Coronavirus RNA Proofreading: Molecular Basis and Therapeutic Targeting.

The coronavirus disease 2019 (COVID-19) that is wreaking havoc on worldwide public health and economies has heightened awareness about the lack of effective antiviral treatments for human coronaviruses (CoVs). Many current antivirals, notably nucleoside analogs (NAs), exert their effect by incorporation into viral genomes and subsequent disruption of viral replication and fidelity. The development of anti-CoV drugs has long been hindered by the capacity of CoVs to proofread and remove mismatched nucleotides during genome replication and transcription. Here, we review the molecular basis of the CoV proofreading complex and evaluate its potential as a drug target. We also consider existing nucleoside analogs and novel genomic techniques as potential anti-CoV therapeutics that could be used individually or in combination to target the proofreading mechanism.

Nucleic Acid-Based Technologies Targeting Coronaviruses.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently creating a global health emergency. This crisis is driving a worldwide effort to develop effective vaccines, prophylactics, and therapeutics. Nucleic acid (NA)-based treatments hold great potential to combat outbreaks of coronaviruses (CoVs) due to their rapid development, high target specificity, and the capacity to increase druggability. Here, we review key anti-CoV NA-based technologies, including antisense oligonucleotides (ASOs), siRNAs, RNA-targeting clustered regularly interspaced short palindromic repeats-CRISPR-associated protein (CRISPR-Cas), and mRNA vaccines, and discuss improved delivery methods and combination therapies with other antiviral drugs.

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.

A Dual Targeting Dendrimer-Mediated siRNA Delivery System for Effective Gene Silencing in Cancer Therapy.

Small interfering RNA (siRNA) is emerging as a novel therapeutic for treating various diseases, provided a safe and efficient delivery is available. In particular, specific delivery to target cells is critical for achieving high therapeutic efficacy while reducing toxicity. Amphiphilic dendrimers are emerging as novel promising carriers for siRNA delivery by virtue of the combined multivalent cooperativity of dendrimers with the self-assembling property of lipid vectors. Here, we report a ballistic approach for targeted siRNA delivery to cancer cells using an amphiphilic dendrimer equipped with a dual targeting peptide bearing an RGDK warhead. According to the molecular design, the amphiphilic dendrimer was expected to deliver siRNA effectively, while the aim of the targeting peptide was to home in on tumors via interaction of its warhead with integrin and the neuropilin-1 receptor on cancer cells. Coating the positively charged siRNA/dendrimer delivery complex with the negatively charged segment of the targeting peptide via electrostatic interactions led to small and stable nanoparticles which were able to protect siRNA from degradation while maintaining the accessibility of RGDK for targeting cancer cells and preserving the ability of the siRNA to escape from endosomes. The targeted system had enhanced siRNA delivery, stronger gene silencing, and more potent anticancer activity compared to nontargeted or covalent dendrimer-based systems. In addition, neither acute toxicity nor induced inflammation was observed. Consequently, this delivery system constitutes a promising nonviral vector for targeted delivery and can be further developed to provide RNAi-based personalized medicine against cancer. Our study also gives new perspectives on the use of nanotechnology based on self-assembling dendrimers in various biomedical applications.

Anticancer drug nanomicelles formed by self-assembling amphiphilic dendrimer to combat cancer drug resistance.

Drug resistance and toxicity constitute challenging hurdles for cancer therapy. The application of nanotechnology for anticancer drug delivery is expected to address these issues and bring new hope for cancer treatment. In this context, we established an original nanomicellar drug delivery system based on an amphiphilic dendrimer (AmDM), which could generate supramolecular micelles to effectively encapsulate the anticancer drug doxorubicin (DOX) with high drug-loading capacity (>40%), thanks to the unique dendritic structure creating large void space for drug accommodation. The resulting AmDM/DOX nanomicelles were able to enhance drug potency and combat doxorubicin resistance in breast cancer models by significantly enhancing cellular uptake while considerably decreasing efflux of the drug. In addition, the AmDM/DOX nanoparticles abolished significantly the toxicity related to the free drug. Collectively, our studies demonstrate that the drug delivery system based on nanomicelles formed with the self-assembling amphiphilic dendrimer constitutes a promising and effective drug carrier in cancer therapy.

Mastering Dendrimer Self-Assembly for Efficient siRNA Delivery: From Conceptual Design to In Vivo Efficient Gene Silencing.

Self-assembly is a fundamental concept and a powerful approach in molecular science. However, creating functional materials with the desired properties through self-assembly remains challenging. In this work, through a combination of experimental and computational approaches, the self-assembly of small amphiphilic dendrons into nanosized supramolecular dendrimer micelles with a degree of structural definition similar to traditional covalent high-generation dendrimers is reported. It is demonstrated that, with the optimal balance of hydrophobicity and hydrophilicity, one of the self-assembled nanomicellar systems, totally devoid of toxic side effects, is able to deliver small interfering RNA and achieve effective gene silencing both in cells - including the highly refractory human hematopoietic CD34(+) stem cells - and in vivo, thus paving the way for future biomedical implementation. This work presents a case study of the concept of generating functional supramolecular dendrimers via self-assembly. The ability of carefully designed and gauged building blocks to assemble into supramolecular structures opens new perspectives on the design of self-assembling nanosystems for complex and functional applications.

The functional landscape of Hsp27 reveals new cellular processes such as DNA repair and alternative splicing and proposes novel anticancer targets.

Previously, we identified the stress-induced chaperone, Hsp27, as highly overexpressed in castration-resistant prostate cancer and developed an Hsp27 inhibitor (OGX-427) currently tested in phase I/II clinical trials as a chemosensitizing agent in different cancers. To better understand the Hsp27 poorly-defined cytoprotective functions in cancers and increase the OGX-427 pharmacological safety, we established the Hsp27-protein interaction network using a yeast two-hybrid approach and identified 226 interaction partners. As an example, we showed that targeting Hsp27 interaction with TCTP, a partner protein identified in our screen increases therapy sensitivity, opening a new promising field of research for therapeutic approaches that could decrease or abolish toxicity for normal cells. Results of an in-depth bioinformatics network analysis allying the Hsp27 interaction map into the human interactome underlined the multifunctional character of this protein. We identified interactions of Hsp27 with proteins involved in eight well known functions previously related to Hsp27 and uncovered 17 potential new ones, such as DNA repair and RNA splicing. Validation of Hsp27 involvement in both processes in human prostate cancer cells supports our system biology-predicted functions and provides new insights into Hsp27 roles in cancer cells.

Arginine-terminated generation 4 PAMAM dendrimer as an effective nanovector for functional siRNA delivery in vitro and in vivo.

Successful therapeutic implementation of RNA interference critically depends on systems able to safely and efficiently deliver small interfering RNA (siRNA). Dendrimers are emerging as appealing nanovectors for siRNA delivery by virtue of their unique well-defined dendritic nanostructure within which is confined an intriguing cooperativity and multivalency. We have previously demonstrated that structurally flexible triethanolamine (TEA) core poly(amidoamine) (PAMAM) dendrimers of high generations are effective nanovectors for siRNA delivery in vitro and in vivo. In the present study, we have developed arginine-terminated dendrimers with the aim of combining and harnessing the unique siRNA delivery properties of the TEA-core PAMAM dendrimer and the cell-penetrating advantages of the arginine-rich motif. A generation 4 dendrimer of this family (G4Arg) formed stable dendriplexes with siRNA, leading to improved cell uptake of siRNA by comparison with its nonarginine bearing dendrimer counterpart. Moreover, G4Arg was demonstrated to be an excellent nanocarrier for siRNA delivery, yielding potent gene silencing and anticancer effects in prostate cancer models both in vitro and in vivo with no discernible toxicity. Consequently, importing an arginine residue on the surface of a dendrimer is an appealing option to improve delivery efficiency, and at the same time, the dendrimer G4Arg constitutes a highly promising nanovector for efficacious siRNA delivery and holds great potential for further therapeutic applications.

Targeted delivery of Dicer-substrate siRNAs using a dual targeting peptide decorated dendrimer delivery system.

Small interfering RNAs (siRNA) are emerging as novel therapeutic agents, providing competent delivery systems that are available. Dendrimers, a special family of synthetic macromolecules, represent an exciting delivery platform by virtue of their well-defined dendritic structure and unique multivalency and cooperativity confined within a nanoscale volume. Here, we report a Dicer-substrate siRNA (dsiRNA) which, when delivered using a structurally flexible triethanolamine-core poly(amidoamine) dendrimer of generation 5 as the nanocarrier, gives rise to a much greater RNAi response than that produced with conventional siRNA. Further decoration of the dsiRNA/dendrimer complexes with a dual targeting peptide simultaneously promoted cancer cell targeting through interacting with integrins and cell penetration via the interaction with neuropilin-1 receptors, which led to improved gene silencing and anticancer activity. Altogether, our results disclosed here open a new avenue for therapeutic implementation of RNAi using dendrimer nanovector based targeted delivery. FROM THE CLINICAL EDITOR: This study demonstrates superior therapeutic properties of siRNA when combined with a dendrimer-based targeted nano-delivery system. Similar approaches may eventually gain clinical utility following additional studies determining safety and efficacy.

Adaptive amphiphilic dendrimer-based nanoassemblies as robust and versatile siRNA delivery systems.

siRNA delivery remains a major challenge in RNAi-based therapy. Here, we report for the first time that an amphiphilic dendrimer is able to self-assemble into adaptive supramolecular assemblies upon interaction with siRNA, and effectively delivers siRNAs to various cell lines, including human primary and stem cells, thereby outperforming the currently available nonviral vectors. In addition, this amphiphilic dendrimer is able to harness the advantageous features of both polymer and lipid vectors and hence promotes effective siRNA delivery. Our study demonstrates for the first time that dendrimer-based adaptive supramolecular assemblies represent novel and versatile means for functional siRNA delivery, heralding a new age of dendrimer-based self-assembled drug delivery in biomedical applications.

Between hope and reality: treatment of genetic diseases through nucleic acid-based drugs.

Rare diseases (RD) affect a small number of people compared to the general population and are mostly genetic in origin. The first clinical signs often appear at birth or in childhood, and patients endure high levels of pain and progressive loss of autonomy frequently associated with short life expectancy. Until recently, the low prevalence of RD and the gatekeeping delay in their diagnosis have long hampered research. The era of nucleic acid (NA)-based therapies has revolutionized the landscape of RD treatment and new hopes arise with the perspectives of disease-modifying drugs development as some NA-based therapies are now entering the clinical stage. Herein, we review NA-based drugs that were approved and are currently under investigation for the treatment of RD. We also discuss the recent structural improvements of NA-based therapeutics and delivery system, which overcome the main limitations in their market expansion and the current approaches that are developed to address the endosomal escape issue. We finally open the discussion on the ethical and societal issues that raise this new technology in terms of regulatory approval and sustainability of production.

Impact of siRNA overhangs for dendrimer-mediated siRNA delivery and gene silencing.

Small interfering RNA (siRNA) have attracted considerable attention, as compelling therapeutics providing safe and competent delivery systems are available. Dendrimers are emerging as appealing siRNA delivery vectors thanks to their unique, well-defined architecture and the resulting cooperativity and multivalency confined within a nanostructure. We have recently disclosed the structurally flexible fifth-generation TEA-core PAMAM dendrimer (G5) as an effective nanocarrier for delivery of sticky siRNA bearing long complementary sequence overhangs (dA)n/(dT)n (n = 5 or 7). Here, using combined experimental/computational approaches, we successfully clarified (i) the underlying mechanisms of interaction between the dendrimer nanovector G5 and siRNA molecules bearing either complementary or noncomplementary sequence overhangs of different length and chemistry and (ii) the impact of siRNA overhangs contributing toward the improved delivery potency. Using siRNA with complementary overhangs offer the best action in term of gene silencing through the formation of concatemers, that is, supramolecular structures resulting from synergistic and cooperative binding via (dA)n/(dT)n bridges (n = 5 or 7). On the other hand, although siRNA bearing long, noncomplementary overhangs (dA)n/(dA)n or (dT)n/(dT)n (n = 5 or 7) are endowed with considerably higher gene silencing potency than normal siRNA with (dT)2/(dT)2, they remain less effective than their sticky siRNA counterparts. The observed gene silencing potency depends on length, nature, and flexibility of the overhangs, which behave as a sort of clamps that hold and interact with the dendrimer nanovectors, thus impacting siRNA delivery performance and, ultimately, gene silencing. Our findings can be instrumental in designing siRNA entities with enhanced capability to achieve effective RNA interference for therapeutic applications.

Targeting TCTP as a new therapeutic strategy in castration-resistant prostate cancer.

Heat shock protein 27 (Hsp27) is highly overexpressed in castration-resistant prostate cancer (CRPC) and an antisense inhibitor (OGX-427) is currently in phase II clinical trials. In order to understand mechanisms of action of Hsp27 and find new therapeutic targets specific of CRPC, we screened for Hsp27 client proteins. Here, we report that translationally controlled tumor protein (TCTP) is a new Hsp27 client protein involved in Hsp27 cytoprotection. We found that TCTP expression is absent or weak in normal prostate cells, moderately expressed in 18.5% of treatment naive PC, and becomes uniformly and strongly expressed in 75% of CRPC. To define TCTP function, we developed and worldwide patented a TCTP antisense oligonucleotide (ASO). Interestingly, we found that CRPC progression correlates with TCTP overexpression and loss of P53. TCTP knockdown restored P53 expression and function, suggesting that castration-sensitivity is directly linked to P53 expression. Collectively, these findings provide a new Hsp27 cytoprotection mechanism in CRPC, and preclinical proof-of-concept that combining ASO-mediated TCTP knockdown with castration and/or docetaxel therapy could serve as a novel strategy to treat CRPC, with no or little toxicity for normal prostate cells.

ZNF703 mRNA-Targeting Antisense Oligonucleotide Blocks Cell Proliferation and Induces Apoptosis in Breast Cancer Cell Lines.

The luminal B molecular subtype of breast cancers (BC) accounts for more than a third of BCs and is associated with aggressive clinical behavior and poor prognosis. The use of endocrine therapy in BC treatment has significantly contributed to the decrease in the number of deaths in recent years. However, most BC patients with prolonged exposure to estrogen receptor (ER) selective modulators such as tamoxifen develop resistance and become non-responsive over time. Recent studies have implicated overexpression of the ZNF703 gene in BC resistance to endocrine drugs, thereby highlighting ZNF703 inhibition as an attractive modality in BC treatment, especially luminal B BCs. However, there is no known inhibitor of ZNF703 due to its nuclear association and non-enzymatic activity. Here, we have developed an antisense oligonucleotide (ASO) against ZNF703 mRNA and shown that it downregulates ZNF703 protein expression. ZNF703 inhibition decreased cell proliferation and induced apoptosis. Combined with cisplatin, the anti-cancer effects of ZNF703-ASO9 were improved. Moreover, our work shows that ASO technology may be used to increase the number of targetable cancer genes.

Castration-Resistant Prostate Cancer: From Uncovered Resistance Mechanisms to Current Treatments.

Prostate cancer (PC) is the second most common cancer in men worldwide. Despite recent advances in diagnosis and treatment, castration-resistant prostate cancer (CRPC) remains a significant medical challenge. Prostate cancer cells can develop mechanisms to resist androgen deprivation therapy, such as AR overexpression, AR mutations, alterations in AR coregulators, increased steroidogenic signaling pathways, outlaw pathways, and bypass pathways. Various treatment options for CRPC exist, including androgen deprivation therapy, chemotherapy, immunotherapy, localized or systemic therapeutic radiation, and PARP inhibitors. However, more research is needed to combat CRPC effectively. Further investigation into the underlying mechanisms of the disease and the development of new therapeutic strategies will be crucial in improving patient outcomes. The present work summarizes the current knowledge regarding the underlying mechanisms that promote CRPC, including both AR-dependent and independent pathways. Additionally, we provide an overview of the currently approved therapeutic options for CRPC, with special emphasis on chemotherapy, radiation therapy, immunotherapy, PARP inhibitors, and potential combination strategies.

TP53INP1 as new therapeutic target in castration-resistant prostate cancer.

BACKGROUND: Prostate cancer (PC) is one of the most common malignancies in industrialized countries, and the second leading cause of cancer-related death in the United States. We recently showed that over-expression of tumor protein 53-induced nuclear protein 1 (TP53INP1), a cell stress response protein, is a worse prognostic factor in PC, particularly predictive of biological cancer relapse. Moreover, treatment of castration-sensitive (CS) LNCaP tumor cells with a TP53INP1 antisense oligonucleotide (TP53INP1 ASO) inhibits proliferation and induces apoptosis. The aim of this study was to investigate variations of TP53INP1 expression in PC during androgen withdrawal therapy and in castration-resistant prostate cancer (CRPC). METHODS: Quantitative measurements of immunohistochemical expression of TP53INP1 using high-throughput densitometry, assessed on digitized microscopic tissue micro-array images were correlated with hormone therapy (HT) status in human PC. Northern blot analysis of TP53INP1 after castration was performed in LNCaP xenograft. Treatment of CR C4-2 tumor cells in vitro with TP53INP1 ASO was analyzed. We also analyzed the effect of TP53INP1 ASO treatment in vivo on tumor xenograft growth. RESULTS: TP53INP1 protein expression decreases during HT and increases after HT in human CRPC. TP53INP1 mRNA increases significantly in CR tumors of LNCaP xenograft. Moreover, treatment of CR C4-2 cells with TP53INP1 ASO downregulates TP53INP1 protein level, inhibits proliferation, and induces apoptosis. Finally, in vivo, TP53INP1 ASO treatment significantly inhibits the tumoral progression of CR C4-2 xenograft and enhances docetaxel cytotoxicity. CONCLUSIONS: These results suggest that TP53INP1 could be considered as a relevant-specific target for molecular therapy of CRPC.

TP53INP1 overexpression in prostate cancer correlates with poor prognostic factors and is predictive of biological cancer relapse.

BACKGROUND: Tumor protein 53-induced nuclear protein 1 (TP53INP1) is a proapoptotic protein involved in cell stress response. Whereas there is an overexpression of TP53INP1 in numerous tissues submitted to stress agents, TP53INP1 is down-expressed in stomach, pancreatic, and inflammation-mediated colic carcinomas. In medullary thyroid carcinomas, TP53INP1 overexpression correlates with poor prognosis. TP53INP1 expression has never been reported in Prostate Cancer (PC). Our aim was to investigate variations of TP53INP1 expression and their correlation to clinicopathological parameters in PC. METHODS: Quantitative measurements of immunohistochemical expression of TP53INP1 using high-throughput densitometry, assessed on digitized microscopic tissue micro-array images, were correlated with clinicopathological parameters in 91 human PC. Treatment of LNCaP tumor cells in vitro with cytokines and with TP53INP1 antisense oligonucleotide (ASO) was also analyzed. RESULTS: In normal prostate tissues, TP53INP1 is only expressed in prostate basal cells. There is a de novo TP53INP1 expression in prostate luminal cells in inflammatory prostate tissues, high grade PIN lesions and in PC. Stimulation of LNCaP cells with inflammatory cytokines enhances the level of TP53INP1 mRNA. In PC, TP53INP1 overexpression correlates with high Gleason grade, unfavorable D'Amico score and lymph node invasion, and is an independent factor of biological cancer relapse. Moreover, treatment of LNCaP cells with a TP53INP1 ASO down-regulates TP53INP1 protein level, inhibits proliferation, and induces apoptosis. CONCLUSION: TP53INP1 overexpression in PC seems to be a worse prognostic factor, particularly predictive of biological cancer relapse. Results in vitro suggest that TP53INP1 could be considered as a relevant target for potential specific therapy.

An efficient mixed-ligand Pd catalytic system to promote C-N coupling for the synthesis of N-arylaminotriazole nucleosides.

Make it unique! A mixed-ligand system of Pd/Synphos/Xantphos promotes effective C-N coupling in the synthesis of various N-arylaminotriazole and N-arylaminopurine nucleoside analogues. This catalytic system is strikingly powerful and efficient, allowing for unparalleled substrate scope and high product yields as well as promotion of C-Cl bond activation for C-N coupling (see scheme).