siRNA Molecules Research Articles

Targeted delivery of small interfering RNA to colon cancer cells using chitosan and PEGylated chitosan nanoparticles

Small interfering RNA (siRNA) molecules specifically target messenger RNA species, decreasing intracellular protein levels. β-Catenin protein concentrations are increased in 70–80% of colon tumors, promoting tumor progression. Chitosan exhibits low levels of toxicity and can be transported across mucosal membranes; therefore, our objective was to develop chitosan and poly(ethylene glycol)-grafted (PEGylated) chitosan nanoparticles, 100–150nm in diameter, encapsulating anti-β-catenin siRNA for transfection into colon cancer cells. Encapsulation efficiencies up to 97% were observed. Confocal microscopy visualized the entry of fluorescently-tagged siRNA into cells. Western blot analysis showed that both chitosan and PEGylated chitosan nanoparticles containing anti-β-catenin siRNA decreased β-catenin protein levels in cultured colon cancer cells. These results indicate that nanoparticles made with chitosan and PEGylated chitosan can successfully enter colon cancer cells and decrease the level of a protein that promotes tumor progression. These or similar nanoparticles may prove beneficial for the treatment of colon cancer in humans.

Engineering Periodic shRNA for Enhanced Silencing Efficacy.

RNA interference (RNAi) provides a versatile therapeutic approach via silencing of specific genes, particularly undruggable targets in cancer and other diseases. However, challenges in the delivery of small interfering RNA (siRNA) have hampered clinical translation. Polymeric or periodic short hairpin RNAs (p-shRNAs)-synthesized by enzymatic amplification of circular DNA-are a recent development that can potentially address these delivery barriers by showing improved stability and complexation to enable nanoparticle packaging. Here, we modify these biomacromolecules via structural and sequence engineering coupled with selective enzymatic digestion to generate an open-ended p-shRNA (op-shRNA) that is cleaved over ten times more efficiently to yield siRNA. The op-shRNA induces considerably greater gene silencing than p-shRNA in multiple cancer cell lines up to 9 days. Furthermore, its high valency and flexibility dramatically improve complexation with a low molecular weight polycation compared to monomeric siRNA. Thus, op-shRNA provides an RNAi platform that can potentially be packaged and efficiently delivered to disease sites with higher therapeutic efficacy.

Inhibition of Gene Expression and Cancer Cell Migration by CD44v3/6-Targeted Polyion Complexes.

In recent years, siRNA technology has emerged as a promising strategy for gene silencing in cancer therapy. We have designed novel CD44-targeted polyion complexes (PICs) composed of poly(ethylene glycol)-block-polyethylenimine (PEG-b-PEI) and laminin-derived peptides (mA5G27D or mA5G27F) for in vivo siRNA delivery and gene silencing in tumors. The full-length A5G27 peptide (RLVSYNGIIFFLK), from which mA5G27D and mA5G27F are derived, binds to CD44v3 and CD44v6 and inhibits tumor cell migration, invasion, and angiogenesis. Thus, when attached to the surface of PICs, A5G27-based peptides can serve both as targeting ligands to navigate siRNA molecules directly to CD44-overexpressing tumors, and as anti-migratory agents to inhibit tumor progression. The mA5G27D- or mA5G27F-harboring PEG-b-PEI copolymers strongly condensed siRNA molecules into nanosized PICs presenting positive surface charges, low in vitro cytotoxicity, and high serum stability. mA5G27D- or mA5G27F-bearing PICs demonstrated high efficacy and selectivity in delivering siRAC1 into CD44-overexpressing cells, thereby silencing RAC1 mRNA and protein levels in such cells. These PICs presented substantial anti-migratory features in vitro and accumulated significantly in SK-OV-3 tumor-bearing mice, following 3 sequential intraperitoneal (i.p.) injections. Treatment of mice with 8 or 9 sequential parenteral (intravenous, (i.v.) or i.p.) injections of mA5G27F-PEG-b-PEI/siRNA efficiently inhibited tumor growth in two different CD44-overexpressing tumor mouse models (A549 and SK-OV-3), regardless of the type of siRNA (siPLK1 or siLUC) used. The results thus reveal the potential utility of this system for targeted delivery of siRNA molecules into solid tumors to prolong the survival time of mice, while at the same time reducing potential toxicity.

Multidimensional Screening Platform for Simultaneously Targeting Oncogenic KRAS and Hypoxia-Inducible Factors Pathways in Colorectal Cancer.

Colorectal cancer (CRC) is a genetic disease, due to progressive accumulation of mutations in oncogenes and tumor suppressor genes. Large scale genomic sequencing projects revealed >100 mutations in any individual CRC. Many of these mutations are likely passenger mutations, and fewer are driver mutations. Of these, activating mutations in RAS proteins are essential for cancer initiation, progression, and/or resistance to therapy. There has been significant interest in developing drugs targeting mutated cancer gene products or downstream signaling pathways. Due to the number of mutations involved and inherent redundancy in intracellular signaling, drugs targeting one mutation or pathway have been either ineffective or led to rapid resistance. We have devised a strategy whereby multiple cancer pathways may be simultaneously targeted for drug discovery. For proof-of-concept, we targeted the oncogenic KRAS and HIF pathways, since oncogenic KRAS has been shown to be required for cancer initiation and progression, and HIF-1α and HIF-2α are induced by the majority of mutated oncogenes and tumor suppressor genes in CRC. We have generated isogenic cell lines defective in either oncogenic KRAS or both HIF-1α and HIF-2α and subjected them to multiplex genomic, siRNA, and high-throughput small molecule screening. We have identified potential drug targets and compounds for preclinical and clinical development. Screening of our marine natural product library led to the rediscovery of the microtubule agent dolastatin 10 and the class I histone deacetylase (HDAC) inhibitor largazole to inhibit oncogenic KRAS and HIF pathways. Largazole was further validated as an antiangiogenic agent in a HIF-dependent manner in human cells and in vivo in zebrafish using a genetic model with activated HIF. Our general strategy, coupling functional genomics with drug susceptibility or chemical-genetic interaction screens, enables the identification of potential drug targets and candidates with requisite selectivity. Molecules prioritized in this manner can easily be validated in suitable zebrafish models due to the genetic tractability of the system. Our multidimensional platform with cellular and organismal components can be extended to larger scale multiplex screens that include other mutations and pathways.

Abstract B31: Combined siRNA and small molecule screening identifies Aurora B kinase as an effective target in MYCN-driven neuroblastoma

Abstract Despite advances in multimodal treatment, neuroblastoma (NB) is often fatal for children with high-risk disease and many survivors need to cope with long-term side effects from high-dose chemotherapy and radiation. To identify new therapeutic targets, we performed a siRNA screen of the druggable genome combined with a small molecule screen of 465 compounds targeting 39 different mechanisms of actions in four NB cell lines. We identified 58 genes as targets, including AURKB, in at least one cell line. In the drug screen, aurora kinase inhibitors (nine molecules) and in particular the AURKB-selective compound, barasertib, were the most discriminatory with regard to sensitivity for MYCN-amplified cell lines. In an expanded panel of NB cell lines, those with MYCN amplification and wild-type TP53 were the most sensitive to low nanomolar concentrations of barasertib. Inhibition of the AURKB kinase activity resulted in decreased phosphorylation of its known target histone H3, and upregulation of p53 pathway in MYCN-amplified NB cells with wild-type TP53. Both wild-type and p53-mutant MYCN-amplified cell lines arrested in G2/M phase upon AURKB inhibition. Additionally, barasertib induced endoreduplication and apoptosis. Treatment of MYCN-amplified/TP53 wild-type neuroblastoma xenografts resulted in profound growth inhibition and tumor regression. Therefore, aurora B kinase inhibition is highly effective in aggressive neuroblastoma and warrants further investigation in clinical trials. Citation Format: Dominik Bogen, Jun S. Wei, David O. Azorsa, Pinar Ormanoglu, Eugen Buehler, Rajarshi Guha, Jonathan M. Keller, Lesley A. Mathews Griner, Marc Ferrer, Young K. Song, Hongling Liao, Arnulfo Mendoza, Berkley E. Gryder, Sivasish Sindri, Jianbin He, Xinyu Wen, Xinyu Wen, Shile Zhang, John F. Shern, Marielle E. Yohe, Sabine Taschner-Mandl, Jason Shohet, Craig J. Thomas, Scott E. Martin, Peter F. Ambros, Javed Khan. Combined siRNA and small molecule screening identifies Aurora B kinase as an effective target in MYCN-driven neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B31.

SiRNA and RNAi optimization.

The discovery and examination of the posttranscriptional gene regulatory mechanism known as RNA interference (RNAi) contributed to the identification of small interfering RNA (siRNA) and the comprehension of its enormous potential for clinical purposes. Theoretically, the ability of specific target gene downregulation makes the RNAi pathway an appealing solution for several diseases. Despite numerous hurdles resulting from the inherent properties of siRNA molecule and proper delivery to the target tissue, more than 50 RNA-based drugs are currently under clinical testing. In this work, we analyze the recent literature in the optimization of siRNA molecules. In detail, we focused on describing the most recent advances of siRNA field aimed at optimize siRNA pharmacokinetic properties. Special attention has been given in describing the impact of RNA modifications in the potential off-target effects (OTEs) such as saturation of the RNAi machinery, passenger strand-mediated silencing, immunostimulation, and miRNA-like OTEs as well as to recent developments on the delivery issue. The novel delivery systems and modified siRNA provide significant steps toward the development of reliable siRNA molecules for therapeutic use. WIREs RNA 2016, 7:316-329. doi: 10.1002/wrna.1337 For further resources related to this article, please visit the WIREs website.

Microfluidics analysis of cancer cell microenvironments and targeted destruction of cancer stem cells by nanomedicine

Tumor microenvironment (TME) of a mature solid tumor is made of three components: tumor cell clusters or nests, stroma and immune cells. Most of the microvessels are located in the stroma, which is made of extracellular matrix and tumor associated fibroblasts (TAF). Circulating nanoparticles (NP) extravasating through the leaky microvessels first encounter the stroma and are taken up by TAF. SomeNPwill also penetrate through the stroma and reach tumor nests, depending on the particle size. In a stroma rich bladder cancer model, TAF are themajor cells taken up small LPC NP which contain ~80% cisplatin. Genome damaged TAF secrete Wnt16 which in a paracrine manner activates tumor cells for drug resistance and for epithelial-to-mesenchymal transition. It also induces neighboring naive fibroblast to become drug resistance and the endothelial cells to undergo angiogenesis. After repeated LPC administration, the bladder tumor becomes highlyWnt16 positive and highly drug resistant. Tumor rapidly regrows soon after LPC dosing is completed. Delivery of Wnt16 siRNA by LPH NP to the tumor can down-regulate Wnt16, inhibit the development of tumor drug resistance and enhance tumor growth inhibition by LPC. In advancedmurine B16F10melanoma, tumor does not contain a lot of stroma, and is relatively resistant to a nano-vaccine. LPH delivery of TGFβ siRNA, PLGA NP delivery of CDDO-Me (an anti-inflammatory) or Sunitinib (a broad-spectrum kinase inhibitor), or PEGylated curcumin micelles, can all downregulate the Th2 type cytokines, inhibit Treg cells and myeloid-derived suppressor cells and enhance tumor infiltrating CD8+ T-cells in the treated tumor. These immune modulators significantly enhance the tumor growth inhibition by a nano vaccine designed to boost the CTL response against the melanoma. Thus, we conclude that both siRNA and small molecule inhibitors can be delivered to modulate TME to facilitate either chemo or immune therapy, or both. Work supported by NIH grants CA149363, CA151652, CA149387 and DK100664.

Improved delivery of anticancer agents via a dual function nanocarrier

Tumor microenvironment (TME) of a mature solid tumor is made of three components: tumor cell clusters or nests, stroma and immune cells. Most of the microvessels are located in the stroma, which is made of extracellular matrix and tumor associated fibroblasts (TAF). Circulating nanoparticles (NP) extravasating through the leaky microvessels first encounter the stroma and are taken up by TAF. SomeNPwill also penetrate through the stroma and reach tumor nests, depending on the particle size. In a stroma rich bladder cancer model, TAF are themajor cells taken up small LPC NP which contain ~80% cisplatin. Genome damaged TAF secrete Wnt16 which in a paracrine manner activates tumor cells for drug resistance and for epithelial-to-mesenchymal transition. It also induces neighboring naive fibroblast to become drug resistance and the endothelial cells to undergo angiogenesis. After repeated LPC administration, the bladder tumor becomes highlyWnt16 positive and highly drug resistant. Tumor rapidly regrows soon after LPC dosing is completed. Delivery of Wnt16 siRNA by LPH NP to the tumor can down-regulate Wnt16, inhibit the development of tumor drug resistance and enhance tumor growth inhibition by LPC. In advancedmurine B16F10melanoma, tumor does not contain a lot of stroma, and is relatively resistant to a nano-vaccine. LPH delivery of TGFβ siRNA, PLGA NP delivery of CDDO-Me (an anti-inflammatory) or Sunitinib (a broad-spectrum kinase inhibitor), or PEGylated curcumin micelles, can all downregulate the Th2 type cytokines, inhibit Treg cells and myeloid-derived suppressor cells and enhance tumor infiltrating CD8+ T-cells in the treated tumor. These immune modulators significantly enhance the tumor growth inhibition by a nano vaccine designed to boost the CTL response against the melanoma. Thus, we conclude that both siRNA and small molecule inhibitors can be delivered to modulate TME to facilitate either chemo or immune therapy, or both. Work supported by NIH grants CA149363, CA151652, CA149387 and DK100664.

Tumor microenvironment and nanodrug delivery

Tumor microenvironment (TME) of a mature solid tumor is made of three components: tumor cell clusters or nests, stroma and immune cells. Most of the microvessels are located in the stroma, which is made of extracellular matrix and tumor associated fibroblasts (TAF). Circulating nanoparticles (NP) extravasating through the leaky microvessels first encounter the stroma and are taken up by TAF. SomeNPwill also penetrate through the stroma and reach tumor nests, depending on the particle size. In a stroma rich bladder cancer model, TAF are themajor cells taken up small LPC NP which contain ~80% cisplatin. Genome damaged TAF secrete Wnt16 which in a paracrine manner activates tumor cells for drug resistance and for epithelial-to-mesenchymal transition. It also induces neighboring naive fibroblast to become drug resistance and the endothelial cells to undergo angiogenesis. After repeated LPC administration, the bladder tumor becomes highlyWnt16 positive and highly drug resistant. Tumor rapidly regrows soon after LPC dosing is completed. Delivery of Wnt16 siRNA by LPH NP to the tumor can down-regulate Wnt16, inhibit the development of tumor drug resistance and enhance tumor growth inhibition by LPC. In advancedmurine B16F10melanoma, tumor does not contain a lot of stroma, and is relatively resistant to a nano-vaccine. LPH delivery of TGFβ siRNA, PLGA NP delivery of CDDO-Me (an anti-inflammatory) or Sunitinib (a broad-spectrum kinase inhibitor), or PEGylated curcumin micelles, can all downregulate the Th2 type cytokines, inhibit Treg cells and myeloid-derived suppressor cells and enhance tumor infiltrating CD8+ T-cells in the treated tumor. These immune modulators significantly enhance the tumor growth inhibition by a nano vaccine designed to boost the CTL response against the melanoma. Thus, we conclude that both siRNA and small molecule inhibitors can be delivered to modulate TME to facilitate either chemo or immune therapy, or both. Work supported by NIH grants CA149363, CA151652, CA149387 and DK100664.

One-pot synthesis of pH-responsive hybrid nanogel particles for the intracellular delivery of small interfering RNA

This report describes a novel, one-pot synthesis of hybrid nanoparticles formed by a nanostructured inorganic silica core and an organic pH-responsive hydrogel shell. This easy-to-perform, oil-in-water emulsion process synthesizes fluorescently-doped silica nanoparticles wrapped within a tunable coating of cationic poly(2-diethylaminoethyl methacrylate) hydrogel in one step. Transmission electron microscopy and dynamic light scattering analysis demonstrated that the hydrogel-coated nanoparticles are uniformly dispersed in the aqueous phase. The formation of covalent chemical bonds between the silica and the polymer increases the stability of the organic phase around the inorganic core as demonstrated by thermogravimetric analysis. The cationic nature of the hydrogel is responsible for the pH buffering properties of the nanostructured system and was evaluated by titration experiments. Zeta-potential analysis demonstrated that the charge of the system was reversed when transitioned from acidic to basic pH and vice versa. Consequently, small interfering RNA (siRNA) can be loaded and released in an acidic pH environment thereby enabling the hybrid particles and their payload to avoid endosomal sequestration and enzymatic degradation. These nanoparticles, loaded with specific siRNA molecules directed towards the transcript of the membrane receptor CXCR4, significantly decreased the expression of this protein in a human breast cancer cell line (i.e., MDA-MB-231). Moreover, intravenous administration of siRNA-loaded nanoparticles demonstrated a preferential accumulation at the tumor site that resulted in a reduction of CXCR4 expression.

Identification of possible siRNA molecules for TDP43 mutants causing amyotrophic lateral sclerosis: In silico design and molecular dynamics study

The DNA binding protein, TDP43 is a major protein involved in amyotrophic lateral sclerosis and other neurological disorders such as frontotemporal dementia, Alzheimer disease, etc. In the present study, we have designed possible siRNAs for the glycine rich region of tardbp mutants causing ALS disorder based on a systematic theoretical approach including (i) identification of respective codons for all mutants (reported at the protein level) based on both minimum free energy and probabilistic approaches, (ii) rational design of siRNA, (iii) secondary structure analysis for the target accessibility of siRNA, (iii) determination of the ability of siRNA to interact with mRNA and the formation/stability of duplex via molecular dynamics study for a period of 15ns and (iv) characterization of mRNA–siRNA duplex stability based on thermo-physical analysis. The stable GC-rich siRNA expressed strong binding affinity towards mRNA and forms stable duplex in A-form. The linear dependence between the thermo-physical parameters such as Tm, GC content and binding free energy revealed the ability of the identified siRNAs to interact with mRNA in comparable to that of the experimentally reported siRNAs. Hence, this present study proposes few siRNAs as the possible gene silencing agents in RNAi therapy based on the in silico approach.

Biokinetic studies of non-complexed siRNA versus nano-sized PEI F25-LMW/siRNA polyplexes following intratracheal instillation into mice

Successful gene therapy requires stability and sufficient bioavailability of the applied drug at the site of action. In the case of RNA interference (RNAi), non-viral vectors play a promising role for delivering intact siRNA molecules. We selected a low molecular weight polyethyleneimine (PEI F25-LMW) and investigated the biokinetics of PEI F25-LMW/siRNA polyplexes in comparison to non-complexed siRNA molecules upon intratracheal application into mice. Additionally, a bronchoalveolar lavage was performed to locate the siRNA within the different lung compartments and to analyse possible inflammatory reactions. Liquid scintillation counting of a 32P-label was used to follow the siRNA within the whole body. During the complete observation time more than 75% of the applied dose was found at the target site. The complexation with PEI F25- LMW prevented the siRNA from being degraded and cleared and prolonged its retention time. A low inflammatory reaction was observed on the basis of cell differentiation. Taken together, PEI F25-LMW meets fundamental requirements on non-viral vectors for local pulmonary siRNA delivery.

SiRNA Delivery: Current Trends and Future Perspectives

2016 Delivery of therapeutic nucleic acids in general and small interfering RNAs (siRNAs) in particular although a relatively nascent area of research, has already overcome several technical and regulatory hurdles over past few years. Some lead formulations undergoing clinical evaluation against wide range of diseases from age-related macular degeneration (AMD) to hypercholesteremia, amyloidosis and cancer. Roughly 4000 diseases are associated with malfunctioning genes in a particular cell type [1]. Gene therapy provides opportunity to modify (inhibit or enhance) the expression of disease-causing genes at the cellular level. Gene correction at the DNA level poses risks of inciting mutations or teratogenicity in comparison with RNA interference (RNAi) approach, which works at the transcription level leading to desired outcomes at the protein production stage such as therapeutic gene silencing of a cancer-causing gene. Currently, the key molecules employed in executing RNAi strategies are short hairpin RNA (shRNA), small interfering RNA (siRNA) or endogenous microRNA, of which siRNA forms the major mode presently under clinical evaluation [2]. siRNA molecules are easy to synthesize and do not need to be delivered into the nucleus as they do not require genome integration or the usage of DNA promoters [3]. They act in a sequence-specific manner against a target messenger RNA (mRNA), which avoids unintended off-target effects. The RNA-induced silencing complex (RISC), which drives RNAi, protects siRNA from degradation by cytosolic nucleases thereby recycling the molecule for many rounds of cleavage activity against the target mRNA. This implies that even few siRNA molecules if delivered stably into the cell cytosol are sufficient to trigger long-lasting gene silencing. In order to reach the desired organ/tissue and subsequently cross the cell membrane, these therapeutic siRNAs need to overcome several physiological barriers such as renal clearance, serum degradation and cellspecific uptake. Following crossing the cell membrane, intracellular barriers in the form of saturation of endogenous RNAi machinery or endosomal escape postendocytosis can reduce the efficiency of the delivered siRNA molecules [4]. Small negatively charged siRNAs can also lead to immunotoxicity and over the past years efforts have been dedicated to develop an efficient carrier modalities to overcome these systemic barriers and toxicity issues, which broadly falls into viral and nonviral strategies. Due to safety concerns on usage of viral vectors, nonviral techniques have taken the center-stage in siRNAs delivery. Delivery vehicles such as liposomes, lipidbased nanoparticles (LNPs) or polymeric nanoparticles are biodegradable, have relatively low toxicity, are stable in storage, easyto scale up and have low production costs – traits favored from an industrial perspective. Moreover, most components used in the development of nanostructures in the form of lipids or polymers are approved by the siRNA delivery: current trends and future perspectives

Antiviral Activity of Specific siRNA against Hemagglutinin and Neuraminidase of Influenza Virus H1N1 in MDCK Cell Culture

H1N1 influenza virus causes respiratory syndrome on the upper respiratory system. Hemagglutinin (HA) and Neuraminidase (NA) proteins are the most important viral proteins that play a role in attachment and facilitates of releasing virus respectively. Short interfering RNA (siRNA) plays notable a roles in the RNA interference (RNAi) pathway and interferes the expression of specific genes with complementary nucleotide sequences by breaking down mRNA function and stopping transcription and translation. To explore shutting down HA and NA, siRNA were designed and genes of H1N1 are inhibited by siRNA molecules in MDCK cell culture. The cells were infected with virus and then treated by different concentration of siRNA. The HA, NA gene expression and the viral load were investigated by Real Time PCR between 24 to 72 hours. The cells were infected with H1N1 virus at a multiplicity of infection (MOI: 3) and then treated with different concentration of siRNA (0.5-6 nmol/L) and monitored for 72 hours post transfection. Interestingly, the viral load extremely decreased at first hours, even the viral load was not detectable after 48 hours. Three nmol/L concentration of siRNA for HA was more effective. In case of siRNA for NA, 3 nmol/L concentrations shows efficacy and also cytopathic effect (CPE) was not observed in 3 nmol/L concentrations in MDCK cells. This study suggests that siRNA against NA and HA, silenced viral genes in H1N1 influenza virus infected cells. Results showed that siRNA is an efficient tool for silencing influenza virus infection. The advantage of siRNA is the specific inhibition of mRNA which can be used for inhibition of other flu viral genes and/or other viruses.

Biodegradable charged polyester-based vectors (BCPVs) as an efficient non-viral transfection nanoagent for gene knockdown of the BCR-ABL hybrid oncogene in a human chronic myeloid leukemia cell line.

First-line therapy of chronic myelogenous leukemia (CML) has always involved the use of BCR-ABL tyrosine-kinase inhibitors which is associated with an abnormal chromosome called Philadelphia chromosome. Although the overall survival rate has been improved by the current therapeutic regime, the presence of resistance has resulted in limited efficacy. In this study, an RNA interference (RNAi)-based therapeutic regime is proposed with the aim to knockdown the BCR-ABL hybrid oncogene using small interfering RNA (siRNA). The siRNA transfection rates have usually been limited due to the declining contact probability among polyplexes and the non-adherent nature of leukemic cells. Our work aims at addressing this limitation by using a biodegradable charged polyester-based vector (BCPV) as a nanocarrier for the delivery of BCR-ABL-specific siRNA to the suspension culture of a K562 CML cell line. BCR-ABL siRNAs were encapsulated in the BCPVs by electrostatic force. Cell internalization was facilitated by the BCPV and assessed by confocal microscopy and flow cytometry. The regulation of the BCR-ABL level in K562 cells as a result of RNAi was analyzed by real-time polymerase chain reaction (RT-PCR). We observed that BCPV was able to form stable nanoplexes with siRNA molecules, even in the presence of fetal bovine serum (FBS), and successfully assisted in vitro siRNA transfection in the non-adherent K562 cells. As a consequence of downregulation of BCR-ABL, BCPV-siRNA nanoplexes inhibited cell proliferation and promoted cell apoptosis. All results were compared with a commercial transfection reagent, Lipofectamine2000™, which served as a positive control. More importantly, this class of non-viral vector exhibits biodegradable features and negligible cytotoxicity, thus providing a versatile platform to deliver siRNA to non-adherent leukemia cells with high transfection efficiency by effectively overcoming extra- and intra-cellular barriers. Due to the excellent in vitro transfection results from BCPV-siRNA, a newly developed biodegradable transfection agent, BCPV, is being probed for transfection performance in an animal model.

In Vivo Magnetic Resonance Imaging of Small Interfering RNA Nanodelivery to Pancreatic Islets.

Pancreatic islet transplantation is a promising therapeutic approach for type 1 diabetes.However, recent advances in islet transplantation are limited by significant graft loss after transplantation. Multiple immunological and nonimmunological factors contribute to this loss. Novel therapies that could target the core reasons for the islet graft loss are desperately needed. Small interfering RNA can be used to inhibit the expression of virtually any gene with single-nucleotide specificity including genes responsible for islet damage. Applying adequate delivery of siRNA molecules to pancreatic islets prior to transplantation holds a great potential for improving the survival of islet grafts. Noninvasive imaging provides means for monitoring the survival of transplanted islets in real time. Here, we summarize the approach that has been developed to deliver siRNA to pancreatic islets in conjunction with tracking of the graft outcome by in vivo magnetic resonance imaging (MRI). We synthesize a nano-sized theranostic agent consisting of magnetic nanoparticles (MN), a reporter for MRI, labeled with Cy5.5 dye for near-infrared fluorescence (NIRF) imaging, and conjugated to siRNA molecule targeting genes that are harmful to islet grafts. Pre-labeling of islets by MN-Cy5.5-siRNA allowed us to monitor the survival of transplanted islet grafts by MRI and NIRF imaging and resulted in efficient silencing of the target genes in vivo. This novel approach combines a therapeutic effect provided by RNA interference technology with in vivo MR imaging and is expected to significantly improve the outcome of islet transplantation in patients with type 1 diabetes.

Insight of In Vitro Small-Interfering RNA Release From Chitosan Nanoparticles Under Enzymolysis With Förster Resonance Energy Transfer Analysis

Small-interfering RNA (siRNA)-mediated gene silencing with the aid of chitosan (CS)-based carriers has shown efficient and reliable outcome in vitro, but the gene silencing efficiency in vivo is still limited. It is of great importance to balance the protection and release of siRNA from nanoparticles (NPs) so as to achieve high efficiency. However, siRNA release profile from CS/siRNA NPs has been rarely concerned. Here, Förster resonance energy transfer technique was adopted for in vitro investigation of siRNA release from CS NPs in lysozyme-contained buffer. The results clearly showed that siRNA molecules experienced a fast and short release phase under lysozyme competition to both CS and siRNA, and then a slow and long release under lysozyme degradation on CS. Moreover, lysozyme competition played more important role than enzymolysis on trigging siRNA release. This preliminary study of siRNA release is the first step to get insight of in vivo siRNA release mechanism from CS/siRNA NPs, which will be helpful to adjust the design of CS/siRNA NPs for balancing the protection and release of siRNA molecules.

Efficient Gene Suppression in Dorsal Root Ganglia and Spinal Cord Using Adeno-Associated Virus Vectors Encoding Short-Hairpin RNA.

RNA interference is a powerful tool used to induce loss-of-function phenotypes through post-transcriptional gene silencing. Small interfering RNA (siRNA) molecules have been used to target the central nervous system (CNS) and are expected to have clinical utility against refractory neurodegenerative diseases. However, siRNA is characterized by low transduction efficiency, insufficient inhibition of gene expression, and short duration of therapeutic effects, and is thus not ideal for treatment of neural tissues and diseases. To address these problems, viral delivery of short-hairpin RNA (shRNA) expression cassettes that support more efficient and long-lasting transduction into target tissues is expected to be a promising delivery tool. Various types of gene therapy vectors have been developed, such as adenovirus, adeno-associated virus (AAV), herpes simplex virus and lentivirus; however, AAV is particularly advantageous because of its relative lack of immunogenicity and lack of chromosomal integration. In human clinical trials, recombinant AAV vectors are relatively safe and well-tolerated. In particular, serotype 9 of AAV (AAV9) vectors show the highest tropism for neural tissue and can cross the blood-brain barrier, and we have shown that intrathecal delivery of AAV9 yields relatively high gene transduction into dorsal root ganglia or spinal cord. This chapter describes how to successfully use AAV vectors encoding shRNA in vivo, particularly for RNA interference in the central and peripheral nervous system.

In Vivo siRNA Delivery Using JC Virus-like Particles Decreases the Expression of RANKL in Rats.

Bone remodeling requires a precise balance between formation and resorption. This complex process involves numerous factors that orchestrate a multitude of biochemical events. Among these factors are hormones, growth factors, vitamins, cytokines, and, most notably, osteoprotegerin (OPG) and the receptor activator for nuclear factor-kappaB ligand (RANKL). Inflammatory cytokines play a major role in shifting the RANKL/OPG balance toward excessive RANKL, resulting in osteoclastogenesis, which in turn initiates bone resorption, which is frequently associated with osteoporosis. Rebalancing RANKL/OPG levels may be achieved through either upregulation of OPG or through transient silencing of RANKL by means of RNA interference. Here, we describe the utilization of a viral capsid-based delivery system for in vivo and in vitro RNAi using synthetic small interfering RNA (siRNA) molecules in rat osteoblasts. Polyoma JC virus-derived virus-like particles are capable of delivering siRNAs to target RANKL in osteoblast cells both in vitro and in a rat in vivo system. Expression levels were monitored using quantitative real-time polymerase reaction and enzyme-linked immunosorbent assay after single and repeated injections over a 14-day period. Our data indicate that this is an efficient and safe route for in vivo delivery of gene modulatory tools to study important molecular factors in a rat osteoporosis model.

Synthesis and Conjugation of Small Interfering Ribonucleic Neutral SiRNNs.

Due to their high potency (EC50 ~1 pM) and exquisite target selectivity for all expressed mRNAs, small interfering RNA (siRNA)-induced RNAi responses hold significant promise as a therapeutic modality. However, the size and high negative charge of siRNAs render them unable to enter cells without assistance from a delivery agent. Most current methods of siRNA delivery rely on encasing siRNA molecules in large nanoparticles or cationic liposomes. However, these approaches suffer from a number of problems, including a poor diffusion coefficient, cytotoxicity, and poor pharmacokinetics. To address the problem of siRNA in vivo delivery, we developed monomeric neutral RNAi prodrugs, termed siRibonucleic neutrals (siRNNs), that directly neutralize the phosphate backbone negative charge by synthesis with bioreversible phosphotriester groups that are enzymatically cleaved off in the cytoplasm of cells. Here we describe the synthesis and purification of siRNN conjugates that induce in vivo target gene knockdown following systemic delivery into mice.