Deep intronic founder mutations identified in the ERCC4/XPF gene are potential therapeutic targets for a high-frequency form of xeroderma pigmentosum.

Xeroderma pigmentosum (XP) is a genodermatosis defined by cutaneous photosensitivity with an increased risk of skin tumors because of DNA repair deficiency. The worldwide prevalence of XP is ~1 to 4 in million, with higher incidence in some countries and regions including Japan (1 in 22,000) and North Africa due to founder mutations and a high degree of consanguinity. Among XP, the complementation group F (XP-F), is a rare form (1% of worldwide XP); however, this is underdiagnosed, because the ERCC4/XPF gene is essential for fetal development and most of previously reported ERCC4/XPF pathogenic variants are hypomorphs causing relatively mild phenotypes. From the largest Japanese XP cohort study, we report 17 XP-F cases bearing two pathogenic variants, both identified in deep intronic regions of the ERCC4/XPF gene. The first variant, located in intron 1, is a Japanese founder mutation, which additionally accounts for ~10% of the entire Japanese XP cases (MAF = 0.00196), causing an aberrant pre-mRNA splicing due to a miss-binding of U1snRNA. The second mutation located in intron eight induces an alternative polyadenylation. Both mutations cause a reduction of the ERCC4/XPF gene expression, resulting in XP clinical manifestations. Most cases developed early-onset skin cancers, indicating that these variants need critical attention. We further demonstrate that antisense oligonucleotides designed for the mutations can restore the XPF protein expression and DNA repair capacity in the patients' cells. Collectively, these pathogenic variants can be potential therapeutic targets for XP.

Endosomal escape of RNA therapeutics: How do we solve this rate-limiting problem?

With over 15 FDA approved drugs on the market and numerous ongoing clinical trials, RNA therapeutics, such as small interfering RNAs (siRNAs) and antisense oligonucleotides (ASOs), have shown great potential to treat human disease. Their mechanism of action is based entirely on the sequence of validated disease-causing genes without the prerequisite knowledge of protein structure, activity or cellular location. In contrast to small molecule therapeutics that passively diffuse across the cell membrane's lipid bilayer, RNA therapeutics are too large, too charged, and/or too hydrophilic to passively diffuse across the cellular membrane and instead are taken up into cells by endocytosis. However, endosomes are also composed of a lipid bilayer barrier that results in endosomal capture and retention of 99% of RNA therapeutics with 1% or less entering the cytoplasm. Although this very low level of endosomal escape has proven sufficient for liver and some CNS disorders, it is insufficient for the vast majority of extra-hepatic diseases. Unfortunately, there are currently no acceptable solutions to the endosomal escape problem. Consequently, before RNA therapeutics can be used to treat widespread human disease, the rate-limiting delivery problem of endosomal escape must be solved in a nontoxic manner.

Metabolic disorders affecting the liver and heart: Therapeutic efficacy of miRNA-based therapies?

Liver and heart disease are major causes of death worldwide. It is known that metabolic alteration causing type 2 diabetes (T2D) and Nonalcoholic fatty liver (NAFLD) coupled with a derangement in lipid homeostasis, may exacerbate hepatic and cardiovascular diseases. Some pharmacological treatments can mitigate organ dysfunctions but the important side effects limit their efficacy leading often to deterioration of the tissues. It needs to develop new personalized treatment approaches and recent progresses of engineered RNA molecules are becoming increasingly viable as alternative treatments. This review outlines the current use of antisense oligonucleotides (ASOs), RNA interference (RNAi) and RNA genome editing as treatment for rare metabolic disorders. However, the potential for small non-coding RNAs to serve as therapeutic agents for liver and heart diseases is yet to be fully explored. Although miRNAs are recognized as biomarkers for many diseases, they are also capable of serving as drugs for medical intervention; several clinical trials are testing miRNAs as therapeutics for type 2 diabetes, nonalcoholic fatty liver as well as cardiac diseases. Recent advances in RNA-based therapeutics may potentially facilitate a novel application of miRNAs as agents and as druggable targets. In this work, we sought to summarize the advancement and advantages of miRNA selective therapy when compared to conventional drugs. In particular, we sought to emphasise druggable miRNAs, over ASOs or other RNA therapeutics or conventional drugs. Finally, we sought to address research questions related to efficacy, side-effects, and range of use of RNA therapeutics. Additionally, we covered hurdles and examined recent advances in the use of miRNA-based RNA therapy in metabolic disorders such as diabetes, liver, and heart diseases.

DDX5 mRNA-targeting antisense oligonucleotide as a new promising therapeutic in combating castration-resistant prostate cancer.

The heat shock protein 27 (Hsp27) has emerged as a principal factor of the castration-resistant prostate cancer (CRPC) progression. Also, an antisense oligonucleotide (ASO) against Hsp27 (OGX-427 or apatorsen) has been assessed in different clinical trials. Here, we illustrate that Hsp27 highly regulates the expression of the human DEAD-box protein 5 (DDX5), and we define DDX5 as a novel therapeutic target for CRPC treatment. DDX5 overexpression is strongly correlated with aggressive tumor features, notably with CRPC. DDX5 downregulation using a specific ASO-based inhibitor that acts on DDX5 mRNAs inhibits cell proliferation in preclinical models, and it particularly restores the treatment sensitivity of CRPC. Interestingly, through the identification and analysis of DDX5 protein interaction networks, we have identified some specific functions of DDX5 in CRPC that could contribute actively to tumor progression and therapeutic resistance. We first present the interactions of DDX5 and the Ku70/80 heterodimer and the transcription factor IIH, thereby uncovering DDX5 roles in different DNA repair pathways. Collectively, our study highlights critical functions of DDX5 contributing to CRPC progression and provides preclinical proof of concept that a combination of ASO-directed DDX5 inhibition with a DNA damage-inducing therapy can serve as a highly potential novel strategy to treat CRPC.

RNA structure in alternative splicing regulation: from mechanism to therapy.

Alternative splicing is a highly intricate process that plays a crucial role in post-transcriptional regulation and significantly expands the functional proteome of a limited number of coding genes in eukaryotes. Its regulation is multifactorial, with RNA structure exerting a significant impact. Aberrant RNA conformations lead to dysregulation of splicing patterns, which directly affects the manifestation of disease symptoms. In this review, the molecular mechanisms of RNA secondary structure-mediated splicing regulation are summarized, with a focus on the complex interplay between aberrant RNA conformations and disease phenotypes resulted from splicing defects. This study also explores additional factors that reshape structural conformations, enriching our understanding of the mechanistic network underlying structure-mediated splicing regulation. In addition, an emphasis has been placed on the clinical role of targeting aberrant splicing corrections in human diseases. The principal mechanisms of action behind this phenomenon are described, followed by a discussion of prospective development strategies and pertinent challenges.

Antisense Oligonucleotides (ASOs) in Motor Neuron Diseases: A Road to Cure in Light and Shade.

Antisense oligonucleotides (ASOs) are short oligodeoxynucleotides designed to bind to specific regions of target mRNA. ASOs can modulate pre-mRNA splicing, increase levels of functional proteins, and decrease levels of toxic proteins. ASOs are being developed for the treatment of motor neuron diseases (MNDs), including spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS) and spinal and bulbar muscular atrophy (SBMA). The biggest success has been the ASO known as nusinersen, the first effective therapy for SMA, able to improve symptoms and slow disease progression. Another success is tofersen, an ASO designed to treat ALS patients with SOD1 gene mutations. Both ASOs have been approved by the FDA and EMA. On the other hand, ASO treatment in ALS patients with the C9orf72 gene mutation did not show any improvement in disease progression. The aim of this review is to provide an up-to-date overview of ASO research in MNDs, from preclinical studies to clinical trials and, where available, regulatory approval. We highlight the successes and failures, underline the strengths and limitations of the current ASO research, and suggest possible approaches that could lead to more effective treatments.

Potential of Cell-Penetrating Peptide-Conjugated Antisense Oligonucleotides for the Treatment of SMA.

Spinal muscular atrophy (SMA) is a severe neuromuscular disorder that is caused by mutations in the survival motor neuron 1 (SMN1) gene, hindering the production of functional survival motor neuron (SMN) proteins. Antisense oligonucleotides (ASOs), a versatile DNA-like drug, are adept at binding to target RNA to prevent translation or promote alternative splicing. Nusinersen is an FDA-approved ASO for the treatment of SMA. It effectively promotes alternative splicing in pre-mRNA transcribed from the SMN2 gene, an analog of the SMN1 gene, to produce a greater amount of full-length SMN protein, to compensate for the loss of functional protein translated from SMN1. Despite its efficacy in ameliorating SMA symptoms, the cellular uptake of these ASOs is suboptimal, and their inability to penetrate the CNS necessitates invasive lumbar punctures. Cell-penetrating peptides (CPPs), which can be conjugated to ASOs, represent a promising approach to improve the efficiency of these treatments for SMA and have the potential to transverse the blood-brain barrier to circumvent the need for intrusive intrathecal injections and their associated adverse effects. This review provides a comprehensive analysis of ASO therapies, their application for the treatment of SMA, and the encouraging potential of CPPs as delivery systems to improve ASO uptake and overall efficiency.

Pathophysiological Aspects and Therapeutic Armamentarium of Alzheimer's Disease: Recent Trends and Future Development.

Alzheimer's disease (AD) is the primary cause of dementia and is characterized by the death of brain cells due to the accumulation of insoluble amyloid plaques, hyperphosphorylation of tau protein, and the formation of neurofibrillary tangles within the cells. AD is also associated with other pathologies such as neuroinflammation, dysfunction of synaptic connections and circuits, disorders in mitochondrial function and energy production, epigenetic changes, and abnormalities in the vascular system. Despite extensive research conducted over the last hundred years, little is established about what causes AD or how to effectively treat it. Given the severity of the disease and the increasing number of affected individuals, there is a critical need to discover effective medications for AD. The US Food and Drug Administration (FDA) has approved several new drug molecules for AD management since 2003, but these drugs only provide temporary relief of symptoms and do not address the underlying causes of the disease. Currently, available medications focus on correcting the neurotransmitter disruption observed in AD, including cholinesterase inhibitors and an antagonist of the N-methyl-D-aspartate (NMDA) receptor, which temporarily alleviates the signs of dementia but does not prevent or reverse the course of AD. Research towards disease-modifying AD treatments is currently underway, including gene therapy, lipid nanoparticles, and dendrimer-based therapy. These innovative approaches aim to target the underlying pathological processes of AD rather than just managing the symptoms. This review discusses the novel aspects of pathogenesis involved in the causation of AD of AD and in recent developments in the therapeutic armamentarium for the treatment of AD such as gene therapy, lipid nanoparticles, and dendrimer-based therapy, and many more.

Facilitators of Organizational Resilience Within South Carolina AIDS Service Organizations: Lessons Learned from the COVID-19 Pandemic.

HIV care services have been interrupted by the COVID-19 pandemic in many states in the U.S. including South Carolina (SC). However, many HIV care facilities demonstrated organizational resilience (i.e., the ability to maintain needed health services amid rapidly changing circumstances) by addressing challenges to maintaining care during the pandemic. This study, therefore, aims to identify key facilitators for organizational resilience among AIDS Services Organizations (ASOs) in SC. In-depth interviews were conducted among 11 leaders, from 8 ASOs, across SC during the summer of 2020. The interviews were recorded after receiving proper consent and then transcribed. Utilizing a codebook based upon the interview guide, a thematic analysis approach was utilized to analyze the data. All data management and analysis were conducted in NVivo 11.0. Our findings demonstrate several facilitators of organizational resilience, including (1) accurate and timely crisis information dissemination; (2) clear and preemptive protocols; (3) effective healthcare system policies, management, and leadership; (4) prioritization of staff psychological wellbeing; (5) stable access to personal protective equipment (PPE); (6) adequate and flexible funding; and (7) infrastructure that supports telehealth. Given the facilitators of organizational resilience among ASOs in SC during the COVID-19 pandemic, it is recommended that organizations implement and maintain coordinated and informed responses based upon preemptive protocols and emerging needs. ASO funders are encouraged to allow a flexibility in spending. The lessons learned from the participating leaders enable ASOs to develop and strengthen their organizational resilience and experience fewer disruptions in the future.

Advances in Nucleic Acid Research: Exploring the Potential of Oligonucleotides for Therapeutic Applications and Biological Studies.

In recent years, nucleic acids have emerged as powerful biomaterials, revolutionizing the field of biomedicine. This review explores the multifaceted applications of nucleic acids, focusing on their pivotal role in various biomedical applications. Nucleic acids, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), possess unique properties such as molecular recognition ability, programmability, and ease of synthesis, making them versatile tools in biosensing and for gene regulation, drug delivery, and targeted therapy. Their compatibility with chemical modifications enhances their binding affinity and resistance to degradation, elevating their effectiveness in targeted applications. Additionally, nucleic acids have found utility as self-assembling building blocks, leading to the creation of nanostructures whose high order underpins their enhanced biological stability and affects the cellular uptake efficiency. Furthermore, this review delves into the significant role of oligonucleotides (ODNs) as indispensable tools for biological studies and biomarker discovery. ODNs, short sequences of nucleic acids, have been instrumental in unraveling complex biological mechanisms. They serve as probes for studying gene expression, protein interactions, and cellular pathways, providing invaluable insights into fundamental biological processes. By examining the synergistic interplay between nucleic acids as powerful biomaterials and ODNs as indispensable tools for biological studies and biomarkers, this review highlights the transformative impact of these molecules on biomedical research. Their versatile applications not only deepen our understanding of biological systems but also are the driving force for innovation in diagnostics and therapeutics, ultimately advancing the field of biomedicine.

Development, validation of a GC-MS method for the simultaneous measurement of amino acids, their PTM metabolites and AGEs in human urine, and application to the bi-ethnic ASOS study with special emphasis to lysine.

A gas chromatography-mass spectrometry (GC-MS) method was developed and validated in relevant concentration ranges for the simultaneous measurement of L-lysine (Lys, L) and its Nε- and Nα-methylated (M), Nε- and Nα-acetylated (Ac), Nε-carboxymethylated (CM) and Nε-carboxyethylated (CE) metabolites in human urine. Analyzed Lys metabolites were the post-translational modification (PTM) products Nε-mono-, di- and trimethyllsine, Nε-MML, Nε-DML, Nε-TML, respectively, Nα-ML, Nε-AcL, Nα-AcL, and its advanced glycation end-products (AGEs) Nε-CML, Nε-CM-[2,4,4-2H3]Lys (d3-CML), Nε-CEL and furosine. AGEs of arginine (Arg) and cysteine (Cys) were also analyzed. De novo synthesized trideutero-methyl esters (R-COOCD3) from unlabelled amino acids and derivatives were used as internal standards. Native urine samples (10 µL aliquots) were evaporated to dryness under a stream of nitrogen. Analytes were esterified using 2 M HCl in methanol (60 min, 80 °C) and subsequently amidated by pentafluoropropionic anhydride in ethyl acetate (30 min, 65 °C). The generated methyl ester-pentafluoropropionyl (Me-PFP) derivatives were reconstituted in borate buffer and extracted immediately with toluene. GC-MS analyses were performed by split-less injection of 1-µL aliquots, oven-programmed separation and negative-ion chemical ionization (NICI). Mass spectra were generated in the scan mode (range, m/z 50-1000). Quantification was performed in the selected-ion monitoring (SIM) mode using a dwell time of 50 or 100 ms for each ion. The GC-MS method was suitable for the measurement of Lys and all of its metabolites, except for the quaternary ammonium cation Nε-TML. The Me-PFP derivatives of Lys, Arg and Cys and its metabolites eluted in the retention time window of 9 to 14 min. The derivatization of Nε-CML, d3-CML and Nε-CEL was accompanied by partial Nε-decarboxylation and formation of the Me-PFP Lys derivative. The lowest derivatization yield was observed for Nε-DML, indicating a major role of the Nε-DML group in Lys derivatization. The GC-MS method enables precise (relative standard deviation, RSD < 20%) and accurate (bias, < ± 20%) simultaneous measurement of 33 analytes in human urine in relevant concentration ranges. We used the method to measure the urinary excretion rates of Lys and its PTM metabolites and AGEs in healthy black (n = 39) and white (n = 41) boys of the Arterial Stiffness in Offspring Study (ASOS). No remarkable differences were found indicating no ethnic-related differences in PTM metabolites and AGEs except for Nε-monomethyllysine and S-(2-carboxymethylcysteine).

Specific and sensitive GC-MS analysis of hypusine, Nε-(4-amino-2-hydroxybutyl)lysine, a biomarker of hypusinated eukaryotic initiation factor eIF5A, and its application to the bi-ethnic ASOS study.

Hypusination is a unique two-step enzymatic post-translational modification of the Nε-amino group of lysine-50 of the eukaryotic initiation factor 5A (eIF5A). We developed a specific and sensitive gas chromatography-mass spectrometry (GC-MS) method for the measurement of biological hypusine (Hyp), i.e., Nε-(4-amino-2-hydroxybutyl)lysine. The method includes a two-step derivatization of Hyp: first esterification with 2 M HCl in CH3OH (60 min, 80 °C) to the methyl ester (Me) and then acylation with penta-fluoro-propionic (PFP) anhydride in ethyl acetate (30 min, 65 °C). Esterification with 2 M HCl in CD3OD was used to prepare the internal standard. The major derivatization product was identified as the un-labelled (d0Me) and the deuterium-labelled methyl esters (d3Me) derivatives: d0Me-Hyp-(PFP)5 and d3Me-Hyp-(PFP)5, respectively. Negative-ion chemical ionization generated the most intense ions with m/z 811 for d0Me-Hyp-(PFP)5 and m/z 814 for the internal standard d3Me-Hyp-(PFP)5. Selected-ion monitoring of m/z 811 and m/z 814 was used in quantitative analyses. Free Hyp was found in spot urine samples (10 µL) of two healthy subjects at 0.60 µM (0.29 µmol Hyp/mmol creatinine) in the female and 1.80 µM (0.19 µmol Hyp/mmol creatinine) in the male subject. The mean accuracy of the method in these urine samples spiked with 1-5 µM Hyp was 91-94%. The limit of detection (LOD) of the method is 1.4 fmol Hyp. The method was applied to measure the urinary excretion rates of Hyp in healthy black (n = 38, age 7.8 ± 0.7 years) and white (n = 41, age 7.7 ± 1.0 years) boys of the Arterial Stiffness in Offspring Study (ASOS). The Hyp concentrations were 3.55 [2.68-5.31] µM (range 0.54-9.84 µM) in the black boys and 3.87 [2.95-5.06] µM (range 1.0-11.7 µM) in the white boys (P = 0.64). The creatinine-corrected excretion rates were 0.25 [0.20-0.29] µmol/mmol (range 0.11-0.36 µmol/mmol) in the black boys and 0.26 [0.21-0.30] µmol/mmol (range 0.10-0.45 µmol/mmol) in the white boys (P = 0.82). These results suggest that there is no ethnic-related difference in the ASOS population in the eIF5A modification. Remarkable differences were found between black and white boys with respect to correlations of urinary Hyp with amino acids and advanced glycation end-products of Lys, Arg and Cys. Deoxyhypusine, formally the direct precursor of Hyp, seems not to be excreted in the urine by healthy subjects.

Prospects of NIR fluorescent nanosensors for green detection of SARS-CoV-2.

The pandemic of the novel coronavirus disease 2019 (COVID-19) is continuously causing hazards for the world. Effective detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can relieve the impact, but various toxic chemicals are also released into the environment. Fluorescence sensors offer a facile analytical strategy. During fluorescence sensing, biological samples such as tissues and body fluids have autofluorescence, giving false-positive/negative results because of the interferences. Fluorescence near-infrared (NIR) nanosensors can be designed from low-toxic materials with insignificant background signals. Although this research is still in its infancy, further developments in this field have the potential for sustainable detection of SARS-CoV-2. Herein, we summarize the reported NIR fluorescent nanosensors with the potential to detect SARS-CoV-2. The green synthesis of NIR fluorescent nanomaterials, environmentally compatible sensing strategies, and possible methods to reduce the testing frequencies are discussed. Further optimization strategies for developing NIR fluorescent nanosensors to facilitate greener diagnostics of SARS-CoV-2 for pandemic control are proposed.

Current state of diagnostic, screening and surveillance testing methods for COVID-19 from an analytical chemistry point of view.

Since December 2019, we have been in the battlefield with a new threat to the humanity known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this review, we describe the four main methods used for diagnosis, screening and/or surveillance of SARS-CoV-2: Real-time reverse transcription polymerase chain reaction (RT-PCR); chest computed tomography (CT); and different complementary alternatives developed in order to obtain rapid results, antigen and antibody detection. All of them compare the highlighting advantages and disadvantages from an analytical point of view. The gold standard method in terms of sensitivity and specificity is the RT-PCR. The different modifications propose to make it more rapid and applicable at point of care (POC) are also presented and discussed. CT images are limited to central hospitals. However, being combined with RT-PCR is the most robust and accurate way to confirm COVID-19 infection. Antibody tests, although unable to provide reliable results on the status of the infection, are suitable for carrying out maximum screening of the population in order to know the immune capacity. More recently, antigen tests, less sensitive than RT-PCR, have been authorized to determine in a quicker way whether the patient is infected at the time of analysis and without the need of specific instruments.

Enhanced Potency of GalNAc-Conjugated Antisense Oligonucleotides in Hepatocellular Cancer Models.

Antisense oligonucleotides (ASOs) are a novel therapeutic approach to target difficult-to-drug protein classes by targeting their corresponding mRNAs. Significantly enhanced ASO activity has been achieved by the targeted delivery of ASOs to selected tissues. One example is the targeted delivery of ASOs to hepatocytes, achieved with N-acetylgalactosamine (GalNAc) conjugation to ASO, which results in selective uptake by asialoglycoprotein receptor (ASGR). Here we have evaluated the potential of GalNAc-conjugated ASOs as a therapeutic approach to targeting difficult-to-drug pathways in hepatocellular carcinoma (HCC). The activity of GalNAc-conjugated ASOs was superior to that of the unconjugated parental ASO in ASGR (+) human HCC cells in vitro, but not in ASGR (-) cells. Both human- and mouse-derived HCC displayed reduced levels of ASGR, however, despite this, GalNAc-conjugated ASOs showed a 5- to 10-fold increase in potency in tumors. Systemically administered GalNAc-conjugated ASOs demonstrated both enhanced antisense activity and antitumor activity in the diethylnitrosamine-induced HCC tumor model. Finally, GalNAc conjugation enhanced ASO activity in human circulating tumor cells from HCC patients, demonstrating the potential of this approach in primary human HCC tumor cells. Taken together, these results provide a strong rationale for a potential therapeutic use of GalNAc-conjugated ASOs for the treatment of HCC.

Intrathecal administration of antisense oligonucleotide against p38α but not p38ß MAP kinase isoform reduces neuropathic and postoperative pain and TLR4-induced pain in male mice.

p38 mitogen-activated protein kinase (MAPK) consists of two major isoforms: p38α and p38ß; however, it remains unclear which isoform is more important for chronic pain development. Recently, we developed potent, long-lasting, and p38 MAPK subtype-specific antisense oligonucleotides (ASOs). We examined the therapeutic effects of isoform-specific ASOs in several chronic pain models following single intrathecal injection (300 µg/10 µl) in CD1 mice. In the chronic constriction injury (CCI) model, p38α MAPK ASO, given on post-operative day 5, reduced CCI-induced mechanical allodynia in male but not female mice. In contrast, mechanical allodynia after CCI in both sexes was not affected by p38ß MAPK ASO. Intrathecal injection of p38α or p38ß ASO resulted in a partial reduction (≈ 50%) of spinal p38α or p38ß mRNA level, respectively, in both sexes at two weeks. In contrast, intrathecal injection of the ASOs did not affect p38α and p38ß MAPK mRNA levels in dorsal root ganglia. Intrathecal p38α ASO also reduced postoperative pain (mechanical and cold allodynia) in male mice after tibia fracture. However, intrathecal p38α ASO had no effect on mechanical allodynia in male mice after paclitaxel treatment. Intrathecal p38α MAPK ASO pre-treatment also prevented TLR4-mediated mechanical allodynia and downregulated levels of p38α MAPK and phosphorylated p38 MAPK following intrathecal treatment of lipopolysaccharide. In summary, our findings suggest that p38α MAPK is the major p38 MAPK isoform in the spinal cord and regulates chronic pain in a sex and model-dependent manner. Intrathecal p38α MAPK ASO may offer a new treatment for some chronic pain conditions.

Reliable and versatile immortal muscle cell models from healthy and myotonic dystrophy type 1 primary human myoblasts.

Primary human skeletal muscle cells (hSkMCs) are invaluable tools for deciphering the basic molecular mechanisms of muscle-related biological processes and pathological alterations. Nevertheless, their use is quite restricted due to poor availability, short life span and variable purity of the cells during in vitro culture. Here, we evaluate a recently published method of hSkMCs immortalization, relying on ectopic expression of cyclin D1 (CCND1), cyclin-dependent kinase 4 (CDK4) and telomerase (TERT) in myoblasts from healthy donors (n=3) and myotonic dystrophy type 1 (DM1) patients (n=2). The efficacy to maintain the myogenic and non-transformed phenotype, as well as the main pathogenetic hallmarks of DM1, has been assessed. Combined expression of the three genes i) maintained the CD56(NCAM)-positive myoblast population and differentiation potential; ii) preserved the non-transformed phenotype and iii) maintained the CTG repeat length, amount of nuclear foci and aberrant alternative splicing in immortal muscle cells. Moreover, immortal hSkMCs displayed attractive additional features such as structural maturation of sarcomeres, persistence of Pax7-positive cells during differentiation and complete disappearance of nuclear foci following (CAG)7 antisense oligonucleotide (ASO) treatment. Overall, the CCND1, CDK4 and TERT immortalization yields versatile, reliable and extremely useful human muscle cell models to investigate the basic molecular features of human muscle cell biology, to elucidate the molecular pathogenetic mechanisms and to test new therapeutic approaches for DM1 in vitro.

RNA helicase A is not required for RISC activity.

It has been shown that siRNAs can compete with each other or with endogenous miRNAs for RISC components. This competition may complicate the interpretations of phenotypes observed through siRNA-mediated knockdown of genes, especially those genes implicated in the RISC pathway. In this study, we re-examined the function of RNA helicase A (RHA), which has been previously proposed to function in RISC loading based on siRNA-mediated knockdown studies. Here we show that reduced RISC activity or loading of siRNAs was observed only in cells depleted of RHA using siRNA, but not using RNaseH-dependent antisense oligonucleotides (ASOs), suggesting that the impaired RISC function stems from the competition between pre-existing and newly transfected siRNAs, but not from reduction of the RHA protein. This view is further supported by the findings that cells depleted of a control protein, NCL1, using siRNA, but not ASO, exhibited similar defects on the loading and activity of a subsequently transfected siRNA. Transfection of RHA or NCL1 siRNAs, but not ASOs, reduced the levels of endogenous miRNAs, suggesting a competition mechanism. As a positive control, we showed that reduction of MOV10 by either siRNA or ASO decreased siRNA activity, confirming its role in RISC function. Together, our results indicate that RHA is not required for RISC activity or loading, and suggest that proper controls are required when using siRNAs to functionalize genes to avoid competition effects.

Generation of stable 3'-mRNA cleavage fragments induced by siRNA in cells with high-levels of duck hepatitis B virus replication.

Therapeutic small interfering RNAs (siRNAs) have attracted a lot of interest both in basic biomedical sciences as well as in translational medicine. Apart from their therapeutic efficacy adverse effects of siRNAs must be addressed. The generation of stable mRNA cleavage fragments and the translation of N-truncated proteins induced by antisense oligodeoxynucleotides (ASOs) have been reported. Similar to ASOs, siRNAs are considered to function via an antisense mechanism that promotes the cleavage of the target mRNA. To further investigate whether the stable mRNA cleavage fragments also occur in siRNA we constructed a short hairpin RNA (shRNA) expression plasmid, pshRNA794, containing the same sequence reported in experiments using ASOs which directly targeted the overlapping region of the pre-genomic mRNA (pgmRNA) and sub-genomic mRNA (sgmRNA) of duck hepatitis B virus (DHBV). The shRNA resulted in a 70.9% and 69.9% reduction of the DHBV mRNAs in LMH and HuH-7 cells, respectively. In addition a 70% inhibition of the DHBV DNA level was observed. Interestingly, 3'-mRNA cleavage fragments were detected in LMH but not in HuH-7 cells. Taken together, our findings demonstrate that the ASO sequence was also effective in siRNA. Importantly, our results provide direct evidence that stable 3'-mRNA fragments were generated by siRNA in cells with high levels of DHBV replication. Whether these can cause adverse RNAi effects needs to be explored further.

Androgen receptor modulatory miR-1271-5p can promote hormone sensitive prostate cancer cell growth.

In most patients with advanced prostate cancer treated with hormonal therapy, androgen independence eventually emerges, leading to death. Androgen receptor signalling remains an important prostate cancer driver, even in the advanced disease stage. MicroRNAs (miRs), non-coding RNAs that regulate gene expression by inhibiting translation and/or promoting degradation of target mRNAs, can act as tumour suppressors or "oncomiRs" and modulate tumour growth. Because of their stability in tissues and in circulation, and their specificity, microRNAs have emerged as potential biomarkers, as well as therapeutic targets in cancer. We identified miR-1271-5p as an androgen receptor modulatory microRNA and we show it can promote hormone sensitive prostate cancer cell growth. Inhibition or overexpression of miR-1271-5p levels affects prostate cancer cell growth, apoptosis and expression of both androgen receptor target genes and other genes that are likely direct targets, dependent on androgen receptor status, and tumour stage. We conclude that miR-1271-5p has the potential to drive progression of hormone-dependent disease and that the use of specific inhibitors of miR-1271-5p may have therapeutic potential in prostate cancer.