of the dexamethasone-loaded lipomers was demonstrated in vitro in an HEK001 human keratinocytes cell culture and the in vitro cytotoxicity of the nanoformulation was investigated.

Deficiency of perforin and hCNT1, a novel inborn error of pyrimidine metabolism, associated with a rapidly developing lethal phenotype due to multi-organ failure.

Pyrimidine nucleotides are essential for a vast number of cellular processes and dysregulation of pyrimidine metabolism has been associated with a variety of clinical abnormalities. Inborn errors of pyrimidine metabolism affecting enzymes in the pyrimidine de novo and degradation pathway have been identified but no patients have been described with a deficiency in proteins affecting the cellular import of ribonucleosides. In this manuscript, we report the elucidation of the genetic basis of the observed uridine-cytidineuria in a patient presenting with fever, hepatosplenomegaly, persistent lactate acidosis, severely disturbed liver enzymes and ultimately multi-organ failure. Sequence analysis of genes encoding proteins directly involved in the metabolism of uridine and cytidine showed two variants c.1528C > T (p.R510C) and c.1682G > A (p.R561Q) in SLC28A1, encoding concentrative nucleotide transporter 1 (hCNT1). Functional analysis showed that these variants affected the three-dimensional structure of hCNT1, altered glycosylation and decreased the half-life of the mutant proteins which resulted in impaired transport activity. Co-transfection of both variants, mimicking the trans disposition of c.1528C > T (p.R510C) and c.1682G > A (p.R561Q) in the patient, significantly impaired hCNT1 biological function. Whole genome sequencing identified two pathogenic variants c.50delT; p.(Leu17Argfs*34) and c.853_855del; p.(Lys285del) in the PRF1 gene, indicating that our patient was also suffering from Familial Hemophagocytic Lymphohistiocytosis type 2. The identification of two co-existing monogenic defects might have resulted in a blended phenotype. Thus, the clinical presentation of isolated hCNT1 deficiency remains to be established.