Special Issue "RNAi-Based Therapeutics"

Guest Editor
Prof. Dr. John J. Rossi
Department of Molecular and Cellular Biology, Irell and Manella Graduate School of Biological Sciences, Beckman Research Int. of City of Hope, 1500 East Duarte Road, Duarte, CA 91010, USA
E-Mail: jrossi@coh.org
Interests: siRNA; shRNA; RNAi; aptamers; small RNA gene activators; silencers

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Sabrina Höbel, Chantal C.M. Appeldoorn, Pieter J. Gaillard and Achim Aigner Article: Targeted CRM197-PEG-PEI/siRNA Complexes for Therapeutic RNAi in Glioblastoma Pharmaceuticals 2011, 4(12), 1591-1606; doi:10.3390/ph4121591 Received: 16 September 2011; in revised form: 8 December 2011 / Accepted: 9 December 2011 / Published: 16 December 2011 Show/Hide Abstract | Download PDF Full-text (337 KB) | Download XML Full-text Abstract: RNA interference (RNAi) allows the specific knockdown of tumor relevant genes. To induce RNAi, the delivery of small interfering RNAs (siRNAs) is of crucial importance. This is particularly challenging for their therapeutic applications in vivo. Low molecular weight branched polyethylenimine (PEI) is safe and efficient for nucleic acid delivery including small RNA molecules, based on its ability to electrostatically complex siRNA molecules, thereby protecting them from nuclease degradation. The nanoscale PEI/siRNA complexes are endocytosed by cells prior to intracellular complex release from the lysosome and cytoplasmic release of the siRNAs from the complexes. Chemical modification and ligand decoration of the complexes aim at introducing target tissue specificity and further increased efficacy of PEI-mediated siRNA delivery. CRM197 is a mutated, non-toxic diphtheria toxin (DT) that binds to the membrane-bound precursor of HB-EGF-like growth factor/diphtheria toxin receptor highly expressed in glioblastoma cells. Likewise, the growth factor pleiotrophin (PTN/HB-GAM/HARP) is overexpressed in glioblastoma and is rate limiting for tumor growth, thus representing an attractive target gene for therapeutic knockdown approaches. PEGylation of PEI was performed to reduce the surface charge, and by CRM197 coupling we prepared a modified PEI for siRNA delivery into glioblastoma cells. The novel PEI conjugates were analyzed for their complexation efficiency and optimal mixing ratios, and complexes were physicochemically characterized regarding stability, size and zeta potential. The biological activity of the complexes was confirmed in cell culture by reporter gene knockdown. For the therapeutic treatment of subcutaneous human gliobastoma xenografts in athymic nude mice, we systemically injected the modified PEI/siRNA complexes targeting PTN. Antitumor effects based on PTN knockdown demonstrated the advantage of tumor-targeted CRM197-PEG-PEI/siRNA over untargeted PEG-PEI polyplexes. Thus, we establish targeted CRM197-PEG-PEI-based complexes for siRNA delivery in vivo, and show therapeutic effects of CRM197-PEG-PEI/siRNA-mediated knockdown of PTN.

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Hidetoshi Arima, Keiichi Motoyama and Taishi Higashi Review: Potential Use of Polyamidoamine Dendrimer Conjugates with Cyclodextrins as Novel Carriers for siRNA Pharmaceuticals 2012, 5(1), 61-78; doi:10.3390/ph5010061 Received: 31 October 2011; in revised form: 20 December 2011 / Accepted: 21 December 2011 / Published: 30 December 2011 Show/Hide Abstract | Download PDF Full-text (572 KB) | Download XML Full-text Abstract: Cyclodextrin (CyD)-based nanoparticles and polyamidoamine (PAMAM) starburst dendrimers (dendrimers) are used as novel carriers for DNA and RNA. Recently, small interfering RNA (siRNA) complex with β-CyD-containing polycations (CDP) having adamantine-PEG or adamantine-PEG-transferrin underwent a phase I study for treatment of solid tumors. Multifunctional dendrimers can be used for a wide range of biomedical applications, including the interaction and intracellular delivery of DNA and RNA. The present review will address the latest developments in dendrimer conjugates with cyclodextrins for siRNA delivery including the novel sustained release system.

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Jiehua Zhou, Ka-To Shum, John C. Burnett and John J. Rossi Review: Nanoparticle-Based Delivery of RNAi Therapeutics: Progress and Challenges Pharmaceuticals 2013, 6(1), 85-107; doi:10.3390/ph6010085 Received: 21 December 2012; in revised form: 8 January 2013 / Accepted: 14 January 2013 / Published: 16 January 2013 Show/Hide Abstract | Download PDF Full-text (222 KB) | Download XML Full-text Abstract: RNA interference (RNAi) is an evolutionarily conserved, endogenous process for post-transcriptional regulation of gene expression. Although RNAi therapeutics have recently progressed through the pipeline toward clinical trials, the application of these as ideal, clinical therapeutics requires the development of safe and effective delivery systems. Inspired by the immense progress with nanotechnology in drug delivery, efforts have been dedicated to the development of nanoparticle-based RNAi delivery systems. For example, a precisely engineered, multifunctional nanocarrier with combined passive and active targeting capabilities may address the delivery challenges for the widespread use of RNAi as a therapy. Therefore, in this review, we introduce the major hurdles in achieving efficient RNAi delivery and discuss the current advances in applying nanotechnology-based delivery systems to overcome the delivery hurdles of RNAi therapeutics. In particular, some representative examples of nanoparticle-based delivery formulations for targeted RNAi therapeutics are highlighted.

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Olivia Perwitasari, Abhijeet Bakre, S. Mark Tompkins and Ralph A. Tripp Review: siRNA Genome Screening Approaches to Therapeutic Drug Repositioning Pharmaceuticals 2013, 6(2), 124-160; doi:10.3390/ph6020124 Received: 20 December 2012; in revised form: 10 January 2013 / Accepted: 22 January 2013 / Published: 28 January 2013 Show/Hide Abstract | Download PDF Full-text (594 KB) | Download XML Full-text Abstract: Bridging high-throughput screening (HTS) with RNA interference (RNAi) has allowed for rapid discovery of the molecular basis of many diseases, and identification of potential pathways for developing safe and effective treatments. These features have identified new host gene targets for existing drugs paving the pathway for therapeutic drug repositioning. Using RNAi to discover and help validate new drug targets has also provided a means to filter and prioritize promising therapeutics. This review summarizes these approaches across a spectrum of methods and targets in the host response to pathogens. Particular attention is given to the utility of drug repurposing utilizing the promiscuous nature of some drugs that affect multiple molecules or pathways, and how these biological pathways can be targeted to regulate disease outcome.

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Yu Fujita, Fumitaka Takeshita, Kazuyoshi Kuwano and Takahiro Ochiya Review: RNAi Therapeutic Platforms for Lung Diseases Pharmaceuticals 2013, 6(2), 223-250; doi:10.3390/ph6020223 Received: 19 December 2012; in revised form: 19 January 2013 / Accepted: 1 February 2013 / Published: 6 February 2013 Show/Hide Abstract | Download PDF Full-text (1276 KB) Abstract: RNA interference (RNAi) is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which affect specific messenger RNA (mRNA) degradation. Two types of small RNA molecules, i.e. small interfering RNAs (siRNAs) and microRNAs (miRNAs), are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including various viral infections and cancers. Lung diseases in general are attractive targets for siRNA therapeutics because of their lethality and prevalence. In addition, the lung is anatomically accessible to therapeutic agents via the intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities, such as inflammation and oncogenesis. Therefore, miRNAs are being targeted for therapeutic purposes. In this review, we present strategies for RNAi delivery and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases.

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Kaleb M. Pauley and Seunghee Cha Review: RNAi Therapeutics in Autoimmune Disease Pharmaceuticals 2013, 6(3), 287-294; doi:10.3390/ph6030287 Received: 31 December 2012; in revised form: 18 February 2013 / Accepted: 27 February 2013 / Published: 5 March 2013 Show/Hide Abstract | Download PDF Full-text (127 KB) | Download XML Full-text Abstract: Since the discovery of RNA interference (RNAi), excitement has grown over its potential therapeutic uses. Targeting RNAi pathways provides a powerful tool to change biological processes post-transcriptionally in various health conditions such as cancer or autoimmune diseases. Optimum design of shRNA, siRNA, and miRNA enhances stability and specificity of RNAi-based approaches whereas it has to reduce or prevent undesirable immune responses or off-target effects. Recent advances in understanding pathogenesis of autoimmune diseases have allowed application of these tools in vitro as well as in vivo with some degree of success. Further research on the design and delivery of effectors of RNAi pathway and underlying molecular basis of RNAi would warrant practical use of RNAi-based therapeutics in human applications. This review will focus on the approaches used for current therapeutics and their applications in autoimmune diseases, including rheumatoid arthritis and Sjögren’s syndrome.

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Luiza I. Hernandez, Katie S. Flenker, Frank J. Hernandez, Aloysius J. Klingelhutz, James O. McNamara and Paloma H. Giangrande Article: Methods for Evaluating Cell-Specific, Cell-Internalizing RNA Aptamers Pharmaceuticals 2013, 6(3), 295-319; doi:10.3390/ph6030295 Received: 11 January 2013; in revised form: 9 February 2013 / Accepted: 1 March 2013 / Published: 14 March 2013 Show/Hide Abstract | Download PDF Full-text (1085 KB) | Download XML Full-text Abstract: Recent clinical trials of small interfering RNAs (siRNAs) highlight the need for robust delivery technologies that will facilitate the successful application of these therapeutics to humans. Arguably, cell targeting by conjugation to cell-specific ligands provides a viable solution to this problem. Synthetic RNA ligands (aptamers) represent an emerging class of pharmaceuticals with great potential for targeted therapeutic applications. For targeted delivery of siRNAs with aptamers, the aptamer-siRNA conjugate must be taken up by cells and reach the cytoplasm. To this end, we have developed cell-based selection approaches to isolate aptamers that internalize upon binding to their cognate receptor on the cell surface. Here we describe methods to monitor for cellular uptake of aptamers. These include: (1) antibody amplification microscopy, (2) microplate-based fluorescence assay, (3) a quantitative and ultrasensitive internalization method (“QUSIM”) and (4) a way to monitor for cytoplasmic delivery using the ribosome inactivating protein-based (RNA-RIP) assay. Collectively, these methods provide a toolset that can expedite the development of aptamer ligands to target and deliver therapeutic siRNAs in vivo.

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Phillip Angart, Daniel Vocelle, Christina Chan and S. Patrick Walton Review: Design of siRNA Therapeutics from the Molecular Scale Pharmaceuticals 2013, 6(4), 440-468; doi:10.3390/ph6040440 Received: 24 January 2013; in revised form: 27 February 2013 / Accepted: 13 March 2013 / Published: 25 March 2013 Show/Hide Abstract | Download PDF Full-text (679 KB) | Download XML Full-text Abstract: While protein-based therapeutics is well-established in the market, development of nucleic acid therapeutics has lagged. Short interfering RNAs (siRNAs) represent an exciting new direction for the pharmaceutical industry. These small, chemically synthesized RNAs can knock down the expression of target genes through the use of a native eukaryotic pathway called RNA interference (RNAi). Though siRNAs are routinely used in research studies of eukaryotic biological processes, transitioning the technology to the clinic has proven challenging. Early efforts to design an siRNA therapeutic have demonstrated the difficulties in generating a highly-active siRNA with good specificity and a delivery vehicle that can protect the siRNA as it is transported to a specific tissue. In this review article, we discuss design considerations for siRNA therapeutics, identifying criteria for choosing therapeutic targets, producing highly-active siRNA sequences, and designing an optimized delivery vehicle. Taken together, these design considerations provide logical guidelines for generating novel siRNA therapeutics.

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Sophie Chabot, Sandrine Pelofy, Justin Teissié and Muriel Golzio Review: Delivery of RNAi-Based Oligonucleotides by Electropermeabilization Pharmaceuticals 2013, 6(4), 510-521; doi:10.3390/ph6040510 Received: 31 December 2012; in revised form: 19 March 2013 / Accepted: 27 March 2013 / Published: 10 April 2013 Show/Hide Abstract | Download PDF Full-text (622 KB) | Download XML Full-text Abstract: For more than a decade, understanding of RNA interference (RNAi) has been a growing field of interest. The potent gene silencing ability that small oligonucleotides have offers new perspectives for cancer therapeutics. One of the present limits is that many biological barriers exist for their efficient delivery into target cells or tissues. Electropermeabilization (EP) is one of the physical methods successfully used to transfer small oligonucleotides into cells or tissues. EP consists in the direct application of calibrated electric pulses to cells or tissues that transiently permeabilize the plasma membranes, allowing efficient in vitro and in vivo. cytoplasmic delivery of exogenous molecules. The present review reports on the type of therapeutic RNAi-based oligonucleotides that can be electrotransferred, the mechanism(s) of their electrotransfer and the technical settings for pre-clinical purposes.

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Hirohiko Hohjoh Review: Disease-Causing Allele-Specific Silencing by RNA Interference Pharmaceuticals 2013, 6(4), 522-535; doi:10.3390/ph6040522 Received: 27 December 2012; in revised form: 28 March 2013 / Accepted: 2 April 2013 / Published: 11 April 2013 Show/Hide Abstract | Download PDF Full-text (270 KB) | Download XML Full-text Abstract: Small double-stranded RNAs (dsRNAs) of approximately 21-nucleotides in size, referred to as small interfering RNA (siRNA) duplexes, can induce sequence-specific posttranscriptional gene silencing, or RNA interference (RNAi). Since chemically synthesized siRNA duplexes were found to induce RNAi in mammalian cells, RNAi has become a powerful reverse genetic tool for suppressing the expression of a gene of interest in mammals, including human, and its application has been expanding to various fields. Recent studies further suggest that synthetic siRNA duplexes have the potential for specifically inhibiting the expression of an allele of interest without suppressing the expression of other alleles, i.e., siRNA duplexes likely confer allele-specific silencing. Such gene silencing by RNAi is an advanced technique with very promising applications. In this review, I would like to discuss the potential utility of allele-specific silencing by RNAi as a therapeutic method for dominantly inherited diseases, and describe possible improvements in siRNA duplexes for enhancing their efficacy.

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Michelle E. Marcus and Joshua N. Leonard Review: FedExosomes: Engineering Therapeutic Biological Nanoparticles that Truly Deliver Pharmaceuticals 2013, 6(5), 659-680; doi:10.3390/ph6050659 Received: 28 March 2013; in revised form: 10 April 2013 / Accepted: 24 April 2013 / Published: 29 April 2013 Show/Hide Abstract | Download PDF Full-text (693 KB) | Download XML Full-text Abstract: Many aspects of intercellular communication are mediated through “sending” and “receiving” packets of information via the secretion and subsequent receptor-mediated detection of biomolecular species including cytokines, chemokines, and even metabolites. Recent evidence has now established a new modality of intercellular communication through which biomolecular species are exchanged between cells via extracellular lipid vesicles. A particularly important class of extracellular vesicles is exosomes, which is a term generally applied to biological nanovesicles ~30–200 nm in diameter. Exosomes form through invagination of endosomes to encapsulate cytoplasmic contents, and upon fusion of these multivesicular endosomes to the cell surface, exosomes are released to the extracellular space and transport mRNA, microRNA (miRNA) and proteins between cells. Importantly, exosome-mediated delivery of such cargo molecules results in functional modulation of the recipient cell, and such modulation is sufficiently potent to modulate disease processes in vivo. It is possible that such functional delivery of biomolecules indicates that exosomes utilize native mechanisms (e.g., for internalization and trafficking) that may be harnessed by using exosomes to deliver exogenous RNA for therapeutic applications. A complementary perspective is that understanding the mechanisms of exosome-mediated transport may provide opportunities for “reverse engineering” such mechanisms to improve the performance of synthetic delivery vehicles. In this review, we summarize recent progress in harnessing exosomes for therapeutic RNA delivery, discuss the potential for engineering exosomes to overcome delivery challenges and establish robust technology platforms, and describe both potential challenges and advantages of utilizing exosomes as RNA delivery vehicles.

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Type of the Paper: Review
Title: Nanoparticles-based Delivery of RNAi Therapeutics: Progress and Challenges
Authors: Jiehua Zhou ^1,†, Kato Shum ^1,†, John J. Rossi ^1,2*
1 Division of Molecular and Cellular Biology, Beckman Research Institute of City of Hope; ² Irell and Manella Graduate School of Biological Sciences, Beckman Research Institute of City of Hope, City of Hope, 1500 East Duarte Rd, Duarte, CA 91010, USA; Email address: Jzhou@coh.org, Kshum@coh.org and Jrossi@coh.org.
Abstract: RNA interference (RNAi) is an evolutionarily conserved, endogenous process for post-transcriptional regulation of gene expression. Although RNAi has recently made it through the pipeline to clinical trials, its application as an ideal, clinical therapeutics require the development of safe and effective delivery systems. Inspired by the immense progress with nanotechnology in drug delivery, efforts have been dedicated to the development of nanoparticles-based RNAi delivery system. For example, a precisely engineered, multifunctional nanocarrier with combined passive and active targeting capabilities may address the delivery challenge to the widespread use of RNAi as a therapy. Therefore, in this review we introduce the major hurdles in achieving efficient RNAi delivery and discuss the current advances in applying nanotechnology-based delivery systems to overcome the delivery drawbacks. In particular, some representative examples of design and apply nanoparticle-based delivery formulations for targeted RNAi therapeutics are highlighted.
Keywords: small interfering (si)RNA; non-viral vector; multifunctional nanoparticle; targeting delivery; cell-specific aptamer.

Type of the Paper: Review
Tentative title: RNAi-based therapy for Diabetic Nephropathy
Authors: Mitsuo Kato & Rama Natarajan
Affiliations: Department of Diabetes, Beckman Research Institute of City of Hope, 1500 East Duarte Road, Duarte, CA 91010, USA; E-Mail: RNatarajan@coh.org
Abstract: Diabetes is associated with significantly increased rates of kidney disease or diabetic nephropathy (DN), a severe microvascular complication that can lead to end-stage renal disease. End-stage renal disease needs to be treated by dialysis or kidney transplantation and also is associated with cardiovascular disease and macrovascular complications. Therefore, effective approaches are needed to reduce the progression of renal dysfunction associated with diabetes. Many contributing factors such as hyperglycemia, hyperlipidemia, advanced glycation end products, growth factors, inflammatory cytokines/chemokines and non-coding RNAs such as microRNAs have been implicated in the pathogenesis of DN. Therefore, targeting genes related to these key factors by small interfering RNAs or chemically-modified oligo-nucleotides can provide new therapeutic modalities for DN. In this review, we will summarize RNA interference-based approaches (including microRNA inhibitors) for the prevention and treatment of DN.

Type of the Paper: Review
Title: New Aspects of Gene-Silencing for Treatment of Cardiovascular Diseases
Authors: Koenig-O, Nolte-A, Walker-T, Schlensak-C, Wendel-HP
Affiliations: Clinical Research Laboratory, Dept. of Thoracic, Cardiac and Vascular Surgery, University Hospital Tuebingen, Calwerstr. 7/1, 72076 Tuebingen, Germany hans-peter.wendel@med.uni-tuebingen.de (H.P.W.
Abstract: RNA interference (RNAi) represents a novel therapeutic strategy due to sequence-specific gene-silencing through the use of small interfering RNA (siRNA). The modulation of gene expression by short RNAs provides a powerful tool to silence every disease-related or disease-promoting gene of interest. In this review we will outline RNAi mechanisms, currently used delivery systems and its possible applications to the cardiovascular system.

Type of the Paper: Review
Title: RNAi-based therapies and drug delivery for lung diseases.
Authors: Yu Fujita ^1,2 , Kazuyoshi Kuwano ² and Takahiro Ochiya 1
¹ Division of Molecular and Cellular Medicine, National Cancer Center Research Institute,Tokyo, Japan.Address; E-Mails: yufujit2@ncc.go.jp; tochiya@ncc.go.jp ² Division of Respiratory Diseases, Department of Internal Medicine, Jikei University School of Medicine,Tokyo, Japan.Address; E-Mail: kkuwano@jikei.ac.jp
Abstract: RNA interference (RNAi) is rapidly becoming an important method for analyzing gene functions in many eukaryotes and holds promise for the development of therapeutic gene silencing. The induction of RNAi relies on small silencing RNAs, which effect the specific messenger RNA degradation. Two types of small RNA molecules – small interfering RNAs (siRNAs) and microRNAs (miRNAs) – are central to RNAi. Drug discovery studies and novel treatments of siRNAs are currently targeting a wide range of diseases, including viral infections and cancer. Lung diseases in general are attractive targets for siRNAs therapeutics because of their lethality and prevalence. In addition, lung is anatomically accessible to therapeutic agents via intrapulmonary route. Recently, increasing evidence indicates that miRNAs play an important role in lung abnormalities such as inflammation and oncogenesis. It is therefore hoped that the targeting of microRNAs also can be exploited for therapeutic purposes. In this review, we present RNAi delivery method strategies and discuss the current state-of-the-art RNAi-based therapeutics for various lung diseases.
Keywords: RNAi; siRNA; miRNA; drug delivery system; lung diseases; lung cancer