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.

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.

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.

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.

HSP27/Menin Expression as New Prognostic Serum Biomarkers of Prostate Cancer Aggressiveness Independent of PSA.

The screening of PCa is based on two tests, the total PSA test and the rectal examination. However, PSA is not specific for PCa stage confirmation, leading in false positive result and involving PCa over-diagnosis and over-treatment. HSP27 and Menin have been found to be overexpressed in a wide range of human cancers. Recent studies showed how HSP27 interacts with and stabilizes Menin to lead PCa progression and treatment resistance. The purpose of our study was to evaluate the correlation of HSP27 and Menin molecular expression, and their prognosis value in PCa with respect to clinicopathological features. Elisa was employed to measure serum HSP27 and Menin concentrations in 73 PCa patients and 80 healthy individuals. Immunohistochemistry (IHC) was used to determine HSP27 and Menin tissue expression in 57 tumors and 4 Benign Prostatic Hyperplasia (BPH) tissues. Serum HSP27 expression correlated with its tissue expression in all PCa patients, whereas serum Menin expression correlated only with tissue expression in aggressive PCa patients. Moreover, the results showed a positive correlation between HSP27 and Menin either in serum (r = 0.269; p = 0.021) or in tissue (r = 0.561; p < 0.0001). In aggressive PCa, serum expression of HSP27 and Menin was positively correlated (r = 0.664; R = 0.441; p = 0.001). The correlation between HSP27 and Menin expression in tissue was found only in patients with aggressive PCa (r = 0.606; R = 0.367; p = 0.004). Statistical analysis showed that the expression of both biomarkers was positively correlated with the hormone resistance or sensitivity, tumor aggressiveness, metastasis, Gleason Score, death and did not significantly correlate with age and PSA. Survival was illustrated by Kaplan−Meier curves; increased HSP27 and Menin expression correlated with shorter survival of PCa patients (p = 0.001 and p < 0.0001, respectively). Accuracy in predicting aggressiveness was quantified by the Area Under the Curve (AUC) of Receiver Operating Characteristic (ROC). We demonstrated that the combination of HSP27/Menin was statistically greater than PSA; it achieved an AUC of 0.824 (95% CI, 0.730−0.918; p < 0.0001). However, HSP27/Menin/PSA combination decreased the diagnostic value with an AUC of 0.569 (95% CI, 0.428−0.710; p = 0.645). Our work suggests the potential role of HSP27/Menin as diagnostic and prognostic biomarkers.

Menin inhibition suppresses castration-resistant prostate cancer and enhances chemosensitivity.

Disease progression and therapeutic resistance of prostate cancer (PC) are linked to multiple molecular events that promote survival and plasticity. We previously showed that heat shock protein 27 (HSP27) acted as a driver of castration-resistant phenotype (CRPC) and developed an oligonucleotides antisense (ASO) against HSP27 with evidence of anti-cancer activity in men with CRPC. Here, we show that the tumor suppressor Menin (MEN1) is highly regulated by HSP27. Menin is overexpressed in high-grade PC and CRPC. High MEN1 mRNA expression is associated with decreased biochemical relapse-free and overall survival. Silencing Menin with ASO technology inhibits CRPC cell proliferation, tumor growth, and restores chemotherapeutic sensitivity. ChIP-seq analysis revealed differential DNA binding sites of Menin in various prostatic cells, suggesting a switch from tumor suppressor to oncogenic functions in CRPC. These data support the evaluation of ASO against Menin for CRPC.

Antisense Oligonucleotide-Based Therapeutic against Menin for Triple-Negative Breast Cancer Treatment.

The tumor suppressor menin has dual functions, acting either as a tumor suppressor or as an oncogene/oncoprotein, depending on the oncological context. Triple-negative breast cancer (TNBC) is characterized by the lack of expression of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (ERBB2/HER2) and is often a basal-like breast cancer. TNBC is associated with a dismal prognosis and an insufficient response to chemotherapies. Previously, menin was shown to play a proliferative role in ER-positive breast cancer; however, the functions of menin in TNBC remain unknown. Here, we have demonstrated that menin is expressed in various TNBC subtypes with the strongest expression in the TNBC Hs 578T cells. The depletion of menin by an antisense oligonucleotide (ASO) inhibits cell proliferation, enhances apoptosis in Hs 578T cells, highlighting the oncogenic functions of menin in this TNBC model. ASO-based menin silencing also delays the tumor progression of TNBC xenografts. Analysis of the menin interactome suggests that menin could drive TNBC tumorigenesis through the regulation of MLL/KMT2A-driven transcriptional activity, mRNA 3'-end processing and apoptosis. The study provides a rationale behind the use of ASO-based therapy, targeting menin in monotherapy or in combination with chemo or PARP inhibitors for menin-positive TNBC treatments.

Hydrogel based lipid-oligonucleotides: a new route to self-delivery of therapeutic sequences.

Synthetic OligoNucleotides (ON) provide promising therapeutic tools for controlling specifically genetic expression in a broad range of diseases from cancers to viral infections. Beside their chemical stability and intracellular delivery, the controlled release of therapeutic sequences remains an important challenge for successful clinical applications. In this work, Lipid-OligoNucleotide (LON) conjugates stabilizing hydrogels are reported and characterized by rheology and cryo-scanning electron microscopy (cryo-SEM). These studies revealed that lipid conjugation of antisense oligonucleotides featuring partial self-complementarity resulted in entangled pearl-necklace networks, which were obtained through micelle-micelle interaction driven by duplex formation. Owing to these properties, the Lipid AntiSense Oligonucleotide (LASO) sequences exhibited a prolonged release after subcutaneous administration compared to the non-lipidic antisense (ASO) one. The LASO self-assembly based hydrogels obtained without adjuvant represent an innovative approach for the sustained self-delivery of therapeutic oligonucleotides.

Nanovectorization of Prostate Cancer Treatment Strategies: A New Approach to Improved Outcomes.

Prostate cancer (PC) is the most frequent male cancer in the Western world. Progression to Castration Resistant Prostate Cancer (CRPC) is a known consequence of androgen withdrawal therapy, making CRPC an end-stage disease. Combination of cytotoxic drugs and hormonal therapy/or genotherapy is a recognized modality for the treatment of advanced PC. However, this strategy is limited by poor bio-accessibility of the chemotherapy to tumor sites, resulting in an increased rate of collateral toxicity and incidence of multidrug resistance (MDR). Nanovectorization of these strategies has evolved to an effective approach to efficacious therapeutic outcomes. It offers the possibility to consolidate their antitumor activity through enhanced specific and less toxic active or passive targeting mechanisms, as well as enabling diagnostic imaging through theranostics. While studies on nanomedicine are common in other cancer types, only a few have focused on prostate cancer. This review provides an in-depth knowledge of the principles of nanotherapeutics and nanotheranostics, and how the application of this rapidly evolving technology can clinically impact CRPC treatment. With particular reference to respective nanovectors, we draw clinical and preclinical evidence, demonstrating the potentials and prospects of homing nanovectorization into CRPC treatment strategies.

Nucleoside-Lipid-Based Nanoparticles for Phenazine Delivery: A New Therapeutic Strategy to Disrupt Hsp27-eIF4E Interaction in Castration Resistant Prostate Cancer.

Heat shock protein 27 (Hsp27) has an established role in tumor progression and chemo-resistance of castration-resistant prostate cancer (CRPC). Hsp27 protects eukaryotic translation initiation factor 4E (eIF4E) from degradation, thereby maintaining survival during treatment. Phenazine derivative compound #14 was demonstrated to specifically disrupt Hsp27/eIF4E interaction and significantly delay castration-resistant tumor progression in prostate cancer xenografts. In the present work, various strategies of encapsulation of phenazine #14 with either DOTAU (N-[5'-(2',3'-dioleoyl)uridine]-N',N',N'-trimethylammonium tosylate) and DOU-PEG2000 (5'-PEG2000-2',3'-dioleoyluridine) nucleolipids (NLs) were developed in order to improve its solubilization, biological activity, and bioavailability. We observed that NLs-encapsulated phenazine #14-driven Hsp27-eIF4E interaction disruption increased cytotoxic effects on castration-resistant prostate cancer cell line and inhibited tumor growth in castration-resistant prostate cancer cell xenografted mice compared to phenazine #14 and NLs alone. Phenazine #14 NL encapsulation might represent an interesting nanostrategy for CRPC therapy.

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.

Enhanced Antisense Oligonucleotide Delivery Using Cationic Liposomes Grafted with Trastuzumab: A Proof-of-Concept Study in Prostate Cancer.

Prostate cancer (PCa) is the second most common cancer in men worldwide and the fifth leading cause of death by cancer. The overexpression of TCTP protein plays an important role in castration resistance. Over the last decade, antisense technology has emerged as a rising strategy in oncology. Using antisense oligonucleotide (ASO) to silence TCTP protein is a promising therapeutic option-however, the pharmacokinetics of ASO does not always meet the requirements of proper delivery to the tumor site. In this context, developing drug delivery systems is an attractive strategy for improving the efficacy of ASO directed against TCTP. The liposome should protect and deliver ASO at the intracellular level in order to be effective. In addition, because prostate cancer cells express Her2, using an anti-Her2 targeting antibody will increase the affinity of the liposome for the cell and optimize the intratumoral penetration of the ASO, thus improving efficacy. Here, we have designed and developed pegylated liposomes and Her2-targeting immunoliposomes. Mean diameter was below 200 nm, thus ensuring proper enhanced permeation and retention (EPR) effect. Encapsulation rate for ASO was about 40%. Using human PC-3 prostate cancer cells as a canonical model, free ASO and ASO encapsulated into either liposomes or anti-Her2 immunoliposomes were tested for efficacy in vitro using 2D and 3D spheroid models. While the encapsulated forms of ASO were always more effective than free ASO, we observed differences in efficacy of encapsulated ASO. For short exposure times (i.e., 4 h) ASO liposomes (ASO-Li) were more effective than ASO-immunoliposomes (ASO-iLi). Conversely, for longer exposure times, ASO-iLi performed better than ASO-Li. This pilot study demonstrates that it is possible to encapsulate ASO into liposomes and to yield antiproliferative efficacy against PCa. Importantly, despite mild Her2 expression in this PC-3 model, using a surface mAb as targeting agent provides further efficacy, especially when exposure is longer. Overall, the development of third-generation ASO-iLi should help to take advantage of the expression of Her2 by prostate cancer cells in order to allow greater specificity of action in vivo and thus a gain in efficacy.

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.

Self-assembly of amphiphilic phospholipid peptide dendrimer-based nanovectors for effective delivery of siRNA therapeutics in prostate cancer therapy.

RNA interference (RNAi) holds great promise for therapeutic applications. However, safe and successful clinical translation essentially requires further advancement of developing efficient delivery systems. Herein, we report that amphiphilic phospholipid peptide dendrimers (AmPPDs) could mediated effective delivery of siRNA targeting Hsp27 for treating castration-resistant prostate cancer (CRPC). AmPPDs bears natural lipid derivative DSPE as the hydrophobic tail and different dendritic l-lysine as the hydrophilic head, capable of compacting siRNA into nanoparticles to protect it from enzymatic degradation. Interestingly, DSPE-KK2, AmPPD bearing smaller hydrophilic dendron, promoting more efficient intracellular uptake and endosome release of the so-formed siRNA complexes, as well as better siRNA releasing ability, ultimately resulting in more potent gene silencing and anticancer effects both in vitro and in vivo. Such outstanding performance of DSPE-KK2 in siRNA delivery may attribute to its optimal balance between the hydrophobic tail and hydrophilic dendritic portion. Our findings provide guidance for the development of safe and effective dendrimer-based siRNA delivery system, thus bringing new hope for combating various diseases.

Integrative proteomic and phosphoproteomic profiling of prostate cell lines.

BACKGROUND: Prostate cancer is a major public health issue, mainly because patients relapse after androgen deprivation therapy. Proteomic strategies, aiming to reflect the functional activity of cells, are nowadays among the leading approaches to tackle the challenges not only of better diagnosis, but also of unraveling mechanistic details related to disease etiology and progression. METHODS: We conducted here a large SILAC-based Mass Spectrometry experiment to map the proteomes and phosphoproteomes of four widely used prostate cell lines, namely PNT1A, LNCaP, DU145 and PC3, representative of different cancerous and hormonal status. RESULTS: We identified more than 3000 proteins and phosphosites, from which we quantified more than 1000 proteins and 500 phosphosites after stringent filtering. Extensive exploration of this proteomics and phosphoproteomics dataset allowed characterizing housekeeping as well as cell-line specific proteins, phosphosites and functional features of each cell line. In addition, by comparing the sensitive and resistant cell lines, we identified protein and phosphosites differentially expressed in the resistance context. Further data integration in a molecular network highlighted the differentially expressed pathways, in particular migration and invasion, RNA splicing, DNA damage repair response and transcription regulation. CONCLUSIONS: Overall, this study proposes a valuable resource toward the characterization of proteome and phosphoproteome of four widely used prostate cell lines and reveals candidates to be involved in prostate cancer progression for further experimental validation.

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.

Correction: Targeting Hsp27/eIF4E interaction with phenazine compound: a promising alternative for castration-resistant prostate cancer treatment.

[This corrects the article DOI: 10.18632/oncotarget.20469.].