Synthetic siRNA Research Articles

Optimized RNA interference therapeutics combined with interleukin-2 mRNA for treating hepatitis B virus infection

This study aimed to develop a pan-genotypic and multifunctional small interfering RNA (siRNA) against hepatitis B virus (HBV) with an efficient delivery system for treating chronic hepatitis B (CHB), and explore combined RNA interference (RNAi) and immune modulatory modalities for better viral control. Twenty synthetic siRNAs targeting consensus motifs distributed across the whole HBV genome were designed and evaluated. The lipid nanoparticle (LNP) formulation was optimized by adopting HO-PEG2000-DMG lipid and modifying the molar ratio of traditional polyethylene glycol (PEG) lipid in LNP prescriptions. The efficacy and safety of this formulation in delivering siHBV (tLNP/siHBV) along with the mouse IL-2 (mIL-2) mRNA (tLNP/siHBVIL2) were evaluated in the rAAV-HBV1.3 mouse model. A siRNA combination (terms “siHBV”) with a genotypic coverage of 98.55% was selected, chemically modified, and encapsulated within an optimized LNP (tLNP) of high efficacy and security to fabricate a therapeutic formulation for CHB. The results revealed that tLNP/siHBV significantly reduced the expression of viral antigens and DNA (up to 3log10 reduction; vs PBS) in dose- and time-dependent manners at single-dose or multi-dose frequencies, with satisfactory safety profiles. Further studies showed that tLNP/siHBVIL2 enables additive antigenic and immune control of the virus, via introducing potent HBsAg clearance through RNAi and triggering strong HBV-specific CD4+ and CD8+ T cell responses by expressed mIL-2 protein. By adopting tLNP as nucleic acid nanocarriers, the co-delivery of siHBV and mIL-2 mRNA enables synergistic antigenic and immune control of HBV, thus offering a promising translational therapeutic strategy for treating CHB.

RNAi-based drug design: considerations and future directions.

More than 25 years after its discovery, the post-transcriptional gene regulation mechanism termed RNAi is now transforming pharmaceutical development, proved by the recent FDA approval of multiple small interfering RNA (siRNA) drugs that target the liver. Synthetic siRNAs that trigger RNAi have the potential to specifically silence virtually any therapeutic target with unprecedented potency and durability. Bringing this innovative class of medicines to patients, however, has been riddled with substantial challenges, with delivery issues at the forefront. Several classes of siRNA drug are under clinical evaluation, but their utility in treating extrahepatic diseases remains limited, demanding continued innovation. In this Review, we discuss principal considerations and future directions in the design of therapeutic siRNAs, with a particular emphasis on chemistry, the application of informatics, delivery strategies and the importance of careful target selection, which together influence therapeutic success.

Abstract 4652: Combined inhibition of MCL-1 and MEK reduces tumor growth in a triple-negative/inflammatory breast cancer, MEK-resistant xenograft mouse model

Abstract Background: Triple-negative breast cancer (TNBC) and inflammatory breast cancer (IBC) are highly aggressive subtypes of invasive breast cancer. Our previous work showed that the MEK inhibitor (MEKi) selumetinib (AZD6244) prevents lung metastasis in a xenograft model. However, clinical studies have shown that MEKi’s as single agents have only modest activity against solid tumors. Using a genome-wide synthetic lethal siRNA screening, we identified myeloid cell leukemia-1 (MCL1) as a potential contributor to selumetinib resistance. MCL-1 is an anti-apoptotic protein associated with cell immortalization, transformation, and chemoresistance in numerous cancers. We hypothesized that MCL-1 promotes MEKi resistance in TNBC/IBC and inhibition of MCL-1 in the presence of AZD6244 overcomes this MEKi resistance. Methods & Results: To understand MCL1’s role in MEKi resistance, we established two AZD6244 -resistant TNBC/IBC cell lines: MDA-MB-231-R and SUM149-R. These cells showed increased cell viability, colony formation, migration, cells in G1 phase, CD44+CD24low, mammosphere count, anchorage-independent growth and high MCL-1 expression. MCL-1’s biological role was evaluated by transient and stably knock down (KD) of MCL-1 in the resistant cells which showed decreased cell viability, colony formation, and increased apoptosis. To understand the mechanism mediating apoptosis, we examined the expression of MCL-1’s binding partners in the MCL-1-KD-R cells in the presence of AZD6244. These cells showed a high expression of the PUMA, NOXA, BAK, and BAX in the presence of AZD6244. Further, we treated resistant cells with MEKi AZD6244 and an MCL-1i (AZD5991) that confirmed restoration of MEK sensitivity in both resistant cell lines, resulting in decreased cell viability, colony formation and increased apoptosis. We compared tumor formation capability between SUM149 parental and SUM149 resistant cells by injecting them in nu/nu mice and observed a time-dependent increase (avg. 4-fold) in tumor growth with SUM149-R cells compared to parental cells (P≤0.0001). Moreover, IHC analysis showed high expression of MCL-1 (P≤0.01), Ki67 (ns), CD44 (P≤0.0001), and ALDHA1 (P≤0.0001) in the mammary gland tumor tissues of SUM149-R-injected mice compared to SUM149-P-injected mice. Next, we evaluated the therapeutic effect of AZD6244 in mice harboring SUM149-R - MCL-1-deficient tumors. AZD6244 treatment significantly reduced tumor development in mice harboring SUM149-R - MCL-1-KD tumors compared to control mice (P<0.0001), suggesting that MCL-1 mediates MEK resistance in triple-negative/inflammatory breast cancer. Conclusion: Our results suggest that MCL-1 is important for inducing acquired MEK resistance and combining an MCL-1i with a MEKi could improve treatment outcomes of patients with TNBC or IBC who have developed resistance to single-agent MEKi. Citation Format: Mohd Mughees, Alex Tan, Lakesla Iles, Michael White, Debu Tripathy, Savitri Krishnamurthy, Jian Wang, Wendy A. Woodward, Geoffrey Bartholomeusz, Chandra Bartholomeusz. Combined inhibition of MCL-1 and MEK reduces tumor growth in a triple-negative/inflammatory breast cancer, MEK-resistant xenograft mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4652.

Rational design of prodrug-type apoB-targeted siRNA for nuclease resistance improvement without compromising gene silencing potency

Synthetic siRNA molecules without chemical modifications are easily degraded in the body, and 2'-O-modifications are frequently introduced to enhance stability. However, such chemical modifications tend to impact the gene knockdown potency of siRNA negatively. To circumvent this problem, we previously developed a prodrug-type siRNA bearing 2'-O-methyldithiomethyl (MDTM) groups, which can be converted into unmodified siRNA under the reductive environment in cells. In this study, we developed a nuclease-resistant prodrug-type 2'-O-MDTM siRNA for deployment in future animal experiments. To rationally design siRNA modified with a minimal number of 2'-O-MDTM nucleotide residues, we identified the sites susceptible to nuclease digestion and tolerant to 2'-O-methyl (2'-OMe) modification in the antisense strand of apolipoprotein B-targeted siRNA. Subsequently, we optimized the positions where the 2'-OMe and 2'-O-MDTM groups should be incorporated. siRNA bearing the 2'-O-MDTM and 2'-OMe groups at their respective optimized positions exhibited efficient knockdown potency in vitro and enhanced stability in serum.

Detection of the effect of synthetic siRNA on efflux pump MexA gene expression and antibiotic resistance in clinical isolates of Pseudomonas aeruginosa

Introduction and Aim: Pseudomonas aeruginosa is a nosocomial infection with an ability to develop high levels of antibiotic resistance. The efflux pump system is one of the mechanisms that is linked to multidrug resistance in P. aeruginosa. In this study, we employed siRNA loaded on gold nanoparticles against the MexA efflux pump gene to decrease the MexA gene expression in P. aeruginosa and estimated antibiotic resistance after gene silencing. Materials and Methods: This study examined four strains of P. aeruginosa isolated from patients in various hospitals in Baghdad. Bacteria isolated were identified by biochemical tests and Vitek compact 2 system. Single-stranded siRNA (33bp) designed in this study was loaded onto gold nanoparticles (AuNPls). Detection of the MexA gene was carried out by conventional PCR technique. The expression of MexA gene was examined by qRT-PCR in order to determine if the siRNA have impacted on MexA gene expression and on the antibiotic resistance in aeruginosa Results: This study showed that the mRNA expression level of the MexA gene exhibited a decrease in fold change CT -2 in P. aeruginosa (isolates numbers 67, 66,49, and PDR(5p)) when examined in vitro. The specific fold change values observed were (0.202, 0.040, 0.063, and 0.163) respectively. The resistance percentages of antibiotics tested was observed to increase after MexA gene silencing. Conclusion: Targeting the MexA gene with synthetic siRNA may be a unique approach to diminish P. aeruginosa resistance to antibiotics. However, many unexpected consequences may occur when utilizing any genetic manipulation in bacteria.

SEMA6B induces macrophage-mediated inflammation and hepatocyte apoptosis in hepatitis B virus-related acute-on-chronic liver failure.

Rationale: Patients with hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF) have a high short-term mortality rate. Semaphorin-6B (SEMA6B) plays a crucial role in the pathogenesis of HBV-ACLF, but its molecular basis remains unclear. This study aimed to elucidate the mechanisms of SEMA6B in HBV-ACLF progression. Methods: A total of 321 subjects with HBV-ACLF, liver cirrhosis (LC), chronic hepatitis B (CHB), and normal controls (NC) from a prospective multicenter cohort were studied. 84 subjects (HBV-ACLF, n = 50; LC, n = 10; CHB, n = 10; NC, n = 14) among them underwent mRNA sequencing using peripheral blood mononuclear cells (PBMCs) to clarify the mechanisms of SEMA6B in HBV-ACLF. These mechanisms were validated through in vitro studies with hepatocytes and macrophages, as well as in vivo using SEMA6B knockout mice and mice treated with synthetic SEMA6B siRNA. Results: Transcriptome analysis of PBMCs showed that SEMA6B was among the most differentially expressed genes when comparing patients with HBV-ACLF to those with LC, CHB, or NC. ROC analysis demonstrated the reliable diagnostic value of SEMA6B for HBV-ACLF in both the sequencing cohort and an external validation cohort (AUROC = 0.9788 and 0.9026, respectively). SEMA6B levels were significantly higher in the HBV-ACLF patients, especially in non-survivors, with high expression mainly observed in macrophages and hepatocytes in liver tissue. Genes significantly associated with highly expressed SEMA6B were enriched in inflammation and apoptosis pathways in HBV-ACLF non-survivors. Overexpression of SEMA6B in macrophages activated systemic inflammatory responses, while its overexpression in hepatocytes inhibited proliferation through G0/G1 cell cycle arrest and induced apoptosis. Knocking out SEMA6B rescued mice with liver failure by improving liver functions, reducing inflammatory responses, and decreasing hepatocyte apoptosis. Transcriptome analysis of liver tissue showed that SEMA6B knockout significantly ameliorated the liver failure signature, significantly downregulating inflammation-related pathways. Importantly, therapeutic delivery of synthetic SEMA6B siRNA also improved liver function, and reduced both inflammation and hepatocyte apoptosis in mice with liver failure. Conclusion: SEMA6B, a potential diagnostic biomarker for HBV-ACLF, exacerbates liver failure through macrophage-mediated systemic inflammation and hepatocyte apoptosis. These findings highlight SEMA6B as a promising early treatment target for HBV-ACLF patients.

A coronavirus-mimic mesoporous silica nanosystem enables efficient targeted delivery of siRNA for anti-SARS-CoV‐2

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been a threat to human beings' health. However, there is still a lack of effective specific antiviral drugs for SARS-CoV-2 at present, which calls for the exploitation of new potent and safe therapeutic agents. Short-interfering RNA (siRNA), as a powerful RNA interference (RNAi) tool, can be designed to target virus-related genes and degrade them with high efficiency, presenting great potential for confronting SARS-CoV-2 infection. Despite the promises, effective delivery of synthetic siRNA into the virus-infected host cells for therapy remains a challenge. Herein, a SARS-CoV-2 spike (S) protein-decorated dendritic mesoporous silica nanoparticle (DMSN) was developed for targeted delivery of the RNA-dependent RNA polymerase (RdRp)-specific siRNA for anti-SARS-CoV‐2. The biocompatible DMSN with a large pore structure and surface area enabled the high siRNA loading and protected siRNA from nuclease-mediated degradation. On the basis of the specific binding between S protein and angiotensin-converting enzyme 2 (ACE2) receptor, the designed nanosystem could imitate coronavirus to target the host cells and promote the cellular internalization of siRNA. The results demonstrated that the designed siRNA delivery nanosystem showed an improved silencing effect on the transfected SARS-CoV-2 RdRp gene in the ACE2-overexpressed host cells. Moreover, such nanosystem could be inhaled and accumulated in the lung of living body through a nebulized administration, offering a promising treatment option for SARS-CoV‐2 infection.

Abstract 3635: TBK1 inhibition potentiates the efficacy of AXL-targeted therapy by modulating tumor microenvironment in aggressive breast cancers

Abstract Introduction: Triple-negative and inflammatory breast cancer (TNBC and IBC) are the most aggressive breast cancer subtypes. Novel actionable targets and complementary therapies are critically needed. AXL, a receptor tyrosine kinase, drives pleiotropic phenotypes of TNBC and IBC aggressiveness. Targeting AXL reduces IBC tumor growth in vivo and inhibits polarization of M2 macrophages. To identify novel tumor microenvironment (TME) targets to enhance the efficacy of AXL-targeted therapy, we conducted a synthetic lethal kinome siRNA screening. We identified TBK1 as a candidate to synergize with AXL growth inhibition. TBK1 is a serine/threonine protein kinase that regulates innate, adaptive responses and antitumor immune responses. We hypothesized that inhibition of TBK1 enhances the antitumor effect of AXL-targeted therapy in aggressive breast cancers. Methods: We used knockdown, knockout (KO) and inhibitors to suppress AXL and TBK1 pathways and tested the synergistic effect of targeting AXL and TBK1 on the growth of human SUM149 and BCX010 IBC cells, and HS578T TNBC cells in vitro. To determine the synergistic effect of targeting both pathways in vivo, we inoculated control or TBK1 KO murine TNBC 4T1.2 cells into BALB/c mice. We assessed the activity of AXL inhibitor TP-0903 in reducing tumor growth. Using multicolor flow cytometry, we studied the effects of targeting AXL and TBK1 on the TME. We tested the activity of TP-0903 combined with a TBK1 inhibitor in another TNBC mouse model, E0771. We used RNA-sequencing and real-time PCR/Western blot to determine the molecular mechanisms of how TBK1 inhibition synergizes AXL-targeted therapy in these cancers. Results: Compared with AXL or TBK1 suppression alone, genomic or pharmacologic suppression of AXL and TBK1 significantly reduced the growth of SUM149, BCX010, and HST578T cells in vitro. In both 4T1.2 and E0771 syngeneic mouse models, TP-0903 was more active in reducing tumor growth in TBK1-inhibited tumors than in control tumors. Tumors with TBK1 inhibition and treated with TP-0903 had a significantly higher population of cytotoxic T cells than control tumors. Depletion of CD8+ T cells blocked the synergistic effect of targeting AXL and TBK1 pathways on reducing tumor growth, suggesting that cytotoxic T cells contributed to the anti-tumor synergy of targeting AXL and TBK1. Mechanistically, TBK1 induced M2 macrophage migration via IRF3-regulated CCL5 secretion in SUM149 and HS578T cells, and AXL KO attenuated the polarization and migration of M2 macrophages by inhibiting the CCR5/CCL5 axis. Conclusions: Targeting TBK1 enhances the efficacy of AXL-targeted therapy in aggressive breast cancer by suppressing the paracrine effect of CCR5/CCL5 axis. This combination represents a novel and effective therapy modulating the TME of aggressive breast cancer, which warrants further investigation in the clinical setting. Citation Format: Lan Phi, Takashi Semba, Ngu V. Trinh, Fang Zou, Jason M. Foulks, Steven L. Warner, Savitri Krishnamurthy, James P. Long, James M. Reuben, Debu Tripathy, Naoto T. Ueno, Xiaoping Wang. TBK1 inhibition potentiates the efficacy of AXL-targeted therapy by modulating tumor microenvironment in aggressive breast cancers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3635.

Effectiveness and safety of Inclisiran in hyperlipidemia treatment: An overview of systematic reviews.

This paper aimed to comprehensively evaluate the effectiveness and safety of Inclisiran in treating hyperlipidemia through an overview of systematic reviews (SRs). The Cochrane Library, EMBASE, PubMed, CNKI, WANGFANG database, VIP database, ClinicalTrials.gov, and ICRT were searched electronically to collect SRs and meta-analysis of Inclisiran in hyperlipidemia treatment from the establishment of the database till May 2022. Two researchers independently screened the relevant literature, then the assessment of multiple systematic reviews tool was made into assess the methodological quality of the included studies. Data extracted were used to perform the study through RevMan5.3 software. The grading of recommendations assessment, development, and evaluation tool was used to grade the quality of the evidence of the outcomes included in the SRs. Prospero ID: CRD 42022326845. A total of 10 relevant SRs were included, involving 7 randomized controlled trials. The assessment results of the assessment of multiple systematic reviews tool suggested that the quality of the SRs included needed to be improved. The reduced level of low-density lipoprotein cholesterol of the experimental group was lower than the control group, and the difference in the amount of effectiveness was statistically significant (MD = -50.13, 95%CI: -56.2 to -44.06, P < .00001). The grading of recommendations assessment, development, and evaluation results showed that out of 27 outcomes, 8 were high-quality, 3 were of medium quality, 6 were of low quality, and 10 were of the most inferior quality. 300mg Inclisiran with 2 injections a year has the best therapeutic effect, which can significantly reduce low-density lipoprotein cholesterol and total cholesterol, and increase high-density lipoprotein cholesterol levels in patients with hyperlipidemia. Inclisiran has a favorable safety profile, with no significant difference in the incidence of adverse reactions compared to a placebo. Most of the adverse effects were associated with the reaction on the injection site.

Effects of Chitosan Nanoparticles with Long Synthetic siRNAs Targeting VEGF in Triple-Negative Breast Cancer Cells

Effects of Chitosan Nanoparticles with Long Synthetic siRNAs Targeting VEGF in Triple-Negative Breast Cancer Cells

Virus-like siRNA construct dynamically responsive to sequential microenvironments for potent RNA interference

Cytoplasmic transportation of therapeutic nucleic acids is deemed as an onerous task with aim of precise knockdown towards the targeted genes. Pertaining to the programed functionalities of natural virus in circumventing the biological barriers, we tailored multifaceted chemistries into manufacture of synthetic siRNA delivery vehicles in resembling the functionalities of viral vectors to dynamically tackle with a sequential of biological obstacles encountered in the journey of systemic anti-tumor RNAi therapy. Once harnessing ligands with RGD motif for specific internalization into subcellular endosomal compartments of the tumor cells, the architecture of the proposed delivery vehicles was subjected to facile transformation responsive to pH stimuli in acidic endosomal compartments. The external biocompatible PEGylation palisade was consequently detached, unveiling the cytomembrane-lytic cationic components to commit disruptive potencies to the anionic endosomal membranes for translocation of siRNA conjugates into cytosol. Eventually, liberation of active siRNA could be accomplished due to its responsiveness to the strikingly high level of glutathione in cytosol, thereby contributing to potent RNAi. Hence, our elaborated virus-mimicking platform has demonstrated significant anti-tumor efficacy through systemic administration of anti-angiogenic RNAi payloads, which inspired prosperous potentials in a variety of therapeutic applications.

Advantages and Disadvantages of Inclisiran: A Small Interfering Ribonucleic Acid Molecule Targeting PCSK9-A Narrative Review.

As dyslipidemias remain one of the main risk factors for developing cardiovascular disease, the question of maintaining optimal lipid levels with pharmacotherapy remains a subject of interest worldwide. In contrast to conventional pharmacotherapy, human monoclonal antibodies directed against proprotein convertase subtilisin/kexin type 9 (PSCK9) and small interfering RNA- (siRNA-) based drug targeting PCSK9 represent a new strategy for managing lipid disorders and reducing cardiovascular risk. Inclisiran is a long-acting, synthetic siRNA that targets hepatic production of PCSK9 and consequently causes a reduction in LDL-C concentrations by approximately 50% compared to placebo. The structural modification of inclisiran has led to better stability and prolonged biological activity of the drug. The main advantage over conventional pharmacotherapy and anti-PCSK9 monoclonal antibodies is its favorable administration regimen (0–90–180 days), which should lead to much better compliance. Clinical trials conducted so far have confirmed the tolerability and efficacy of inclisiran in long-term PCSK9 and LDL-C level reductions. Moreover, a short-term follow-up on the safety of inclisiran showed a relatively good safety profile of the drug. However, it is still of great importance for ongoing and forthcoming clinical trials to be continued on a larger group of patients in order to assess long-term tolerability, efficacy, and safety of inclisiran.

Engineering miRNA features into siRNAs: Guide-strand bulges are compatible with gene repression

Synthetic siRNA guide strands are typically designed with perfect complementarity to the passenger strand and the target mRNA. We examined whether siRNAs with intentional guide-strand bulges are functional in vitro and in vivo. Importantly, this was done by systematic shortening of the passenger strand, evaluating identical 19-mer guide-strand sequences but forcing them into conformations with 1- to 4-nt bulges after annealing. We demonstrate that guide-strand bulges can be well tolerated at several positions of unmodified and modified siRNAs. Beyond that, we show that GalNAc-conjugated siRNAs with bulges at certain positions of the guide strand repress transthyretin in murine primary hepatocytes and in vivo in mice. In vivo, a GalNAc-conjugated siRNA with a 1-nt bulge at position 14 of the guide strand was as active as the perfectly complementary siRNA. Finally, in a luciferase reporter system, mRNA target sequences were systematically shortened so that RNA-induced silencing complex activity could only occur with a guide-strand bulge. Here, luciferase reporters were repressed when 1- and 2-nt deletions of the reporter were applied to the edges of the sequence. We conclude that some guide-strand bulges versus target transcript can result in target repression and therefore should be evaluated as off-target risks.

Treatment of bladder cancer by geoinspired synthetic chrysotile nanocarrier-delivered circPRMT5 siRNA

BackgroundCircular RNAs (circRNAs) have important functions in many fields of cancer biology. In particular, we previously reported that the oncogenic circRNA, circPRMT5, has a major role in bladder cancer progression. Therapy based on circRNAs have good prospects as anticancer strategies. While anti-circRNAs are emerging as therapeutics, the specific in vivo delivery of anti-circRNAs into cancer cells has not been reported and remains challenging.MethodsSynthesized chrysotile nanotubes (SCNTs) with a relatively uniform length (~ 200 nm) have been designed to deliver an siRNA against the oncogenic circPRMT5 (si-circPRMT5) inhibit circPRMT5. In addition, the antitumor effects and safety evaluation of SCNTs/si-circPRMT5 was assessed with a series of in vitro and in vivo assays.ResultsThe results showed that SCNTs/si-circPRMT5 nanomaterials prolong si-circPRMT5’s half-life in circulation, enhance its specific uptake by tumor cells, and maximize the silencing efficiency of circPRMT5. In vitro, SCNTs encapsulating si-circPRMT5 could inhibit bladder cancer cell growth and progression. In vivo, SCNTs/si-circPRMT5 inhibited growth and metastasis in three bladder tumor models (a subcutaneous model, a tail vein injection lung metastatic model, and an in situ model) without obvious toxicities. Mechanistic study showed that SCNTs/si-circPRMT5 regulated the miR-30c/SNAIL1/E-cadherin axis, inhibiting bladder cancer growth and progression.ConclusionThe results highlight the potential therapeutic utility of SCNTs/si-circPRMT5 to deliver si-circPRMT5 to treat bladder cancer.

Inhibition of Breast Tumour Growth with Intravenously Administered PRKCA siRNA- and PTEN Tumour Suppressor Gene-Loaded Carbonate Apatite Nanoparticles

Gene therapy aims to silence an oncogene through RNA interference, or replace an abnormal tumour suppressor via gene augmentation. In this study, we intended RNA interference for PRKCA oncogene and gene augmentation for PTEN tumour suppressor with a view to reduce tumour growth in a mouse model of breast cancer. Inorganic carbonate apatite nanoparticles (CA NPs) were utilized to deliver the synthetic siRNA and the purified gene-carrying plasmid DNA both in vitro and in vivo. Effects of PRKCA siRNA- and PTEN plasmid-loaded NPs on viability of MCF-7, MDA-MB-231 and 4T1 breast cancer cells were assessed by MTT assay. The cell viability data in MCF-7 cell line demonstrated that combined delivery of PRKCA specific siRNA and PTEN plasmid with CA NPs had an additive effect to significantly decrease cellular growth compared to individual treatments. In addition, we observed a similar pattern of cumulative influence for combined treatment in triple negative MDA-MB-231 breast cancer cell line. Upon treatment with PRKCA siRNA+PTEN plasmid-loaded NPs, a remarkable decrease in the phosphorylated form of AKT protein of PI3K/AKT pathway was observed in Western blot, indicative of diminished proliferative signal. Moreover, in vivo study in MCF-7 xenograft breast cancer mouse model demonstrated that the rate of growth and final tumour volume were reduced significantly in the mouse group that received intravenous treatment of PRKCA siRNA+NPs, and PTEN plasmid+NPs. Our findings demonstrated that PRKCA siRNA and PTEN plasmid loaded into CA NPs attenuated breast tumour growth, suggesting their therapeutic potential in the treatment of breast cancer.

Building siRNAs with Cubes: Synthesis and Evaluation of Cubane-Modified siRNAs.

Cubane molecules hold great potential for medicinal chemistry applications due to their inherent stability and low toxicity. In this study, we report the synthesis of a cubane derivative phosphoramidite for the incorporation of cubane into small interfering RNAs (siRNAs). Synthetic siRNAs rely on chemical modifications to improve their pharmacokinetic profiles. However, they are still able to mediate sequence-specific gene silencing via the endogenous RNA interference pathway. We designed a library of siRNAs bearing cubane at different positions within the sense and antisense strands. All siRNAs showed excellent gene-silencing activity, with IC50 values ranging from 45.4 to 305 pM. Incorporating the cubane modification in both the sense and antisense strand led to viable duplexes with good biological activity. To the best of our knowledge, this is the first report of siRNAs bearing a cubane derivative within the backbone.

New therapeutic option for acute hepatic porphyrias

Givosiran is a small synthetic double-stranded siRNA (small interfering RNA) conjugated with N-acetyl-galactosamine (GalNAc) for specific hepatocyte targeting via the asialoglycoprotein receptor. A prospective randomized multicenter study (Envision) demonstrated the clinical efficacy of monthly subcutaneous injection of Givosiran for the prevention of attacks of acute hepatic porphyria (AHP). This leads to highly selective transcriptional inhibition of the key hepatic enzyme, aminolaevulinate synthase 1, that is overexpressed in AHP. The success of the Envision study has led to the approval of Givosiran in the US and Europe for the treatment of severe AHP. This innovative guided siRNA therapy has opened up the possibility to selectively inhibit the expression of any hepatocyte gene whose overexpression that causes pathology, which can be considered a milestone development in hepatology. However, currently this treatment with givosiran is very costly. Moreover, since some patients experience worsening of kidney function and elevated aminotransferases, monthly monitoring of these parameters is necessary in the first half year of treatment.

In vivo self-assembled small RNAs as a new generation of RNAi therapeutics

RNAi therapy has undergone two stages of development, direct injection of synthetic siRNAs and delivery with artificial vehicles or conjugated ligands; both have not solved the problem of efficient in vivo siRNA delivery. Here, we present a proof-of-principle strategy that reprogrammes host liver with genetic circuits to direct the synthesis and self-assembly of siRNAs into secretory exosomes and facilitate the in vivo delivery of siRNAs through circulating exosomes. By combination of different genetic circuit modules, in vivo assembled siRNAs are systematically distributed to multiple tissues or targeted to specific tissues (e.g., brain), inducing potent target gene silencing in these tissues. The therapeutic value of our strategy is demonstrated by programmed silencing of critical targets associated with various diseases, including EGFR/KRAS in lung cancer, EGFR/TNC in glioblastoma and PTP1B in obesity. Overall, our strategy represents a next generation RNAi therapeutics, which makes RNAi therapy feasible.

Novel vector preS1-tp fusion protein effectively inhibits hepatitis B virus replication and cccDNA synthesis by mediating hepatitis B virus targeting sequence small interfering RNA

Objective: To study the use of preS1-tp fusion protein as a novel vector to mediate the entry of small interfering RNA (siRNA) targeting the carboxy-terminal nuclear localization signal (NLS) region of hepatitis B virus (HBV) core protein into HBV-infected hepatocytes, and to further explore the HBV replication inhibition and covalently closed circular DNA synthesis. Methods: HepG2.2.15 cells expressing the human sodium taurocholate co-transporting polypeptide were established on the basis of lentivirus infection system. siRNA against HBV NLS region was designed and synthesized. PreS1-tp fusion protein expression and purification was observed to test its ability to cell entry and DNA binding. NLS siRNA were delivered into HepG2.2.15- sodium taurocholate co-transporting polypeptide cells by preS1-tp fusion protein as a vector to observe the effects of NLS siRNA on HBV replication and covalently closed circular DNA levels. Analysis of variance was used for comparison between multiple groups, and the measurement data differences between groups were analyzed by t-test. Results: HepG2.2.15-sodium taurocholate co-transporting polypeptide cell line was successfully constructed. Screened synthetic HBV NLS siRNA had significantly inhibited HBV replication. The preS1-tp fusion protein was expressed and purified on a large-scale. The fusion protein as a vector for HBV NLS siRNA had targeted delivery. The result showed that the fusion protein had effectively targeted siRNA to Hepg2.2.15-sodium taurocholate co-transporting polypeptide cell, which not only had effectively inhibited the expression of HBV mRNA, HBsAg and HBeAg, but also had significantly reduced the levels of HBV DNA and covalently closed circular DNA. Conclusion: The preS1-tp fusion protein constructed in this study uses the dual functional characteristics of preS1 binding to hepatocyte HBV receptor, and tp binding to nucleic acids, and targets HBV NLS siRNA against HBV-infected cells and block rcDNA from being transported to nucleus. siRNA plays a role in inhibiting HBV replication and covalently close circular DNA synthesis, providing a new strategy for the treatment of chronic hepatitis B caused by HBV infection, and a new research perspective for the complete elimination of HBV from the body.

Combined high-throughput library screening and next generation RNA sequencing uncover microRNAs controlling human cardiac fibroblast biology

BackgroundMyocardial fibrosis is a hallmark of the failing heart, contributing to the most common causes of deaths worldwide. Several microRNAs (miRNAs, miRs) controlling cardiac fibrosis were identified in recent years; however, a more global approach to identify miRNAs involved in fibrosis is missing. Methods and resultsFunctional miRNA mimic library screens were applied in human cardiac fibroblasts (HCFs) to identify annotated miRNAs inducing proliferation. In parallel, miRNA deep sequencing was performed after subjecting HCFs to proliferating and resting stimuli, additionally enabling discovery of novel miRNAs. In-depth in vitro analysis confirmed the pro-fibrotic nature of selected, highly conserved miRNAs miR-20a-5p and miR-132-3p. To determine downstream cellular pathways and their role in the fibrotic response, targets of the annotated miRNA candidates were modulated by synthetic siRNA. We here provide evidence that repression of autophagy and detoxification of reactive oxygen species by miR-20a-5p and miR-132-3p explain some of their pro-fibrotic nature on a mechanistic level. ConclusionWe here identified both miR-20a-5p and miR-132-3p as crucial regulators of fibrotic pathways in an in vitro model of human cardiac fibroblast biology.