Synthetic lethal therapy for KRAS mutant non-small-cell lung carcinoma with nanoparticle-mediated CDK4 siRNA delivery.

The KRAS mutation is present in ~20% of lung cancers and has not yet been effectively targeted for therapy. This mutation is associated with a poor prognosis in non-small-cell lung carcinomas (NSCLCs) and confers resistance to standard anticancer treatment drugs, including epidermal growth factor receptor tyrosine kinase inhibitors. In this study, we exploited a new therapeutic strategy based on the synthetic lethal interaction between cyclin-dependent kinase 4 (CDK4) downregulation and the KRAS mutation to deliver micellar nanoparticles (MNPs) containing small interfering RNA targeting CDK4 (MNPsiCDK4) for treatment in NSCLCs harboring the oncogenic KRAS mutation. Following MNPsiCDK4 administration, CDK4 expression was decreased, accompanied by inhibited cell proliferation, specifically in KRAS mutant NSCLCs. However, this intervention was harmless to normal KRAS wild-type cells, confirming the proposed mechanism of synthetic lethality. Moreover, systemic delivery of MNPsiCDK4 significantly inhibited tumor growth in an A549 NSCLC xenograft murine model, with depressed expression of CDK4 and mutational KRAS status, suggesting the therapeutic promise of MNPsiCDK4 delivery in KRAS mutant NSCLCs via a synthetic lethal interaction between KRAS and CDK4.

Co-silence of the coatomer ß and v-ATPase A genes by siRNA feeding reduces larval survival rate and weight gain of cotton bollworm, Helicoverpa armigera.

Coatomer and v-ATPase are two genes expressed in insect midgut epithelial cells and their knockdown is lethal to insect larvae. To investigate the RNAi response mediated by multiple siRNA duplexes, partial length cDNA of Helicoverpa armigera coatomer ß and v-ATPase A was cloned and siRNA feeding-based RNAi was performed. Simultaneous ingestion of siRNAs specific to the H. armigera coatomer ß and v-ATPase A led to co-silencing of the target genes and reduction in larval survival rate and weight gain. These results suggest that silencing two genes by feeding of multiple siRNAs is a good RNAi strategy.

Targeted nanodelivery of siRNA against KRAS G12D inhibits pancreatic cancer.

Pancreatic cancer (PC) stands as a most deadly malignancy due to few effective treatments in the clinics. KRAS G12D mutation is a major driver for most PC cases, and silencing of KRAS G12D is considered as a potential therapeutic strategy for PC, which is nevertheless crippled by lacking a pragmatic delivery system for siRNA against KRAS G12D (siKRAS). Here, we report that cRGD peptide-modified bioresponsive chimaeric polymersomes (cRGD-BCP) mediate highly efficient siKRAS delivery to PANC-1 tumor, potently silencing KRAS G12D mRNA in tumor cells and effectively suppressing PC tumor growth in mice. cRGD-BCP exhibited remarkable encapsulation of siKRAS (loading content > 14 wt.%, loading efficiency > 90%) to form stable and uniform (ca. 68 nm) nanovesicles (cRGD-BCP-siKRAS). Of note, cRGD density greatly impacted the cellular uptake and silencing efficiency of cRGD-BCP-siKRAS in PANC-1 cells, in which an optimal cRGD density of 15.7 mol.% achieved 3.7- and 3.6-fold enhancement of internalization and gene silencing, respectively, compared with non-targeted BCP-siKRAS. cRGD-BCP-siKRAS was practically intact after 3-week storage at 4°C. Intriguingly, cRGD-BCP-siKRAS markedly enhanced the uptake of siKRAS in PANC-1 tumor, and at a siKRAS dose of 3 mg/kg knocked down 90% KRAS G12D gene, resulting in potent tumor inhibition and extraordinary survival benefits (median survival time: 101 days versus 38 (PBS group) and 59 days (BCP-siKRAS)) with 40% mice achieved complete regression. It appears that cRGD-mediated nanodelivery of siKRAS provides a potential cure for pancreatic cancer. STATEMENT OF SIGNIFICANCE: Small interfering RNA (siRNA) emerges as a specific and powerful biopharmaceuticals against cancers; however, inefficient in vivo delivery impedes its clinical translation. In spite of the fact that KRAS G12D mutation has been identified as a major driver for most pancreatic cancer, its notorious non-druggability renders little success on development of molecular targeted drugs. Pancreatic cancer is deemed as current king-of-cancer. Here, we show that cyclic RGD peptide installed bioresponsive polymersomes are able to efficiently deliver siRNA against KRAS G12D to pancreatic tumor, resulting in 90% gene knock-down and effective tumor inhibition. Strikingly, two out of five mice have been cured. This targeted nanodelivery of siRNA provides a high-efficacy treatment strategy for pancreatic cancer.

Targeting SOD1 via RNAi with PEGylated graphene oxide nanoparticles in platinum-resistant ovarian cancer.

Acquired platinum resistance poses a significant therapeutic impediment to ovarian cancer patient care, accounting for more than 200,000 deaths annually worldwide. We previously identified that overexpression of the antioxidant superoxide dismutase 1 (SOD1) in ovarian cancer is associated with a platinum-resistant phenotype via conferring oxidative stress resistance against platinum compounds. We further demonstrated that enzymatic inhibition using small-molecule inhibitors or silencing of SOD1 via RNA interference (RNAi) increased cisplatin sensitivity and potency in vitro. We launched this study to explore the potential therapeutic applications of SOD1 silencing in vivo in order to reverse cisplatin resistance using a graphene-based siRNA delivery platform. PEGylated graphene oxide (GO) polyethyleneimine (GOPEI-mPEG) nanoparticle was complexed with SOD1 siRNA. GOPEI-mPEG-siSOD1 exhibited high biocompatibility, siRNA loading capacity, and serum stability, and showed potent downregulation of SOD1 mRNA and protein levels. We further observed that cisplatin and PEI elicited mitochondrial dysfunction and transcriptionally activated the mitochondrial unfolded protein response (UPRmt) used as a reporter for their respective cytotoxicities. SOD1 silencing was found to augment cisplatin-induced cytotoxicity resulting in considerable tumour growth inhibition in cisplatin-sensitive A2780 and cisplatin-resistant A2780DDP subcutaneous mouse xenografts. Our study highlights the potential therapeutic applicability of RNAi-mediated targeting of SOD1 as a chemosensitizer for platinum-resistant ovarian cancers.

Co-delivery of gemcitabine and paclitaxel plus NanoCpG empowers chemoimmunotherapy of postoperative "cold" triple-negative breast cancer.

Triple-negative breast cancer (TNBC) due to lack of clear target and notorious "cold" tumor microenvironment (TME) is one of the most intractable and lethal malignancies. Tuning "cold" TME into "hot" becomes an emerging therapeutic strategy to TNBC. Herewith, we report that integrin-targeting micellar gemcitabine and paclitaxel (ATN-mG/P, ATN sequence: Ac-PhScNK-NH2) cooperating with polymersomal CpG (NanoCpG) effectively "heated up" and treated TNBC. ATN-mG/P exhibited greatly boosted apoptotic activity in 4T1 cells, induced potent immunogenic cell death (ICD), and efficiently stimulated maturation of bone marrow-derived dendritic cells (BMDCs). Remarkably, in a postoperative TNBC model, ATN-mG/P combining with NanoCpG promoted strong anti-cancer immune responses, showing a greatly augmented proportion of mature DCs and CD8+ T cells while reduced immune-suppressive myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Treg), which led to complete inhibition of lung metastasis and 60% mice tumor-free. The co-delivery of gemcitabine and paclitaxel at desired ratio in combination with NanoCpG provides a unique platform for potent chemoimmunotherapy of "cold" tumors like TNBC.

Down-regulation of spinal D-amino acid oxidase expression blocks formalin-induced tonic pain.

A series of inhibitors of d-amino acid oxidase (DAAO) are specific in blocking chronic pain, including formalin-induced tonic pain, neuropathic pain and bone cancer pain. This study used RNA interference technology to further validate the notion that spinal DAAO mediates formalin-induced pain. To target DAAO, a siRNA/DAAO formulated in polyetherimide (PEI) complexation and a shRNA/DAAO (shDAAO, with the same sequence as siRNA/DAAO after intracellular processing) expressed in recombinant adenoviral vectors were designed. The siRNA/DAAO was effective in blocking DAAO expression in NRK-52E rat kidney tubule epithelial cells, compared to the nonspecific oligonucleotides. Furthermore, multiple-daily intrathecal injections of both siRNA/DAAO and Ad-shDAAO for 7 days significantly inhibited spinal DAAO expression by 50-80% as measured by real-time quantitative PCR and Western blot, and blocked spinal DAAO enzymatic activity by approximately 60%. Meanwhile, both siRNA/DAAO and Ad-shDAAO prevented formalin-induced tonic phase pain by approximately 60%. Multiple-daily intrathecal injections of siRNA/DAAO and Ad-shDAAO also blocked more than 30% spinal expression of GFAP, a biomarker for the activation of astrocytes. These results further suggest that down-regulation of spinal DAAO expression and enzymatic activity leads to analgesia with its mechanism potentially related to activation of astrocytes in the spinal cord.

Comprehensive analysis of sequence-specific stability of siRNA.

Double-stranded small interfering RNAs (siRNAs) are important modulators of biological processes and hold great promise for therapeutic applications. However, serum processing of synthetic siRNAs is still largely unknown. To address this issue, serum degradation assays of 125 siRNAs were first performed in this study. Four siRNA categories of distinct serum stability were identified, including a group of siRNAs that were stable in their native form for both in vitro and in vivo assays. Fine mapping of the cleavage events occurring in serum treatment demonstrated that most occurred at two vulnerable sites, leading to a speculation that rational modification of these sites might protect most siRNAs from serum degradation. For proof of concept, an exhaustive siRNA modification study was performed. In addition to the consistent stabilization pattern revealed at these sites, our study further showed that a single modification made at the cleavage site stabilized the siRNAs to a large extent, highlighting the importance of these sites in siRNA degradation. In summary, the present study provided a comprehensive picture of serum processing of siRNA as well as a starting point for a rational siRNA modification strategy, both of which are of great importance to in vivo and therapeutic applications of siRNA.

Targeted delivery of antisense inhibitor of miRNA for antiangiogenesis therapy using cRGD-functionalized nanoparticles.

MiRNAs are viable therapeutic targets for cancer therapy, but the targeted delivery of miRNA or its anti-miRNA antisense oligonucleotides (AMOs) remains a challenge. We report here a PEGylated LPH (liposome-polycation-hyaluronic acid) nanoparticle formulation modified with cyclic RGD peptide (cRGD) for specific and efficient delivery of AMO into endothelial cells, targeting α(v)ß3 integrin present on the tumor neovasculature. The nanoparticles effectively delivered anti-miR-296 AMO to the cytoplasm and downregulated the target miRNA in human umbilical vein endothelial cells (HUVECs), which further efficiently suppressed blood tube formulation and endothelial cell migration, owing to significant upregulation of hepatocyte growth factor-regulated tyrosine kinase substrate (HGS), whereas nanoparticles without cRGD modification showed only little AMO uptake and miRNA silencing activity. In vivo assessment of angiogenesis using Matrigel plug assay also demonstrated that cRGD modified LPH nanoparticles have potential for antiangiogenesis in miRNA therapeutics. With the delivery of anti-miR-296 AMO by targeted nanoparticles, significant decrease in microvessel formulation within Matrigel was achieved through suppressing the invasion of CD31-positive cells into Matrigel and prompting HGS expression in angiogenic endothelial cells.

Gold nanoparticles capped with polyethyleneimine for enhanced siRNA delivery.

An efficient and safe delivery system for small interfering RNA (siRNA) is required for clinical application of RNA interfering therapeutics. Polyethyleneimine (PEI)-capped gold nanoparticles (AuNPs) are successfully manufactured using PEI as the reductant and stabilizer, which bind siRNA at an appropriate weight ratio by electrostatic interaction and result in well-dispersed nanoparticles with uniform structure and narrow size distribution. With siRNA binding, PEI-capped AuNPs induce more significant and enhanced reduction in targeted green fluorescent protein expression in MDA-MB-435s cells, though more internalized PEI/siRNA complexes in cells are evidenced by confocal laser scanning microscopy observation and fluorescence-activated cell sorting analyses. PEI-capped AuNPs/siRNA targeting endogenous cell-cycle kinase, an oncogene polo-like kinase 1 (PLK1), display significant gene expression knockdown and induce enhanced cell apoptosis, whereas it is not obvious when the cells are treated with PLK1 siRNA using PEI as the carrier. Without exhibiting cellular toxicity, PEI-capped AuNPs appear to be suitable as a potential carrier for intracellular siRNA delivery.

The design, synthesis and evaluation of hypoxia-activated pro-oligonucleotides.

Hypoxia-activated pro-oligonucleotides were synthesized through the commercial phosphoramidite method, and could be readily cleaved to form normal oligos with good hypoxia selectivity in vitro under the effect of reductases, as well as in tumor cell extract.

RNA interference in mammalian cells by siRNAs modified with morpholino nucleoside analogues.

siRNAs modified with morpholino nucleoside analogues were synthesized and their biological properties were examined in details. The gene silence abilities of modified siRNAs were correlated to the positions of the modifications, some of which appeared to be more potent than the native siRNA. The 3'-end modification improved the stability of siRNAs in serum significantly. Furthermore, the dose-response and time-course experiments demonstrated that the siRNAs with 3'-end modification have potent gene silence activity at lower concentration and prolonged action time. These favorable properties make the morpholino modified siRNA a potentially useful tool in therapeutic applications.

Aberrant expression of microRNAs in peripheral blood mononuclear cells as candidate biomarkers in patients with axial spondyloarthritis.

OBJECTIVES: Axial spondyloarthritis (axSpA) is a chronic inflammatory arthritis involving the axial skeleton. Recent evidence suggests that microRNAs (miRNAs) play a critical role in ankylosing spondylitis (AS). In this study, we aimed to investigate whether miR-17-5p, miR-27a, miR-29a and miR-126-3p can be verified as potential biomarkers of axSpA. METHODS: Peripheral blood mononuclear cell (PBMC) miRNA expression was evaluated by quantitative real-time polymerase chain reaction among 43 patients with AS, 26 patients with non-radiographic axSpA (nr-axSpA) and 39 healthy controls. Detailed clinical histories were recorded and the correlation of miRNAs and clinical features were analyzed. RESULTS: When compared to controls, both patients with AS and nr-axSpA had significantly higher expression levels of miR-17-5p, miR-27a, miR-29a and miR-126-3p. MiR-27a was negatively correlated with Ankylosing Spondylitis Disease Activity Score as well as C-reactive protein in patients with nr-axSpA (r = -0.51, P < 0.01 and r = -0.42, P = 0.034 respectively). No other clinical features were found to correlate with the four miRNAs in patients with AS. Mir-29a showed highest area under the curve with 0.952 and these four miRNAs may be potential biomarkers in patients with axSpA. CONCLUSIONS: We reported elevated miR-17-5p, miR-27a, miR-29a and miR-126-3p expression in PBMCs of patients with axSpA, and the expression of these four miRNAs might be used as useful diagnostic markers in axSpA.

A Detailed Protein-SELEX Protocol Allowing Visual Assessments of Individual Steps for a High Success Rate.

As a nucleic acid alternative to traditional antibody, aptamer holds great potential in various fields of biology and medicine such as targeted gene therapy, drug delivery, bio-sensing, and laboratory medicine. Over the past decades, the conventional Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method has undergone dramatic modifications and improvements owing to developments in material sciences and analytical techniques. However, many of the recently developed strategies either require complex materials and instruments or suffer from low efficiency and high failure rates in the selection of desired aptamers. Accordingly, the development of aptamers against new or novel targets is still a major obstacle for aptamer-based research and application. Here, an improved protein-SELEX procedure is presented for simplified and highly efficient isolation of aptamers against protein targets. Approaches are described that ensure a high success rate in aptamer selection by simplifying polymerase chain reaction procedures, introducing denature gel, utilizing an electro-elution-based single-stranded DNA separation strategy, as well as an enzyme-linked immunosorbent assay-based highly sensitive binding assay. In addition, a simplified sample preparation method for MiSeq-based next-generation sequencing is also introduced. While a recombinant protein as a bait protein for SELEX is discussed here, this protocol will also be invaluable for researchers wishing to develop aptamers against targets other than proteins such as small molecules, lipids, carbohydrates, cells, and micro-organisms for future gene therapy and/or diagnostics.

Circulating HER-2 mRNA in the peripheral blood as a potential diagnostic and prognostic biomarker in females with breast cancer.

Breast cancer is a prevalent malignant cancer worldwide, and a lack of defined biomarkers for early prognostication contributes to its high associated mortality rate, especially in human epidermal growth factor receptor 2 (HER-2)-positive breast cancer. In the present study, HER-2 mRNA levels in patients were detected prior to surgery and during neoadjuvant chemotherapy to explore its potential diagnostic and prognostic value. Blood samples were collected from 70 patients with breast cancer, including 50 HER-2-negative and 20 HER-2-positive patients, prior to and following surgery (postoperative, n=13; neoadjuvant chemotherapy, n=5); the control group included 35 samples from healthy individuals. The relative mRNA level of HER-2 in blood was determined by one-step reverse transcription-quantitative polymerase chain reaction. HER-2 expression curves of measurements taken during neoadjuvant chemotherapy were compared with the tumor size. A significant difference in the blood HER-2 mRNA level was observed between healthy women and patients with breast cancer (P<0.0001). A cutoff value of 1.512 was established for the circulating HER-2 level in healthy subjects based on the upper 95% confidence interval value of samples from the control group. The level of HER-2 mRNA in blood was associated with the HER-2 status, Ki-67 expression, and lymphovascular invasion in primary tumor tissue samples; however, there was no association with the lymph node status, tumor stage, tumor grade, tumor size, patient age, estrogen or progesterone receptor status of the primary tumor. HER-2 mRNA levels were associated with the response rate, as determined by primary tumor size, in patients who received neoadjuvant chemotherapy. In conclusion, baseline and early changes in peripheral blood HER-2 mRNA indicated that HER-2 mRNA may be a potential diagnostic biomarker for breast cancer and a prognostic marker for predicting the efficacy of neoadjuvant therapy.

Aptamer-mediated survivin RNAi enables 5-fluorouracil to eliminate colorectal cancer stem cells.

The development of chemoresistance and inability in elimination of cancer stem cells are among the key limitations of cancer chemotherapy. Novel molecular therapeutic strategies able to overcome such limitations are urgently needed for future effective management of cancer. In this report, we show that EpCAM-aptamer-guided survivin RNAi effectively downregulated survivin both in colorectal cancer cells in vitro and in a mouse xenograft model for colorectal cancer. When combined with the conventional chemotherapeutic agents, the aptamer-guided survivin RNAi was able to enhance the sensitivity towards 5-FU or oxaliplatin in colorectal cancer stem cells, increase apoptosis, inhibit tumour growth and improve the overall survival of mice bearing xenograft colorectal cancer. Our results indicate that survivin is one of the key players responsible for the innate chemoresistance of colorectal cancer stem cells. Thus, aptamer-mediated targeting of survivin in cancer stem cells in combination with chemotherapeutic drugs constitutes a new avenue to improve treatment outcome in oncologic clinics.

Transforming doxorubicin into a cancer stem cell killer via EpCAM aptamer-mediated delivery.

Chemotherapy-resistant cancer stem cells (CSCs) are a major obstacle to the effective treatment of many forms of cancer. To overcome CSC chemo-resistance, we developed a novel system by conjugating a CSC-targeting EpCAM aptamer with doxorubicin (Apt-DOX) to eliminate CSCs. Incubation of Apt-DOX with colorectal cancer cells resulted in high concentration and prolonged retention of DOX in the nuclei. Treatment of tumour-bearing xenograft mice with Apt-DOX resulted in at least 3-fold more inhibition of tumour growth and longer survival as well as a 30-fold lower frequency of CSC and a prolonged longer tumourigenic latency compared with those receiving the same dose of free DOX. Our data demonstrate that a CSC-targeting aptamer is able to transform a conventional chemotherapeutic agent into a CSC-killer to overcome drug resistance in solid tumours.

Disorders of MicroRNAs in Peripheral Blood Mononuclear Cells: As Novel Biomarkers of Ankylosing Spondylitis and Provocative Therapeutic Targets.

BACKGROUND: MicroRNAs can potentially regulate every aspect of cellular activity. In this study, we investigated whether AS pathogenesis involves microRNAs disorders. RESULT: The expression of 2 microRNAs, hsa-miR-126-3p and hsa-miR-29a, was significantly lower in active AS group before etanercept therapy than in control group. Marched fold changes of them were 3.76 and 16.22. Moreover, expressions of hsa-miR-126-3p and hsa-miR-29a were dramatically upregulated after 12-weeks etanercept treatment. Fold changes were 2.20 and 3.18. All regulations of microRNAs expression mentioned before were statistically significant (fold change >2 and P < 0.05). The expression disorders of the 2 microRNAs did not statistically significantly correlated with BASDAI, CRP, and ESR. CONCLUSION: AS pathogenesis involved dysregulation of microRNAs. Hsa-miR-126-3p and hsa-miR-29a will probably become the potential biomarkers and provocative therapeutic targets of AS.

Silencing herpes simplex virus type 1 capsid protein encoding genes by siRNA: a promising antiviral therapeutic approach.

Herpes simplex virus type 1 (HSV-1), a member of the herpesviridae, causes a variety of human viral diseases globally. Although a series of antiviral drugs are available for the treatment of infection and suppression of dissemination, HSV-1 remains highly prevalent worldwide. Therefore, the development of novel antiviral agents with different mechanisms of action is a matter of extreme urgency. During the proliferation of HSV-1, capsid assembly is essential for viral growth, and it is highly conserved in all HSV-1 strains. In this study, small interfering RNAs (siRNAs) against the HSV-1 capsid protein were screened to explore the influence of silencing capsid expression on the replication of HSV-1. We designed and chemically synthesized siRNAs for the capsid gene and assessed their inhibitory effects on the expression of target mRNA and the total intracellular viral genome loads by quantitative real-time PCR, as well as on the replication of HSV-1 via plaque reduction assays and electron microscopy. Our results showed that siRNA was an effective approach to inhibit the expression of capsid protein encoding genes including UL18, UL19, UL26, UL26.5, UL35 and UL38 in vitro. Interference of capsid proteins VP23 (UL18) and VP5 (UL19) individually or jointly greatly affected the replication of clinically isolated acyclovir-resistant HSV-1 as well as HSV-1/F and HSV-2/333. Plaque numbers and intracellular virions were significantly reduced by simultaneous knockdown of UL18 and UL19. The total intracellular viral genome loads were also significantly decreased in the UL18 and UL19 knockdown groups compared with the viral control. In conclusion, interfering with UL18 and UL19 gene expression could inhibit HSV-1 replication efficiently in vitro. Our research offers new targets for an RNA interference-based therapeutic strategy against HSV-1.

Investigation of the endovascularly treated and untreated unruptured vertebrobasilar artery aneurysms.

BACKGROUND AND PURPOSE: The present study aimed to investigate the endovascular indication with close monitoring of both endovascularly treated and untreated unruptured vertebrobasilar aneurysms. MATERIALS AND METHODS: In the past 2 years, 36 patients [7 women and 29 men with a mean age of 48.5 years (range, 13-75 years)] who had 40 unruptured vertebrobasilar artery aneurysms were reviewed. The decision of the endovascular treatment was not randomized. In both endovascularly untreated and treated patients, overall mortality and morbidity corresponding to Glasgow outcome scale (GOS) score III or worse was counted as unruptured aneurysm related event. The ratio of event free was compared between endovascularly treated and untreated patients using Log-rank test. In a univariate analysis, poor outcome was predicted by endovascularly untreated. RESULTS: Thirty-six patients were followed with a mean period of 10.7 months (range, 0.7 months-21 months). In untreated cases, new neurological deficits caused by the aneurysm were noted in 2 cases. The annual deterioration rate of the untreated cases was 9.1%. In endovascularly treated cases, there was no mortality and morbidity. There was no SAH in all cases during follow-up period. When ratio of event free was compared, significant advantage of endovascular treatment was noted (X(2)=4.429, P=0.035). CONCLUSIONS: The present results indicated that endovascular treatment has a benefit for the unruptured vertebrobasilar artery aneurysms.