siRNA Molecules Research Articles

Inclisiran: A New Promising Agent in the Management of Hypercholesterolemia.

The discovery of proprotein convertase subtilisin-kexin type 9 (PCSK9), a serine protease which binds to the low-density lipoprotein (LDL) receptors and targets the receptors for lysosomal degradation, offered an additional route through which plasma LDL-cholesterol (LDL-C) levels can be controlled. Initially, the therapeutic approaches to reduce circulating levels of PCSK9 were focused on the use of monoclonal antibodies. To that effect, evolocumab and alirocumab, two human monoclonal antibodies directed against PCSK9, given on a background of statin therapy, have been shown to markedly decrease LDL-C levels and significantly reduce cardiovascular risk. The small interfering RNA (siRNA) molecules have been used recently to target the hepatic production of PCSK9. siRNA interferes with the expression of specific genes with complementary nucleotide sequences by affecting the degradation of mRNA post-transcription, thus preventing translation. Inclisiran is a long-acting, synthetic siRNA directed against PCSK9 and it has been shown to significantly decrease hepatic production of PCSK9 and cause a marked reduction in LDL-C levels. This review aims to present and discuss the current clinical and scientific evidence pertaining to inclisiran, which is a new promising agent in the management of hypercholesterolemia.

Abstract 3969: Lipocalin-2-targeted small interference RNA for inflammatory breast cancer treatment

Abstract Inflammatory Breast Cancer (IBC) is an aggressive form of invasive breast cancer with low survival rate. Available therapies to treat this type of cancer are almost limited to surgery and radiation therapy. Potential therapeutic targets for IBC treatment include the oncogene Her2 and the Epidermal Growth Factor Receptor (EGFR). However, these therapies have not been successful since not all IBC tumors are Her2-amplified and development of resistance to treatment has also been observed. Hence, there is a critical need for novel therapeutic targets for IBC treatment. One of such possibilities is Lipocalin-2 (LNC2). LCN-2 is a secreted glycoprotein and an adipokine superfamily member that posses roles in transport of small lipophilic molecules in circulation, innate immunity, and iron homeostasis. High levels of LCN2 have been observed in invasive breast cancer human tissue. Additionally, LCN2 has been linked with breast cancer tumorigenesis and metastasis. Therefore, the purpose of our study is to assess if LCN2 is a plausible therapeutic target to treat IBC. Western blot analysis was used to assess the LCN2 protein level in a panel of IBC (MDA-IBC-3, SUM-149) and non-IBC cell lines (MCF-7, MDA-MB-231, MDA-MB-438). IBC cell lines showed a significantly higher expression of the LCN2 protein in comparison to the non-IBC cell lines. We decreased the expression of the LCN2 protein with small interference RNA (siRNA) molecules. Silencing of IBC cells with LCN2-targeted siRNA molecules decreased the LCN2 protein expression levels in almost fifty percent. Colony formation assay demonstrated that siRNA-mediated LCN2 silencing in MDA-IBC-3 and SUM-149 cell lines resulted in a significant decrease in cell proliferation. Taken together these results suggest that LCN2 may become a right target for IBC treatment. Ongoing experiments include cell invasion assays to determine if LCN2-targeted siRNAs reduce the metastatic potential, increase apoptosis and block cell cycle progression. Acknowledgements: This work is supported by the University of Puerto Rico Comprehensive Cancer Center and the U54 partnership with MD Anderson Cancer Research Center. Citation Format: Ginette S. Santiago-Sanchez, Fatma Valiyeva, Bisrat Debeb, Pablo E. Mejia. Lipocalin-2-targeted small interference RNA for inflammatory breast cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3969.

Abstract 3836: Imbalanced nucleotide metabolism sensitizes breast cancer cells to anthracyclines

Abstract Introduction. Triple negative breast cancer (TNBC) makes up 15% of breast cancers and is associated with poor prognosis. TNBC treatment remains hampered by early visceral metastasis and lymph node involvement at the time of diagnosis and limited effective therapeutic options. Advances in treatment that translate to significant improvements in outcome have been painstakingly incremental, despite advances in research technologies and TNBC subtyping. We have identified deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) as a critical gatekeeper that protects tumor DNA from the genotoxic misincorporation of uracil during treatment with anthracyclines that are commonly used in the FEC chemotherapy regimen. dUTPase catalyses the hydrolytic dephosphorylation of deoxyuridine triphosphate (dUTP) to deoxyuridine monophosphate (dUMP). This reaction has the dual function of providing dUMP for thymidylate synthase (TS) as part of the thymidylate biosynthesis pathway and of maintaining low intracellular dUTP pools. This is crucial as DNA polymerase cannot distinguish between dUTP and deoxythymidylate triphosphate (dTTP), leading to dUTP misincorporated into DNA. dUTPase is being evaluated as a target in cancer therapy alongside TS-targeted therapies and uracil misincorporation is believed to be a potential mechanism of cytotoxicity. Hypothesis: Targeting dUTPase and inducing uracil misincorporation during the repair of DNA damage induced by anthracyclines represents a novel strategy to induce TNBC cell lethality. Methods. Inhibition of dUTPase (DUTi) was carried out using SMARTpool siRNA or small molecule inhibition. The effect of DUTi in combination with anthracyclines was determined by growth inhibition and clonogenics assays. DNA damage was assessed by Western blot, immune-fluorescent foci detection, and flow cytometry. Nucleotide metabolites were quantified by LC/MS. Results. DUTi significantly sensitised TNBC cell lines to doxorubicin and epirubicin. In the MDA-MB-231 cancer cells, loss of dUTPase expression resulted in a synergistic 80% reduction in survival compared to 0.075µM doxorubicin alone at 40%. This decrease in survival correlated with increased activation of proteins involved in DNA damage response, including γ-H2AX(Ser139), a marker for dsDNA breaks. We anticipate that quantification of nucleotides will show an imbalance of dUTP:dTTP, subsequently leading to uracil misincorporation and resulting cell death. Discussion. These results suggest that repair of anthracycline-induced DNA damage requires dUTPase to prevent uracil misincorporation and that inhibition of dUTPase is a potential novel strategy to enhance the efficacy of anthracyclines. This shows the potential advantage of a dUTPase inhibitor being added to current chemotherapy regimens for TNBC. Future work will elucidate the mechanism of sensitisation and in vivo translation of combination therapy for TNBC. Citation Format: Craig Davison, Olivier P. Chevallier, Catherine Knowlson, Melanie McKechnie, Robbie Carson, Jaime Esteve, Richard Wilson, Robert D. Ladner, Melissa J. LaBonte (Wilson). Imbalanced nucleotide metabolism sensitizes breast cancer cells to anthracyclines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3836.

Abstract 1308: Post translational crosstalk networks identify strategies to overcome EMT-mediated resistance to EGFR inhibitors

Abstract Epithelial-mesenchymal transition (EMT) mediates intrinsic and acquired resistance to epidermal growth factor receptor (EGFR) inhibitors. This becomes a major hurdle in lung cancer treatment due to the lack of effective therapeutic strategies. We hypothesized that decoding the EMT signaling network could provide insights into the specific combinatorial logic associated with EMT signaling and identify new therapeutic strategies to combat EGFRi resistance. To test this hypothesis, we applied sequential enrichment of post-translational modifications (SEPTM) proteomics to analyze proteomes of expressed proteins and multiple post-translational modifications (PTM) including phosphorylation, ubiquitination, and acetylation in erlotinib sensitive cells (HCC4006) and matched erlotinib resistant cells after EMT (HCC4006ER). We conducted integrative informatics to characterize EMT associated proteins, PTMs, pathways, cross-talk among PTMs and signaling networks from our data. We used siRNA and small molecules to functionally interrogate our results by assaying cell viability and migration. We identified 6,641 proteins, 2,418 unique pSTY sites, 784 unique UbK-sites and 713 unique AcK-sites respectively. We found 377 proteins increased and 1377 proteins decreased (p<0.05, fold>2) in HCC4006ER cells compared to parent HCC4006 cells. We constructed an EMT signaling network, composed of 206 proteins with PTM changes including pSTY-sites (141 increase, 191 decrease), UbK-sites (29 increase, 32 decrease) and AcK-sites (14 increase, 46 decrease). Of 206 differentially modified proteins, 88 proteins are reported to be associated with EMT. Pathway analysis enriched 284 pathways from this EMT signaling network. We identified small molecule inhibitors associated with various pathways and tested for their effects on resistant cells. Inhibitors targeting 17 pathways and 3 major transcription factors were found to have effects on H4006ER viability, with inhibitors targeting DDR1, WNT and CDK signaling pathways demonstrating the most impact. Using RNAi, we found that that loss-of-function of 8 of 88 EMT-associated proteins (TAGLN2, STMN1, FYN, HNRNPA2B1, DDR1, INPPL1, OSMR and PRKAR2A) decreased HCC4006ER cell viability. Finally, integrative informatics revealed cross-talk among PTMs within EMT signaling network. From this analysis, we found that inhibiting GLI induced transcription sensitizes H4006ER cells to both EGFR inhibitor and Casein Kinase inhibitor. Collectively, SEPTM proteomics allows decoding the complex interplay in PTM modulation associated with EMT-mediated resistance. Our results suggest DDR1 as a potential actionable target for EMT driven resistance, which can serve as an example for combinatorial targeting of EMT proteins and signaling pathways as a strategy for overcoming EMT-mediated drug resistance. Citation Format: Guolin Zhang, Karen Ross, Bin Fang, Jun-Min Zhou, Paul A. Stewart, Emma Adhikari, Eric A. Welsh, Xuefeng Wang, John M. Koomen, Cathy H. Wu, Eric B. Haura. Post translational crosstalk networks identify strategies to overcome EMT-mediated resistance to EGFR inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1308.

Abstract 5917: A novel lipid nanoparticle (NBF-006) encapsulating glutathione S-transferase P (GSTP) siRNA for the treatment of KRAS-driven non-small cell lung cancer

Abstract Non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancers and KRAS mutation occurs in 25-30% of NSCLC. Therapeutics directly targeting KRAS mutant proteins are currently not available. We developed a novel siRNA therapeutic against Glutathione S-Transferase P (GSTP), a gene target that is highly expressed in KRAS mutant NSCLC and is also involved in modulating RAS signaling pathway proteins. GSTP has been known as a Phase II detoxification enzyme and a modulator of MAP kinase-related cell-signaling pathways. Therefore, developing a GSTP inhibiting siRNA may be an effective therapeutic approach to treat KRAS mutant NSCLC. The lead drug product candidate (NBF-006) is a proprietary siRNA-based lipid nanoparticle (LNP) with a size of less than 70 nm, comprising GSTP siRNA (NDT-05-1040) and a novel ionizable, non-immunogenic, biodegradable lipid developed to target tumors in the lung. Our studies evaluated biodistribution and in vivo antitumor activities of NBF-006 in KRAS mutant NSCLC xenograft models. NBF-006 delivered siRNA molecules favorably to the lungs and tumor as compared to other tissues, with 70-80% of tumor cells showing siRNA uptake. Significant tumor growth inhibition (P <0.05) was observed in xenograft models derived from A549 (KRASMUT/TP53WT) and H23 (KRASMUT/TP53MUT) NSCLC cells, with notable tumor regression in A549 tumors and highly significant tumor regression in the H23 tumors following once-a-week administration. In the bioluminescent orthotopic lung tumor model, the average luminescent signal response in the NBF-006 treatment group was markedly lower than the vehicle control group (14.3 vs. 69.6 million photon/second, P <0.05). Additionally, in a surgically implanted orthotopic lung tumor model, the survival rate of the NBF-006 treatment group was significantly prolonged (P <0.005) at a 4 mg/kg dose compared to the vehicle control group. Finally, NBF-006 treatment was well tolerated in the preclinical efficacy models. In summary, we conclude that NBF-006 is selectively delivered to lung tumor tissue and its considerable uptake by tumor cells results in significant tumor growth inhibition and overall survival benefit as demonstrated in our preclinical models of KRAS mutant NSCLC. *These authors contributed equally. Citation Format: Zhihong O'Brien*, Li Wang*, Bharat Majeti*, Jeanpierre Clamme, Robiel Baclig, James Chu, Steven Fong, Jens Harborth, Jose Ibarra, Haiqing Yin, Jing Yu, Clayton Zhang, Roger Adami, Sonya Zabludoff, Wenbin Ying. A novel lipid nanoparticle (NBF-006) encapsulating glutathione S-transferase P (GSTP) siRNA for the treatment of KRAS-driven non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5917.

Engineered Exosomes for Targeted Transfer of siRNA to HER2 Positive Breast Cancer Cells.

Exosomes are the best options for gene targeting, because of their natural, nontoxic, non-immunogenic, biodegradable, and targetable properties. By engineering exosome-producing cells, ligands can be expressed fusing with exosomal surface proteins for targeting cancer cell receptors. In the present study, HER2-positive breast cancer cells were targeted with a modified exosome producing engineered HEK293T cell. For this purpose, the HEK293T cells were transduced by a lentiviral vector bearing-LAMP2b-DARPin G3 chimeric gene. Stable cells expressing the fusion protein were selected, and the exosomes produced by these cells were isolated from the culture medium, characterized, and then loaded with siRNA for subsequent delivery to the SKBR3 cells. Our results showed that stable HEK293T cells produced DARPin G3 on the surface of exosomes. These exosomes can bind specifically to HER2/Neu and are capable of delivering siRNA molecules against TPD52 gene into SKBR3 cell line down-regulating the gene expression up to 70%. Present approach is envisaged to facilitate genes and drugs transfer to HER2 cancer cells providing additional option for gene therapy and drug delivery.

Toward Understanding Liposome-Based siRNA Delivery Vectors: Atomic-Scale Insight into siRNA-Lipid Interactions.

Liposome carriers for delivering small interfering RNA (siRNA) into target cells are of tremendous importance because the siRNA-based therapy offers a completely new approach for treating a wide range of diseases, including cancer and viral infections. In this paper, we employ the state-of-the-art computer simulations to get an atomic-scale insight into the interactions of siRNA with zwitterionic (neutral) lipids. Our computational findings clearly demonstrate that siRNA does adsorb on the surface of a neutral lipid bilayer. The siRNA adsorption, being rather weak and unstable, is driven by attractive interactions of overhanging unpaired nucleotides with choline moieties of lipid molecules. It is the presence of the unpaired terminal nucleotides that underlies a drastic difference between siRNA and DNA; the latter is not able to bind to the zwitterionic lipid bilayer. We also show that adding divalent Ca ions leads to the formation of stable siRNA-lipid system complexes; these complexes are stabilized by Ca-mediated aggregates of siRNA and lipid molecules rather than by the overhanging siRNA nucleotides. Furthermore, the molecular mechanism of interactions between siRNA and the lipid bilayer in the presence of divalent cations seems to involve exchange of Ca ions between the outer mouth of the major groove of siRNA and the lipid/water interface. Overall, our findings contribute significantly to a deeper understanding of the structure and function of liposome carriers used for siRNA delivery and can be used as a theoretical basis for further development of siRNA-based therapeutics.

The Unfolded Protein Response: A Novel Therapeutic Target for Poor Prognostic BRAF Mutant Colorectal Cancer.

BRAFV600E mutations occur in ∼10% of colorectal cancer cases, are associated with poor survival, and have limited responses to BRAF/MEK inhibition with or without EGFR inhibition. There is an unmet need to understand the biology of poor prognostic BRAFMT colorectal cancer. We have used differential gene expression and pathway analyses of untreated stage II and stage III BRAFMT (discovery set: n = 31; validation set: n = 26) colorectal cancer, and an siRNA screen to characterize the biology underpinning the BRAFMT subgroup with poorest outcome. These analyses identified the unfolded protein response (UPR) as a novel and druggable pathway associated with the BRAFMT colorectal cancer subgroup with poorest outcome. We also found that oncogenic BRAF drives endoplasmic reticulum (ER) stress and UPR pathway activation through MEK/ERK. Furthermore, inhibition of GRP78, the master regulator of the UPR, using siRNA or small molecule inhibition, resulted in acute ER stress and apoptosis, in particular in BRAFMT colorectal cancer cells. In addition, dual targeting of protein degradation using combined Carfilzomib (proteasome inhibitor) and ACY-1215 (HDAC6-selective inhibitor) treatment resulted in marked accumulation of protein aggregates, acute ER stress, apoptosis, and therapeutic efficacy in BRAFMT in vitro and xenograft models. Mechanistically, we found that the apoptosis following combined Carfilzomib/ACY-1215 treatment is mediated through increased CHOP expression. Taken together, our findings indicate that oncogenic BRAF induces chronic ER stress and that inducers of acute ER stress could be a novel treatment strategy for poor prognostic BRAFMT colorectal cancer. Mol Cancer Ther; 17(6); 1280-90. ©2018 AACR.

Synthesis of 4′-C-aminoalkyl-2′-O-methyl modified RNA and their biological properties

In this paper, we describe the synthesis of 4′-C-aminoalkyl-2′-O-methylnucleosides and the properties of RNAs containing these analogs. Phosphoramidites of 4′-C-aminoethyl and 4′-C-aminopropyl-2′-O-methyluridines were prepared using glucose as starting material, and RNAs containing the analogs were synthesized using the phosphoramidites. Thermal denaturation studies revealed that these nucleoside analogs decreased the thermal stabilities of double-stranded RNAs (dsRNAs). Results of NMR, molecular modeling, and CD spectra measurements suggested that 4′-C-aminoalkyl-2′-O-methyluridine adopts an C2′-endo sugar puckering in dsRNA. The 4′-C-aminoalkyl modifications in the passenger strand and the guide strand outside the seed region were well tolerated for RNAi activity of siRNAs. Single-stranded RNAs (ssRNAs) and siRNAs containing the 4′-C-aminoethyl and 4′-C-aminopropyl analogs showed high stability in buffer containing bovine serum. Thus, siRNAs containing the 4′-C-aminoethyl and 4′-C-aminopropyl analogs are good candidates for the development of therapeutic siRNA molecules.

FusionHub: A unified web platform for annotation and visualization of gene fusion events in human cancer.

Gene fusion is a chromosomal rearrangement event which plays a significant role in cancer due to the oncogenic potential of the chimeric protein generated through fusions. At present many databases are available in public domain which provides detailed information about known gene fusion events and their functional role. Existing gene fusion detection tools, based on analysis of transcriptomics data usually report a large number of fusion genes as potential candidates, which could be either known or novel or false positives. Manual annotation of these putative genes is indeed time-consuming. We have developed a web platform FusionHub, which acts as integrated search engine interfacing various fusion gene databases and simplifies large scale annotation of fusion genes in a seamless way. In addition, FusionHub provides three ways of visualizing fusion events: circular view, domain architecture view and network view. Design of potential siRNA molecules through ensemble method is another utility integrated in FusionHub that could aid in siRNA-based targeted therapy. FusionHub is freely available at https://fusionhub.persistent.co.in.

State‐of‐the‐Art Design and Rapid‐Mixing Production Techniques of Lipid Nanoparticles for Nucleic Acid Delivery

AbstractLipid nanoparticles (LNPs) are currently the most clinically advanced nonviral carriers for the delivery of small interfering RNA (siRNA). Free siRNA molecules suffer from unfavorable physicochemical characteristics and rapid clearance mechanisms, hampering the ability to reach the cytoplasm of target cells when administered intravenously. As a result, the therapeutic use of siRNA is crucially dependent on delivery strategies. LNPs can encapsulate siRNA to protect it from degradative endonucleases in the circulation, prevent kidney clearance, and provide a vehicle to deliver siRNA in the cell and induce its subsequent release into the cytoplasm. Here, the structure and composition of LNP–siRNA are described including how these affect their pharmacokinetic parameters and gene‐silencing activity. In addition, the evolution of LNP–siRNA production methods is discussed, as the development of rapid‐mixing platforms for the reproducible and scalable manufacturing has facilitated entry of LNP–siRNA into the clinic over the last decade. Finally, the potential of LNPs in delivering other nucleic acids, such as messenger RNA and CRISPR/Cas9 components, is highlighted alongside how a design‐of‐experiment approach may be used to improve the efficacy of LNP formulations.

1342 Polycomb repressive complex 2 controls differentiation program in human epidermal progenitor cells

Polycomb repressive complex 2 (PRC2) catalyses methylation of Lys-27 of histone H3 leading to transcriptional repression of target genes. In mouse skin, Ezh2, a catalytic subunit of PRC2, prevents premature differentiation of epidermal progenitor cells and their ectopic differentiation into Merkel cells. However, the role of PRC2 in human skin homeostasis remains unclear. Here, we show an increased EZH2 and H2K27me3 expression in differentiated suprabasal keratinocytes in human epidermis. Similarly, Ca2+-induced differentiation markedly up-regulated EZH2 expression in primary human epidermal keratinocytes (NHEK). Interestingly, knockdown of PRC2 subunits EZH1, EZH2 and EED using siRNA and small molecule inhibitors GSK126 and UNC1999 up-regulated expression of KRT1, LOR and FLG genes and suppressed cell proliferation in NHEKs cultured in low Ca2+ condition. However, disruption of the PRC2 function in already differentiated keratinocytes did not affect expression of the terminal differentiation genes. Bioinformatic analysis of genome-wide EZH2 and H3K27me3 distribution identified distinct sets of PRC2 targets in undifferentiated progenitor cells and their differentiated progeny. Taken together, these results highlight a critical role for PRC2 in human epithelial progenitor cells proliferation and differentiation.

A review on current status of antiviral siRNA.

SummaryViral diseases like influenza, AIDS, hepatitis, and Ebola cause severe epidemics worldwide. Along with their resistant strains, new pathogenic viruses continue to be discovered so creating an ongoing need for new antiviral treatments. RNA interference is a cellular gene‐silencing phenomenon in which sequence‐specific degradation of target mRNA is achieved by means of complementary short interfering RNA (siRNA) molecules. Short interfering RNA technology affords a potential tractable strategy to combat viral pathogenesis because siRNAs are specific, easy to design, and can be directed against multiple strains of a virus by targeting their conserved gene regions. In this review, we briefly summarize the current status of siRNA therapy for representative examples from different virus families. In addition, other aspects like their design, delivery, medical significance, bioinformatics resources, and limitations are also discussed.

RNA interference-based silencing of the alpha-amylase (amy1) gene in Aspergillus flavus decreases fungal growth and aflatoxin production in maize kernels.

Expressing an RNAi construct in maize kernels that targets the gene for alpha-amylase in Aspergillus flavus resulted in suppression of alpha-amylase (amy1) gene expression and decreased fungal growth during in situ infection resulting in decreased aflatoxin production. Aspergillus flavus is a saprophytic fungus and pathogen to several important food and feed crops, including maize. Once the fungus colonizes lipid-rich seed tissues, it has the potential to produce toxic secondary metabolites, the most dangerous of which is aflatoxin. The pre-harvest control of A. flavus contamination and aflatoxin production is an area of intense research, which includes breeding strategies, biological control, and the use of genetically-modified crops. Host-induced gene silencing, whereby the host crop produces siRNA molecules targeting crucial genes in the invading fungus and targeting the gene for degradation, has shown to be promising in its ability to inhibit fungal growth and decrease aflatoxin contamination. Here, we demonstrate that maize inbred B104 expressing an RNAi construct targeting the A. flavus alpha-amylase gene amy1 effectively reduces amy1 gene expression resulting in decreased fungal colonization and aflatoxin accumulation in kernels. This work contributes to the development of a promising technology for reducing the negative economic and health impacts of A. flavus growth and aflatoxin contamination in food and feed crops.

Inclisiran for the treatment of dyslipidemia

ABSTRACTIntroduction: Dyslipidemia is one of the most important risk factors for cardiovascular disease. Insufficient reduction in LDL-C from existing therapies in patients at high risk of atherogenic cardiovascular disease is an unmet clinical need. Circulating PCSK9 causes hypercholesterolemia by reducing LDL receptors in hepatocytes.Areas covered: PCSK9 inhibition has emerged as a promising new therapeutic strategy to reduce LDL-C. Inclisiran, a novel, synthetic, siRNA molecule, inhibits PCSK9 synthesis in hepatocytes. Inclisiran targets intracellular PCSK9 synthesis specifically, resulting in a dose-dependent, long-term, significant reduction in LDL-C. Inclisiran has been well tolerated and safe, without severe adverse events so far. This review discusses current PCSK9 inhibitors and the results of phase I and II clinical trials of inclisiran.Expert opinion: Plasma PCSK9 enhances the degradation of LDL receptor, resulting in accumulation of LDL-C in the circulation. Current approaches with monoclonal antibodies sequester circulating PCSK9 but require frequent injections. Inclisiran inhibits translation of PCSK9 mRNA and thus switches off PCSK9 production and provides advantages over monoclonal antibodies with an infrequent dosing interval of twice a year to reduce LDL-C by over 50%. Ongoing studies will establish the long-term safety of inclisiran in patients with high cardiovascular risk and an elevated LDL-C.

Computational Design of Potential Sirna Molecules for Silencing nucleoprotein Gene of Rabies Virus

Aim: Rabies virus infections are a global threat to human and animal health, yet no progressive curative therapy has been developed. In this study, the nucleoprotein gene of rabies virus which is responsible for viral infection was used as a target to design our desired siRNA. Methods: The conserved regions were analyzed by doing alignment of sequences from different strains. Subsequently, different computational tools were used for designing and validation of siRNA molecules. Results: We identified four probable siRNA molecules from twelve different strains of rabies virus which may silence the nucleoprotein gene and inhibit the multiplication of the virus. Conclusion: Our study may help to take an effective therapeutic approach against rabies virus and lead to better control of rabies in humans.

Characterization of modified mesoporous silica nanoparticles as vectors for siRNA delivery

Gene therapy using siRNA molecules is nowadays considered as a promising approach. For successful therapy, development of a stable and reliable vector for siRNA is crucial. Non-viral and non-organic vectors like mesoporous silica nanoparticles (MSN) are associated with lack of most viral vector drawbacks, such as toxicity, immunogenicity, but also generally a low nucleic acid carrying capacity. To overcome this hurdle, we here modified the pore walls of MSNs with surface-hyperbranching polymerized poly(ethyleneimine) (hbPEI), which provides an abundance of amino-groups for loading of a larger amount of siRNA molecules via electrostatic adsorption. After loading, the particles were covered with a second layer of pre-polymerized PEI to provide better protection of siRNA inside the pores, more effective cellular uptake and endosomal escape. To test the transfection efficiency of PEI covered siRNA/MSNs, MDA-MB 231 breast cancer cells stably expressing GFP were used. We demonstrate that PEI-coated siRNA/MSN complexes provide more effective delivery of siRNAs compared to unmodified MSNs. Thus, it can be concluded that appropriately surface-modified MSNs can be considered as prospective vectors for therapeutic siRNA delivery.

Prognostic and therapeutic significance of ribonucleotide reductase small subunit M2 in prostate cancer.

240 Background: DNA-repair defects are common in advanced prostate cancer (PC) and high-risk localized tumors. Besides deficiency of DNA repair, overexpression of DNA repair genes could also contribute to poorer outcomes for PC patients. The nucleotide metabolism enzyme ribonucleotide reductase (RNR) plays the key role in DNA synthesis and repair. RRM2, the rate-limiting RNR subunit, is frequently up-regulated in cancer, and its overexpression leads to chemoresistance. Although targeting RRM2 by siRNA and small molecules has been applied in clinical trials in multiple cancers, limited knowledge of RRM2 function in PC delays potential clinical application of RRM2 inhibition. Methods: We leveraged publically available PC clinical cohorts to examine RRM2 levels and clinical outcomes. siRNAs was applied to knockdown of RRM2 in multiple PC cell lines. Cell growth, cell cycle and apoptosis were analyzed to determine siRRM2 or RRM2 inhibitor (COH29)-induced phenotypes. RNA-seq and protein array were performed to identify downstream targets of RRM2. Immunohistochemistry staining was applied to determine prevalence of RRM2 protein expression in PC tissues microarrays (TMAs). Results: In PC cohorts, increased RRM2 expression was associated with a higher likelihood of metastasis, poorer disease-free survival, and increased risk of development of lethal disease (N = 1200, PHS/HPFS cohorts). In PC cells, Inhibition of RRM2 induced remarkable cell growth inhibition, cell cycle arrest (at S phase) and apoptosis. DNA damage was observed in siRRM2/COH29-treated PC cells with increased activation of DNA damage markers. GSEA analysis of the RNA-seq dataset revealed multiple biological processes were affected by inhibition of RRM2, such as cell cycle, apoptosis, and DNA damage response. Intriguingly, MYC oncogenic signaling is the major downstream targets of RRM2. Furthermore, inhibition of RRM2 can block multiple oncogenic signaling including mTOR/AKT, SFK, and STAT signaling by repressing the key phospho-kinases in PC cells. Among 121 cases on the PC TMAs, 20% showed strong RRM2 protein expression. Conclusions: RRM2 may serve as a prognostic biomarker and novel therapeutic target in PC.

Cellular uptake mechanism and comparative in vitro cytotoxicity studies of monomeric LMWP-siRNA conjugate

The covalent attachment of CPPs to siRNA molecules offers great potential for CPP-mediated siRNA delivery. We recently reported a concise and high-yield synthesis strategy of the cell-permeable, cytosol-dissociable LMWP-siRNA covalent conjugate. Herein, cell uptake mechanism and cellular toxicity studies of this conjugate were performed to evaluate the potential of LMWP-siRNA conjugate for clinical translation. Cellular uptake mechanism study indicated that the conjugate could be taken up by cells via multiple pathways, including direct penetration of the plasma membrane and clathrin- and caveolae-independent endocytosis. In vitro cytotoxicity study revealed that the conjugation promoted internalization in a low-toxic fashion.

Covalent Strategies for Targeting Messenger and Non-Coding RNAs: An Updated Review on siRNA, miRNA and antimiR Conjugates.

Oligonucleotide-based therapy has become an alternative to classical approaches in the search of novel therapeutics involving gene-related diseases. Several mechanisms have been described in which demonstrate the pivotal role of oligonucleotide for modulating gene expression. Antisense oligonucleotides (ASOs) and more recently siRNAs and miRNAs have made important contributions either in reducing aberrant protein levels by sequence-specific targeting messenger RNAs (mRNAs) or restoring the anomalous levels of non-coding RNAs (ncRNAs) that are involved in a good number of diseases including cancer. In addition to formulation approaches which have contributed to accelerate the presence of ASOs, siRNAs and miRNAs in clinical trials; the covalent linkage between non-viral vectors and nucleic acids has also added value and opened new perspectives to the development of promising nucleic acid-based therapeutics. This review article is mainly focused on the strategies carried out for covalently modifying siRNA and miRNA molecules. Examples involving cell-penetrating peptides (CPPs), carbohydrates, polymers, lipids and aptamers are discussed for the synthesis of siRNA conjugates whereas in the case of miRNA-based drugs, this review article makes special emphasis in using antagomiRs, locked nucleic acids (LNAs), peptide nucleic acids (PNAs) as well as nanoparticles. The biomedical applications of siRNA and miRNA conjugates are also discussed.