Dose Of siRNA Research Articles

Ionic liquid combined with cationic liposome co-delivers microphthalmia-associated transcription factor small interfering RNA to regulate melanogenesis

Suppressing allele-specific genes using small interfering RNAs (siRNAs) can effectively whiten skin by influencing cellular gene and protein expression. Topical delivery of siRNA is a promising alternative to injections for RNA interference. However, the barrier function of the skin hinders the effective penetration of siRNA. Here, we report, a novel approach to achieve the transdermal delivery of effective siRNA doses using a complementary synergistic strategy of an ionic liquid (IL) and cationic liposome (CL). Microphthalmia-associated transcription factor (MITF) siRNA molecules were formed through electrostatic adsorption of the IL and CL to form positively charged nanocomposites, which were named IL-CL/p-siM. IL-CL/p-siM has a particle size of 171.47 nm, ζ-potential of 29.94 mV, high encapsulation rate of 92.11 %, and pH-sensitive release properties. In vitro studies on porcine skin confirmed the additive/synergistic effect of this strategy in enhancing epidermal and dermal penetration. This combination enabled superior transfection efficiency and cell viability while inhibiting melanin synthesis in skin melanocytes by downregulating the expression of genes downstream of MITF, namely tyrosinase-related protein-1, tyrosinase, and tyrosinase-related protein-2, which are associated with the melanocortin 1 receptor. We also conducted clinical studies that demonstrated its potential in treating melasma and its anti-melanotic efficacy. To summarize, IL-CL/p-siM represents a simple, personalized, and scalable platform for effective local delivery of siRNA to treat skin complications.

RNA Therapy for Oncogenic NRAS-Driven Nevi Induces Apoptosis

RAS proteins regulate cell division, differentiation and apoptosis via multiple downstream effector pathways. Oncogenic RAS variants are the commonest drivers in cancers, however they also drive many benign lesions predisposing to malignancy, such as melanocytic naevi, thyroid nodules, and colonic polyps. Reversal of these benign lesions could reduce cancer incidence, however the effects of oncogenic RAS have been notoriously difficult to target with downstream pathway inhibitors. Here we show effective suppression of oncogenic and currently undruggable NRASQ61K in primary cells from melanocytic naevi using siRNA targeted to the recurrent causal variant. This results in striking reduction in expression of ARL6IP1, a known inhibitor of endoplasmic reticulum stress-induced apoptosis not previously linked to NRAS. We go on to show that a single dose of siRNA in primary cells triggers an apoptotic cascade, in contrast to treatment with a MEK inhibitor. Protective packaging of the targeted siRNA into lipid nanoparticles permits successful delivery into a humanised mouse model of melanocytic naevi, and results in variant NRAS knockdown in vivo. These data show that RAS-induced protection from apoptosis is involved in persistence of NRAS-driven melanocytic naevi and anticipate that targeted siRNA could form the basis of clinical trials for RAS-driven benign tumours.

Investigating the effect of LncRNA DLGAP1-AS2 suppression on chemosensitivity of gastric cancer to chemotherapy.

Gastric cancer, as the fifth most frequent disease and the fourth foremost cause of cancer-related death worldwide, remains a main clinical challenge due to its poor prognosis, limited treatment choices, and ability to metastasize. Combining siRNAs to suppress lncRNA with chemotherapeutic medications is a novel treatment approach that eventually increases the therapeutic efficacy of the drug while lessening its adverse effects. This study was performed with the purpose of examining the impact of inhibiting DLGAP1-AS2 expression on gastric cancer cells' drug chemosensitivity. AGS cells were cultured as the study cell line and were transfected with an optimum dose of DLGAP1-AS2 siRNA and then treated with oxaliplatin. Cell viability was examined using the MTT technique. Apoptosis and cell cycle were evaluated using Annexin V/PI staining and flow cytometry. Later, the scratch test was conducted to investigate the ability of cells to migrate, and the inhibition of the stemness of AGS cells was further investigated through the colony formation method. Finally, the qRT-PCR technique was used to assess the expression of Bax, Bcl-2, Caspase-3, p53, MMP-2, and CD44 genes. The MTT test indicated the effect of gene therapy with siRNA and oxaliplatin in combination reduced the chemotherapy drug dose to 29.92µM and increased AGS cells' sensitivity to oxaliplatin. Also, the combination therapy caused a significant increase in apoptosis. However, it reduced the stemness feature, the rate of cell viability, proliferation, and metastasis compared to the effect of each treatment alone; the results also showed the arrest of the cell cycle in the Sub G1 phase after the combined treatment and a further reduction in the number and size of the formed colonies. Suppressing the expression of lncRNA DLGAP1-AS2 by siRNA followed by treatment with oxaliplatin can be utilized as an effective and new therapeutic technique for gastric cancer therapy.

Abstract 4607: Targeted nanoparticle delivery of irinotecan and Ewing sarcoma-specific siRNA is an effective combination therapy for Ewing sarcoma

Abstract Background: Ewing sarcoma (ES) is an aggressive bone and soft tissue tumor affecting children and adolescents with an overall survival rate of 50%. Irinotecan (IR) is an effective chemotherapeutic agent but is associated with significant toxicity. Using an anti-CD99 targeted hybrid polymerized lipid nanoparticle containing IR, NV103, in pre-clinical mouse xenograft model of the disease, tumor ablation was achieved at 2mg/kg of IR versus 50mg/kg for free IR. All ES harbor a chromosomal translocation resulting in expression of a fusion EWS-FLI1 protein or equivalent. This chimeric gene is a transcriptional regulator that upregulates the FEZF1 and FEZF1-AS1 genes. Both EWS-FLI1 and FEZF1-AS1 genes are unique to ES and are not expressed in normal adult tissues. EWS-FLI1 promotes cell growth while the FEZF1-AS1 complex promotes tumor metastasis, a hallmark of ES. In vitro RNAi targeting of EF and FEZF1-AS1 results in tumor cell death. We hypothesized that combining EWS-FLI1 and/or FEZF1-AS1 targeted RNAi therapy with NV103 cytotoxic therapy would be more effective than any single modality therapy. Methods: NV103 was provided by NanoValent Pharmaceuticals. siRNA containing lipid nanoparticles (LNP-siRNA) were generated using PreciGenome NanoGenerator. siRNA was quantified using the ribogreen assay. Nanoparticles were analyzed for size and particle numbers by NanoSight. siRNA was used to target the following genes- EWS-FLI1, FEZF1, FEZF1-AS1, or GFP. A673 cells expressing GFP were used for this in vitro study. Various doses of IR and siRNA were used in combination and cell growth/death was imaged live using Incucyte. ATP assay was used to confirm live cells and to determine minimum effective dose (MED) over 72 hours of treatment. Results: LNP-siRNA, mean diameter 70nm and PDI<0.2, encapsulated 90% of the siRNA in solution. LNP are stable at 4°C for at least 4 weeks with minimal-to-no loss of activity. NV103 had a MED of 1µM. MED of various siRNA was 30 to 50nM. Combination of NV103 and LNP-siRNA reduced MED of IR to 250nM. One treatment dose of LNP-siRNA followed by NV103 treatment three days later maintained significant cell death over 10 days post treatment initiation. Control cells were 97% more confluent than NV103/EWS-FLI1 siRNA treated ones, 85% more than NV103/FEZF1 siRNA cells and 92% more than NV103/FEZF1-AS1 treated cells. siRNA combinations of EWS-FLI1 and FEZF1 or FEZF1-AS1 along with NV103 added no further benefit. Conclusion: Combination nanoparticle therapy of IR and ES-specific siRNA reduces IR MED by four-fold. LNP delivery of siRNA enables tumor-specific gene targeting. Most have no available small molecule therapy. Targeting ES cells with both a low dose (250nM) of IR and 30-50nM of siRNA against unique tumor biomarkers results in increased cell death with no IR toxicity. Further animal studies are required to determine the effectiveness of such therapy in vivo. Citation Format: Sheetal A. Mitra, Jean-Hugues Parmentier, Hyung-Gyoo Kang, Timothy J. Triche. Targeted nanoparticle delivery of irinotecan and Ewing sarcoma-specific siRNA is an effective combination therapy for Ewing sarcoma [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 4607.

Evaluation of mAb 2C5-modified dendrimer-based micelles for the co-delivery of siRNA and chemotherapeutic drug in xenograft mice model

Combination therapy with small interfering RNA (siRNA) and chemotherapeutic drug is proven to be effective in downregulating cancer resistance proteins, such as P-glycoprotein (P-gp). These proteins are involved in multidrug resistance (MDR) of tumors. A targeted formulation capable of delivering siRNA and chemotherapeutic drug will not only downregulate P-gp but also increase the concentration of the chemotherapeutic drug at the site of tumor thereby increasing the therapeutic effect and lowering the systemic exposure. In this study, monoclonal antibody 2C5-modified dendrimer-based micelles were used to co-deliver siRNA and doxorubicin (DOX) to the tumor site in both male and female xenograft mouse model. The nucleosome-specific 2C5 antibody recognizes the cancer cells via the cell-surface bound nucleosomes. The ability of ability of the 2C5-modified formulation to affect the metastasis of highly aggressive triple negative breast cancer cell migration in (MDA-MB-231) was assessed by a wound healing. Further, the therapeutic efficacy of the formulation was assessed by measuring the tumor volume progression in which the 2C5-modified nanoparticle group had a similar tumor volume to the free drug group at the end of the study, although a 50% increase in DOX concentrations in blood was observed after the last dose of nanoparticle. The free drug group on the other hand showed body weight reduction as well as the visible irritation around the injection spot. The treatment group with 2C5-modified micelles has shown to be safe at the current dose of DOX and siRNA. Furthermore, the siRNA mediated P-gp downregualtion was studied using western blotting assay. We observed a 29% reduction of P-gp levels in both males and females with respect to the control (BHG). We also conclude that the dose of DOX and siRNA should be further optimized to have a better efficacy in a metastatic tumor model, which will be the subject of our future studies.Graphical abstract

Milk exosomes anchored with hydrophilic and zwitterionic motifs enhance mucus permeability for applications in oral gene delivery.

Exosomes have emerged as a promising tool for the delivery of drugs and genetic materials, owing to their biocompatibility and non-immunogenic nature. However, challenges persist in achieving successful oral delivery due to their susceptibility to degradation in the harsh gastrointestinal (GI) environment and impeded transport across the mucus-epithelium barrier. To overcome these challenges, we have developed high-purity bovine milk exosomes (mExo) as a scalable and efficient oral drug delivery system, which can be customized by incorporating hydrophilic and zwitterionic motifs on their surface. In our study, we observed significantly improved transport rates by 2.5-4.5-fold in native porcine intestinal mucus after the introduction of hydrophilic and zwitterionic surface modifications, as demonstrated by transwell setup and fluorescence recovery after photobleaching (FRAP) analysis. Remarkably, mExo functionalized by a block peptide (BP), consisting of cationic and anionic amino acids arranged in blocks at the two ends, demonstrated superior tolerability in the acidic gastric environment (with a protein recovery rate of 84.8 ± 7.7%) and exhibited a 2.5-fold increase in uptake by intestinal epithelial cells. Furthermore, both mExo and mExo-BP demonstrated successful intracellular delivery of functional siRNA, resulting in up to 65% suppression of the target green fluorescence protein (GFP) gene expression at a low dose of siRNA (5 pmol) without causing significant toxicity. These findings highlight the immense potential of modifying mExo with hydrophilic and zwitterionic motifs for effective oral delivery of siRNA therapies.

Drp1 is essential for PINK1/Parkin signaling in H9c2 cardiomyocytes.

Drp1 is essential for PINK1/Parkin signaling in H9c2 cardiomyocytes.

Regulation effect of siRNA on β-globin in erythrocytes of hemoglobin H disease

目的研究靶向siRNA对体外定向分化培养的红系细胞β-珠蛋白的调控作用，为血红蛋白H（Hemoglobin H, HbH）病的基因治疗提供新的理论支持。方法①根据β-珠蛋白基因表达结果，在红系细胞中筛选出最佳siRNA序列及其有效作用剂量，检测有效剂量的最佳siRNA对红系细胞β-珠蛋白表达的调控和细胞凋亡的影响。②将有效剂量的最佳siRNA作用于HbH病红系细胞，检测转染后细胞β-珠蛋白表达、活性氧（ROS）和细胞凋亡率，综合评估转染siRNA对HbH病红系细胞的影响。结果①siRNA2可在转染后96 h内显著下调体外培养的红系细胞β-珠蛋白的表达，但对α-珠蛋白无明显调控作用。siRNA2的沉默效应和效应持续时间均与作用剂量有关。②siRNA2可下调HbH病红系细胞β-珠蛋白表达，减少细胞内ROS生成，并减少细胞凋亡率。结论靶向性siRNA可下调HbH病红系细胞β-珠蛋白表达，减少细胞内ROS产生，下调细胞凋亡率。

Targeted RNAi of BIRC5/Survivin Using Antibody-Conjugated Poly(Propylene Imine)-Based Polyplexes Inhibits Growth of PSCA-Positive Tumors.

Delivery of siRNAs for the treatment of tumors critically depends on the development of efficient nucleic acid carrier systems. The complexation of dendritic polymers (dendrimers) results in nanoparticles, called dendriplexes, that protect siRNA from degradation and mediate non-specific cellular uptake of siRNA. However, large siRNA doses are required for in vivo use due to accumulation of the nanoparticles in sinks such as the lung, liver, and spleen. This suggests the exploration of targeted nanoparticles for enhancing tumor cell specificity and achieving higher siRNA levels in tumors. In this work, we report on the targeted delivery of a therapeutic siRNA specific for BIRC5/Survivin in vitro and in vivo to tumor cells expressing the surface marker prostate stem cell antigen (PSCA). For this, polyplexes consisting of single-chain antibody fragments specific for PSCA conjugated to siRNA/maltose-modified poly(propylene imine) dendriplexes were used. These polyplexes were endocytosed by PSCA-positive 293TPSCA/ffLuc and PC3PSCA cells and caused knockdown of reporter gene firefly luciferase and Survivin expression, respectively. In a therapeutic study in PC3PSCA xenograft-bearing mice, significant anti-tumor effects were observed upon systemic administration of the targeted polyplexes. This indicates superior anti-tumor efficacy when employing targeted delivery of Survivin-specific siRNA, based on the additive effects of siRNA-mediated Survivin knockdown in combination with scFv-mediated PSCA inhibition.

Plasma polymerized nanoparticles effectively deliver dual siRNA and drug therapy in vivo

Multifunctional nanocarriers (MNCs) promise to improve therapeutic outcomes by combining multiple classes of molecules into a single nanostructure, enhancing active targeting of therapeutic agents and facilitating new combination therapies. However, nanocarrier platforms currently approved for clinical use can still only carry a single therapeutic agent. The complexity and escalating costs associated with the synthesis of more complex MNCs have been major technological roadblocks in the pathway for clinical translation. Here, we show that plasma polymerized nanoparticles (PPNs), synthesised in reactive gas discharges, can bind and effectively deliver multiple therapeutic cargo in a facile and cost-effective process compatible with up scaled commercial production. Delivery of siRNA against vascular endothelial growth factor (siVEGF) at extremely low concentrations (0.04 nM), significantly reduced VEGF expression in hard-to-transfect cells when compared with commercial platforms carrying higher siRNA doses (6.25 nM). PPNs carrying a combination of siVEGF and standard of care Paclitaxel (PPN-Dual) at reduced doses (< 100 µg/kg) synergistically modulated the microenvironment of orthotopic breast tumors in mice, and significantly reduced tumor growth. We propose PPNs as a new nanomaterial for delivery of therapeutics, which can be easily functionalised in any laboratory setting without the need for additional wet-chemistry and purification steps.

Pharmacokinetics and Biodistribution Analysis of Small Interference RNA for Silencing Tissue Transglutaminase-2 in Celiac Disease After Oral Administration in Mice Using Gelatin-Based Multicompartmental Delivery Systems.

Background: RNA interference (RNAi) therapy has tremendous potential in treating diseases that are characterized by overexpression of genes. However, the biggest challenge to utilize the therapy is to engineer delivery systems that can efficiently transport small interfering RNA (siRNA) to appropriate target sites. Our objective in this study was to develop and evaluate multi-compartmental systems for the oral delivery of siRNA that targets the overexpressed TG2 gene (TG2-siRNA) in the small intestine for the treatment of celiac disease (CD). Materials and Methods: Two types of multicompartmental systems were developed and evaluated: (1) a solid-in-solid multicompartmental system featuring "nanoparticle in microsphere oral system (NiMOS)" where type B gelatin nanoparticles containing TG2-siRNA (TG2-NiMOS) were encapsulated within poly(ɛ-caprolactone) (PCL) based microspheres, and (2) a solid-in-liquid multicompartmental system, "Nanoparticle-in-Emulsion (NiE)" consisting of type-B gelatin nanoparticles containing TG2-siRNA encapsulated within safflower oil containing water-in-oil-in-water (W/O/W) multiple emulsion (TG2-NiE). Results: Evaluation of the biodistribution and pharmacokinetics (PK) after a single oral dose of siRNA containing multicompartmental systems to C57BL/6 mice showed that TG2-siRNA was delivered to the small intestine (duodenum, jejunum and ileum), and colon with minimal systemic exposure via both TG2-NiE and TG2-NiMOS systems. TG2-siRNA exposure (AUC0-t) in the duodenum, jejunum, ileum and colon was 56.4-, 34.3-, 85.5- and 35.5-fold greater for the TG2-NiMOS formulation, relative to the TG2-NiE formulation. Conclusion: The results of this study suggest that TG2-NiMOS formulation was more superior than TG2-NiE formulation in facilitating intestinal delivery of siRNA via the oral route of administration and can be potentially used in the treatment of CD.

Topical delivery of siRNA into skin using ionic liquids

Skin diseases such as lupus, cancer, psoriasis, and hyperhidrosis can potentially be treated effectively by suppressing allele-specific genes using small interfering RNA (siRNA). Injections of siRNA into skin, though effective, are painful and cover small surface areas and thus are not suitable as a long-term treatment option. Topical delivery of siRNA is an attractive alternative option to mediate RNA interference (RNAi). However, the barrier function of the epidermis impedes effective permeation of siRNA into the skin. Herein, we describe topical delivery of siRNA using ionic liquids (ILs) capable of complexing with siRNA non-covalently and delivering it effectively. Using complementary and synergistic strategies of ionic liquids, we report delivery of effective doses of siRNA into skin. The first strategy involved the use of hydrophobic cations to robe the siRNA and the second strategy involved the use of choline-geranic acid ionic liquid (CAGE) to enhance its dermal penetration. In vitro studies in porcine skin confirmed the synergistic effect of these strategies in enhancing epidermal and dermal penetration. In vivo application of siRNA formulation to SKH-1E hairless mice significantly suppressed GAPDH expression with no clinical evidence of toxicity. This is a simple, personalized, and scalable platform for effective topical delivery of siRNA for treating genetic skin diseases.

Reduction of the therapeutic dose of silencing RNA by packaging it in extracellular vesicles via a pre-microRNA backbone.

A small percentage of the short interfering RNA (siRNA) delivered via passive lipid nanoparticles and other delivery vehicles reaches the cytoplasm of cells. The high doses of siRNA and delivery vehicle that are thus required to achieve therapeutic outcomes can lead to toxicity. Here, we show that the integration of siRNA sequences into a Dicer-independent RNA stem-loop based on pre-miR-451 microRNA-which is highly enriched in small extracellular vesicles secreted by many cell types-reduces the expression of the genes targeted by the siRNA in the liver, intestine and kidney glomeruli of mice at siRNA doses that are at least tenfold lower than the siRNA doses typically delivered via lipid nanoparticles. Small extracellular vesicles that efficiently package siRNA can significantly reduce its therapeutic dose.

A systematic comparison of lipopolymers for siRNA delivery to multiple breast cancer cell lines: In vitro studies

Small interfering RNA (siRNA) therapy is a promising approach for treatment of a wide range of cancers, including breast cancers that display variable phenotypic features. To explore the general utility of siRNA therapy to control aberrant expression of genes in breast cancer, we conducted a detailed analysis of siRNA delivery and silencing response in vitro in 6 separate breast cancer cell models (MDA-MB-231, MDA-MB-231-KRas-CRM, MCF-7, AU565, MDA-MB-435 and MDA-MB-468 cells). Using lipopolymers for siRNA complexation and delivery, we found a large variation in siRNA delivery efficiency depending on the specific lipopolymer used for siRNA complexation and delivery. Some lipopolymers were effective in all cell types used in this study, indicating the possibility of universal carriers for siRNA therapy. The delivery efficiency for effective lipopolymers was not correlated with dextran uptake in the cells tested, which indicated a receptor-mediated internalization for siRNA complexes with lipopolymers, unlike fluid-phase transfer associated with dextran uptake. Consistent with this, specific inhibitors involved in clathrin- and caveolin-mediated endocytosis significantly (>50%) reduced the internalization of siRNA complexes in all cell types. Using JAK2 and STAT3 silencing in MDA-MB-231 and MDA-MB-468 cells, a general correlation between the uptake and silencing efficiency at the mRNA level was evident, but it appeared that the choice of the target rather than the cell type was more critical for consistent silencing. We conclude that siRNA therapy with lipopolymers can be undertaken in multiple breast cancer cell phenotypes with similar efficiency, indicating the general applicability of non-viral RNAi in clinical management of molecularly heterogeneous breast cancers. Statement of significanceThe manuscript investigated the efficacy of siRNA carriers across multiple breast cancer cell lines. The lipopolymeric carriers were capable of delivering effective dose of siRNA to a range of breast cancer cells. Despite some differences in uptake efficiency among cell types, the mechanism of delivery was similar, with CME and CvME significantly involved in the internalization of polyplexes, while fluid-phase endocytosis was not significant. Specific target silencing was correlated to delivery efficiency, but we did notice the presence of lipopolymers that achieved high silencing with minimal siRNA delivery. Silencing specific targets in different cell types were more uniformly achieved as compared to targeting different targets in the same cells. Our studies enhance the feasibility of delivering siRNA to different types of breast cancer cells.

LncRNA ANRIL knockdown ameliorates retinopathy in diabetic rats by inhibiting the NF-κB pathway.

To evaluate the impacts of long non-coding ribonucleic acid (lncRNA) antisense non-coding RNA in the INK4 locus (ANRIL) on diabetic retinopathy (DR) in rats and the underlying mechanism. A total of 60 adult male Sprague Dawley (SD) rats were randomly divided into 3 groups: the Sham group (n=20), DR group (n=20) and DR + lncRNA ANRIL knockdown group [DR + lncRNA ANRIL small interfering RNA (siRNA) group, n=20]. DR model in rats was established by intraperitoneal injection of streptozocin (STZ; 60 mg/kg). Meanwhile, a certain dose of lncRNA ANRIL siRNA was added dropwise into rat eyes of DR + lncRNA ANRIL siRNA group during model induction to downregulate lncRNA ANRIL expression in the retina. 16 weeks later, rat retinal tissues were taken to extract total RNA and protein. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was applied to detect the expression levels of lncRNA ANRIL, interleukin-1 (IL-1), IL-6 and monocyte chemotactic protein-1 (MCP-1) in each group of the rat retina. Pathological structure of rat retinal tissues in each group was observed via hematoxylin and eosin (H&E) staining. Immunohistochemistry was adopted to measure the expression levels of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax) and P65 in each group of retinal tissues. In addition, the retinal vascular permeability in each group of rats was detected by fluorescent staining. Finally, Western blotting was utilized to determine the expressions of genes in the P65 signaling pathway. Compared with rats in the Sham group, lncRNA ANRIL was upregulated in rat retinal tissues harvested from the DR + lncRNA ANRIL siRNA group (p<0.05). After knockdown of lncRNA ANRIL in the retinal tissues of DR rats, pathologic damage was alleviated notably, and the levels of inflammatory markers (IL-1, IL-10 and MCP-1) were lowered markedly (p<0.05). The protein expressions of Bax and P65 decreased evidently, while the protein level of Bcl-2 increased markedly (p<0.05) in DR rats with ANRIL knockdown. Furthermore, Western blotting results revealed that inhibition of lncRNA ANRIL could prominently repress the phosphorylation level of P65 in the retinal tissues of DR rats (p<0.05). LncRNA ANRIL knockdown can significantly ameliorate the retinopathy in diabetic rats by blocking the nuclear factor-kappa B (NF-κB) signaling pathway.

SiRNA-mediated BCR-ABL silencing in primary chronic myeloid leukemia cells using lipopolymers

Despite development of effective tyrosine kinase inhibitors for treatment of chronic myeloid leukemia (CML), some patients do not effectively respond to the therapy and can display resistance in response to the drug therapy. To develop an alternative approach to CML therapy, we are exploring siRNA mediated silencing of the primary CML oncogene, BCR-ABL, by using non-viral (polymeric) delivery systems. In this study, a group of lipopolymers derived from low molecular PEIs substituted with linoleic acid (LA), α-linolenic acid (αLA) and cholesterol (Chol) was investigated for the first time for siRNA delivery to CML primary samples. The delivery efficiency in primary cells was equivalent to CML K562 cell line, and the lipopolymers gave effective internalization of siRNA depending on the nature of lipid substituent. The PEI-αLA (2.5 αLA/PEI), PEI-Chol (2.2 Chol/PEI), and PEI-LA (2.6 LA/PEI) lipopolymers used as BCR-ABL siRNA carriers (at 60 nM siRNA) reduced the BCR-ABL mRNA expression by 17% to 45%, and inhibited the formation of colonies by 24% to 41% in comparison with control siRNA in mononuclear cells. BCR-ABL siRNA treatment reduced the BCR-ABL mRNA expression by 50% in one of two CD34+ samples tested, and combination of BCR-ABL siRNA with imatinib (IM) treatment decreased the colony formation by 65% in one of two samples evaluated. The fact that no single polymer was universally effective in all patient samples may suggest patient-to-patient variability in terms of therapeutic responses to siRNA therapy. These results showed that a low dose of BCR-ABL siRNA could be used with lipopolymers to reduce BCR-ABL mRNA expression, CML cell survival and colony formation. This proof of principle study in CML primary cells can be applied to silencing of other therapeutic targets besides BCR-ABL and a study with larger patient samples is warranted for better identification of effective siRNA carriers.

The silencing of indoleamine 2,3-dioxygenase 1 (IDO1) in dendritic cells by siRNA-loaded lipid nanoparticles enhances cell-based cancer immunotherapy

Cell-based therapy using dendritic cells (DC) represents a potent cancer immunotherapy. However, activated DC express indoleamine 2,3-dioxygenase 1 (IDO1), a counter-regulatory and tolerogenic molecule, leading to the inhibition of T cell activation and the promotion of T cell differentiation into regulatory T cells. Silencing the IDO1 gene in DC by small interfering RNA (siRNA) represents a potent therapeutic strategy. We report on the successful and efficient introduction of a siRNA targeting IDO1 into mouse DCs by a means of a multifunctional envelope-type nanodevice (MEND) containing a YSK12-C4 (YSK12-MEND). The YSK12-C4 has both fusogenic and cationic properties. The YSK12-MEND induced an effective level of gene silencing of IDO1 at siRNA doses in the range of 1–20 nM, a concentration that commercially available transfection reagents are not able to silence. The YSK12-MEND mediated IDO1 silencing had no effect on the characteristic determinants of DC phenotype such as CD11c, CD80 and MHC class II. The silencing of IDO1 in DC by the YSK12-MEND significantly enhanced the antitumor effect against E.G7-OVA tumor. Moreover, a decrease in the numbers of regulatory T cells in the tumor was observed in mice that were treated with the IDO1-silenced DC. The YSK12-MEND appears to be a potent delivery system for IDO1-silenced DC based cancer immunotherapy.

Design of a Novel PEGylated Liposomal Vector for Systemic Delivery of siRNA to Solid Tumors.

A small interfering RNA (siRNA) delivery system using dioleylphosphate-diethylenetriamine conjugate (DOP-DETA)-based liposomes (DL) was assessed for systemic delivery of siRNA to tumors. DL carrying siRNA capable of inducing efficient gene silencing with low doses of siRNA were modified with polyethylene glycol (PEG-DL/siRNA) for systemic injection of siRNA. The biodistribution of DL and siRNA in the PEG-DL/siRNA was studied by using radiolabeled DL and fluorescence-labeled siRNA, respectively. DL in the PEG-DL/siRNA showed a high retention in the plasma, accumulation in the tumor, and low accumulation in the liver and spleen after intravenous injection. The in vivo effects of PEGylation were observed only when distearoylphosphatidylethanolamine (DSPE)-PEG but not distearoylglycerol (DSG)-PEG were used. This result suggests that the electrostatic interaction between lipid molecules on the surface of PEG-DL/siRNA was a critical determinant for the in vivo effect of PEGylation. When PEG-DL/siRNA (0.1 mg/kg siRNA) was intravenously injected into tumor-bearing mice, in vivo gene silencing was observed in subcutaneous tumors. These results indicate that PEG-DL/siRNA designed in this study is a promising formulation for systemic use of siRNA.

Protein silencing to stop a “silent killer”

Delivery of a single dose of siRNA to the placenta silences proteins and may help prevent preeclampsia.

PTPN22 Silencing in Human Acute T-Cell Leukemia Cell Line (Jurkat Cell) and its Effect on the Expression of miR-181a and miR-181b.

Purpose: T-cell acute lymphoblastic leukemia (T-ALL) is one of the most common malignancies associated with T-lymphocytes, accounting for 10 to 15 percent of ALL cases in children and 25 percent in adults. Innovative therapeutic approaches that overcome ineffective treatments on tumor cells may be a potential source of improvement in therapeutic approaches. Suppression of gene expression at transfusion level is one of the important strategies in gene therapy. The expression of PTPN22 and miR-181 genes in all types of hematologic malignancies increases and is likely to contribute to the survival and death of cells by affecting a variety of signaling pathways. The purpose of this study was to determine the role of PTPN22 inhibition by siRNA, and alteration in miR-181a and miR-181b in Jurkat cell line.Methods: Jurkat cells were transfected with 80 pmol of siRNA to inhibit PTPN22. After that, expression of PTPN22 mRNA and transcript levels of miR-181a and miR-181b were measured with Real-time PCR after 48hrs.Results: Experiments demonstrated that siRNA transfection resulted in significant downregulation of PTPN22 mRNA after 48 hrs in 80 pmol dose of siRNA. Moreover, transcript levels of both miR-181a and miR-181b was decreased after transfection.Conclusion: PTPN22, miR-181a and miR-181b might be involved in progression of Jurkat cells and targeting these molecules by RNAi might confer promising tool in treatment of T-ALL.