Development of RNAi technology for targeted therapy — A track of siRNA based agents to RNAi therapeutics

COREL-07159; No of Pages 12 Journal of Controlled Release xxx (2014) xxx–xxx

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Development of RNAi technology for targeted therapy — A track of siRNA based agents to RNAi therapeutics Yinjian Zhou, Chunling Zhang, Wei Liang ⁎ Protein and Peptide Pharmaceutical Laboratory, National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

a r t i c l e

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Article history: Received 12 March 2014 Accepted 23 April 2014 Available online xxxx Keywords: RNAi therapeutics siRNA delivery Barriers Off-target effect Clinical trials

a b s t r a c t RNA interference (RNAi) was intensively studied in the past decades due to its potential in therapy of diseases. The target specificity and universal treatment spectrum endowed siRNA advantages over traditional small molecules and protein drugs. However, barriers exist in the blood circulation system and the diseased tissues blocked the actualization of RNAi effect, which raised function versatility requirements to siRNA therapeutic agents. Appropriate functionalization of siRNAs is necessary to break through these barriers and target diseased tissues in local or systemic targeted application. In this review, we summarized that barriers exist in the delivery process and popular functionalized technologies for siRNA such as chemical modification and physical encapsulation. Preclinical targeted siRNA delivery and the current status of siRNA based RNAi therapeutic agents in clinical trial were reviewed and finally the future of siRNA delivery was proposed. The valuable experience from the siRNA agent delivery study and the RNAi therapeutic agents in clinical trial paved ways for practical RNAi therapeutics to emerge early. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Since the disclosure of the RNAi mechanism by Andrew Fire and Crag C. Mello in 1998 [1], RNAi became an attractive tool in cell culture, living organ and disease models, due to the specific and robust suppression to the targeted gene [2]. RNAi is a natural process of double strand ribonucleic acid (dsRNA) regulating specific gene activity, which occurs in most eukaryotic cells. RNAi mediators encompass microRNAs (miRNAs) and small interference RNAs (siRNAs) [3]. Either siRNA or miRNA could be associate into the RNA induced silencing complex (RISC). siRNA mediates sequencespecific degradation of the corresponding mRNA, however, miRNA can recognize their target mRNA in sequence-specific manner or via binding as little as 6–8 nucleotides in the seed region at the 5′ end of miRNA, in this perspective, one miRNA may have multiple different mRNA targets, is a combinatorial regulation. In this review, RNAi is referred to siRNA related RNA interference. siRNAs are classes of double strand nucleotide molecules, 19–27 base pairs in length, most preferred to be 21 nt siRNAs with a structure of 19 nt duplex region and 2 nt 3′ overhangs. In mammalian cells, siRNAs can be generated from processing of long double-strand RNAs (dsRNAs) or short hairpin RNAs (shRNAs) by the RNase III endonuclease Dicer. By taking the advantage of the RNAi machinery, synthetic siRNAs can also be introduced to silence targeted

⁎ Corresponding author at: 15 Datun Road, Chaoyang District, Beijing 100101, China. Tel.: +86 10 64889861; fax: +86 10 64845388. E-mail address: [email protected] (W. Liang).

genes [4]. In the cytoplasm, siRNA interacts with multifunctional protein Argonaute-2, and forms the activated RISC while releasing the sense/passenger strand of siRNA. The activated RISCs hunt down messenger RNAs (mRNAs) which are perfect or near perfect complimentary to the siRNA antisense strand [5]. After releasing fragments of degraded mRNA, the RISC was regenerated for new round of mRNA cleavage. The mRNA cleavage extent directly affects the gene suppression or relative protein expression level (see Fig. 1). The great therapeutic potential of RNAi lies in the treatment of diseases caused by gene disorder [6], viral infection [7], cancer [8] etc. With the accomplishment of the Human Genome Project, pathogenic gene could be discovered and selected for gene therapy. Sequencing of these genomes provided the necessary information for designing therapeutic siRNAs. Different from the traditional small molecule and protein/antibody based drugs, siRNAs own their advantages. The chief advantage of siRNAs over traditional drugs is their universal disease targets, including those ‘non-druggable’ targets. As therapeutics, siRNAs antagonize their targets whereas traditional drugs take effect mainly by blocking or activating their targeted function. Moreover, compared to traditional drugs, the discovery and identification of gene specific siRNAs are more rapid, less study time needed and capital saving, moreover, their selectivity and potency are much higher [9]. Appropriately delivered synthetic siRNAs are capable of knocking down the targeted gene in vivo. Since the first siRNA based agents entered clinical trial in 2004 [10], studies in developing RNAi therapeutics were arising, these included applying naked siRNA, chemical modified or excipient mediated siRNAs to treat local or systemic diseases [11].

http://dx.doi.org/10.1016/j.jconrel.2014.04.044 0168-3659/© 2014 Elsevier B.V. All rights reserved.

Please cite this article as: Y. Zhou, et al., Development of RNAi technology for targeted therapy — A track of siRNA based agents to RNAi therapeutics, J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.04.044

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2.1. Barriers from intrinsic nature of siRNA siRNA intrinsic characteristics endow them as RNAi agents, meanwhile, impede their effectiveness in vivo. siRNAs are subjected to degradation by endogenous enzymes, and their small size (Mw, 13 kDa) made them easily filtered off by kidneys when administered intravenously. Once on the cell surface, their molecular weight is too large and their surface is too negatively charged to cross cellular membranes. The fact is that the half life of most naked siRNA molecules is within 30 min after administered intravenously [13–15]. Chemical modification and physical encapsulation to siRNAs that altered their behavior in vivo are strategies to improve their therapeutic potency. 2.2. Barriers from blood circulation system

Fig. 1. siRNA silencing mechanism: exogenous shRNAs or endogenous dsRNAs cleaved by Dicer to generate endogenous siRNAs, endogenous siRNA and synthetic siRNA complex with RISC proteins to form RISC, the activated RISC enters a cycle of cleavage complementary mRNAs.

Despite the great therapeutic potential in clinical trial, RNAi application is still hindered by multiple barriers. The in vivo effectiveness of siRNA based agents only emerges when intact siRNAs are delivered into the targeted cell cytoplasm [9]. Chemical modified, carrier encapsulation [12] was outfitting siRNAs with characteristics to resist the endogenous RNase degradation, renal clearance, blood component opsonization and RES filtration, as well as facilitate their cellular uptake and endosome escape. In this review, we summarize the major barriers that exist in the transformation process of siRNA from a biomolecule to siRNA based therapeutic, as are approaches to mitigate them, as well as popular functionalized technologies for siRNAs such as chemical modification and physical encapsulation. Preclinical studies on local and systemic targeted deliveries of siRNA based RNAi agents are reviewed. Finally, the current status of siRNA based RNAi therapeutics in clinical trial and their future prospect are discussed. 2. Barriers to siRNA therapeutics As ectogenic materials, synthetic siRNAs are facing multiple barriers, including barriers from siRNA themselves, circulating systems and targeted tissues (see Fig. 2).

2.2.1. Renal clearance Small particles in the blood circulation system with a diameter of less than 6–8 nm or molecular weight less than 30–50 kDa are subject to quick excretion by the kidney through the urine [16]. Naked siRNAs with small molecule weight are suffering quick renal clearance. Proper delivery formulation or chemical modification is necessary to increase siRNA retention time in the blood circulatory system. For example, radioactively labeled Chol–siRNAs had an elimination half life of 95 min, whereas unconjugated siRNA half life is 6 min [13]. siRNA–cationic copolymer complexes exhibited 100-fold increase in circulation time [17]. In addition to size, surface cationically charged RNAi nanoparticles (NPs) also suffer renal clearance. The cause is mainly from their interaction with anionically charged proteins on the glomerular capillary wall. In the neutralization process, siRNA is released into the glomerular capillary bed and subsequently excreted. PEgylation to cationic RNAi NP surface reduces their renal clearance. 2.2.2. Protein binding To achieve high loading density of negatively charged siRNA, positive charge to polymers is an essential requirement. In blood circulation, the plasma opsonin proteins and cell membrane proteins were negatively charged components. Surface positively charged RNAi NPs can be heavily opsonized by opsonin protein or unspecifically bound with the cell membrane proteins. The opsonins are mainly blood complement proteins and immunoglobulins. Opsonins bind with surface cationically charged RNAi NPs via electrostatic interaction. The opsonization process takes place anywhere in the blood stream and last from seconds to days to complete. The opsonized NPs undergo RES system recognition and elimination [18], and their cell surface binding usually led to inflammation effect [19], which greatly reduced their therapeutic concentration. Surface treatments are regular

Fig. 2. Representative barriers to siRNA based RNAi agents from the point of intravenous administration to endosome escape: 1) serum protein opsonization; 2) RES filtration; 3) immunostimulation (TLR: Toll-like receptor); 4) renal clearance; 5) extracellular matrix (ECM); 6) cellular uptake; 7) endosome escape; etc.

Please cite this article as: Y. Zhou, et al., Development of RNAi technology for targeted therapy — A track of siRNA based agents to RNAi therapeutics, J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.04.044

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approaches to interfere the interaction of opsonins with RNAi NPs. Coating of cationically charged NPs with hydrophilic polymers effectively reduced their opsonization and unspecific binding [20], such as PEG and some synthetic polymers. For example, a palmitate-avidin containing PLGA particle adsorbs PEG–biotin on their surface, and the 10 kDa PEG coated particle reduced protein adsorption by 75% [21]. 2.2.3. RES filtration Reticuloendothelial system (RES)/mononuclear phagocytic system (MPS) containing tissues, such as the spleen and liver are pervaded with fenestrated capillaries and phagocytic cells, primary monocytes and macrophages [22]. Followed by intravenous administration, NPs that suffered from serum protein opsonization are removed by the macrophages in the RES tissues via cell surface bound opsonins or nonspecific adhesion. Parameters to NPs such as size, geometry, surface charge and hydrophobicity affect their fate in these tissues. Surface cationically charged or hydrophobic RNAi NPs with a size larger than the renal threshold become sequestered in the RES organs, typically, RNAi NPs with larger size than 100 nm often rapidly uptake and concentrate into the RES organs. Uptake of particles from blood flow to the RES is fairly fast, however, the processing and excretion of these particles are relatively slow, which often result in prolonged retention in filtering organs. RNAi NP surface adsorption or grafting with hydrophilic polymers reduces their plasma protein opsonization and retention time [23]. However, RES filtration is favored for siRNA based agents to target these RES containing tissues [24]. 2.3. Barrier from targeted tissue Evading the main systemic barriers, RNAi NPs that travel near the targeted diseased tissue are still facing new series of barriers from the targeted tissue. 2.3.1. Tumor extracellular matrix RNAi particles in the blood circulating system need to get into the extracellular matrix (ECM) before coming to the cell surface of inner tissues. The dense polysaccharides and fibrous integrin network resist further transportation of macromolecule and particles [12]. However, in tumor tissues, the highly heterogeneous vascular architecture throughout the tumor is poorly aligned with defective endothelial cell and wide fenestrations, uneven muscle layer or intervention, furthermore, tumor tissues usually lack effective lymphatic drainage. RNAi NPs extravasated from these vascular more effectively accumulate in tumor than in normal tissue, achieving targeted accumulation. This is the so called Enhanced Permeability and Retention effect (EFR) phenomenon [25]. The broad gap that exists in the vascular endothelial cells of inflammatory tissues is also favored for NP targeted accumulation [26]. EPR effect is the basic mechanism of RNAi NP enrichment in tumor and inflammatory tissues [27]. The low level expressed integrin αvβ3 sub-type in normal tissue epithelia cells is over-expressed in most tumors. αvβ3 integrins specifically bind to some ECM protein through their Arg-Gly-Asp (RGD) peptide [28]. The RNAi NP surface is decorated with RGD peptide or its analogues increase its tumor ECM binding and accumulate in inner tumor tissues [29]. 2.3.2. Cellular uptake Followed by passive or active targeted enrichment in the tumor tissue matrix, RNAi particles need to stride over the cell membrane to enter inside the cells [30]. Except for the phagocytosis mechanism of phagocytes, diverse pinocytosis based mechanisms occur in mammalian cells. Uptake of RNAi NPs could be achieved in several manners, including clathrin-mediated endocytosis (CME), caveolae-mediated endocytosis (CvME), macropinocytosis and clathrin and caveolaeindependent endocytosis [31].

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When RNAi NPs are taken up in the CME pathway, the particles are engulfed in clathrin coated pit forming early endosomes. Some of the early endosomes release their content into the cytosol via tubules, but most early endosomes experience a drop in pH process from early endosome, late endosome and finally exposure to acid and nuclease degradation in lysosome. Small size and ligand (such as transferrin and endothelial growth factor) decorated RNAi NPs are preferentially taken up in the CME pathway [32]. CvME is a clathrin-independent pathway, and it plays a less important role in NP cellular uptake. Different from CME pathway, particles internalized via the CvME pathway escape the drop in pH and nuclease degradation process [33]. CvME is favored for its larger size particles. Some ligands such as folic acid, albumin and cholesterol conjugated NPs preferentially internalized through the CvME pathway [34]. Particles internalizing mechanism and intracellular routing strongly depend on particle size. Size itself of ligand-free particles can determine the mode of their cellular entry. Spherical particles with a diameter less than 200 nm predominantly internalized via the CME pathway. With the increasing particle size, the mechanism shifted to caveolaemediated internalization, for particles with size around 500 nm, caveolae-mediated internalization was the predominant pathway [35]. Surface charge is another important concern for NP cell internalization. Positively charged surface of NPs is favored for their association with negatively charged cell membrane and cell internalization. For examples, positive cross-linked chitosan NPs were efficiently taken up by Caco-2 cells [36], and cationic lipid stearylamine coated PEG–PLA NPs showed faster uptake than uncoated PEG–PLA NPs [37]. Hydrophilic polymer coating to NPs significantly affected their cellular endocytosis. PEGylated poly(hexadecylcyano-acrylate) NPs interacted with brain endothelial cells and translocated into the brain after intravenous injection, while unPEGylated polyhexadecylcyanoacrylate nanoparticles did not [38]. Moreover, high degree of PEGylation reduced the internalization of transferrin or antibody conjugated complexes to a similar level to ligand free NPs [39].

2.3.3. Endosome escape RNAi NPs internalized via the endosome need to escape from the jailed compartment earlier before degradation. Pore formation and membrane disruption on endosome membrane are common strategies for RNAi particle escape from endosome. Fusogenic lipids [40], pH buffering polymer [41], chemical agents [42] and cell penetrating peptide [43] are materials frequently used in RNAi NP endosome escape. Short cationic amphiphilic peptides (AMPs) bind to endosome lipid bilayer, which lead to internal membrane stress or internal membrane tension, and the strong membrane tension finally created pores in the endosome lipid membrane [44]. AMP modified siRNA lipoplexes or polyplexes improved siRNA endosome escape [45]. Polycationic polymers and pH sensitive lipidic carriers in the endosome with high proton buffering capacity and flexibility induce extensive inflow of ions and water into the endosome, which lead to endosome membrane rupture and release of the internalized cargos. Under endosome pH, amine groups rich liposomes in the endosome compartment enhanced interaction with the endosome membranes and lead to endosome membrane rupture, subsequently released siRNA to cytoplasm [46]. PEI could also achieve efficient siRNA delivery in various cell lines, its highly protonable amino groups ruptured endosome membrane via the proton sponges effect over a pH range of 5.0– 7.0, which lead to siRNA release from endosome [47]. Histidine rich amphiphilic, pH-sensitive peptide disrupted endosome membrane after the protonation of the imidazole groups at low pH, which resulted in effective release of siRNA into the cytosol [48]. Some chemical agents also improved the siRNA escape from endosome compartment via membrane disruption manner, such as ammonium chloride, Ca2+, sucrose and photosensitizer [49].

Please cite this article as: Y. Zhou, et al., Development of RNAi technology for targeted therapy — A track of siRNA based agents to RNAi therapeutics, J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.04.044

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2.4. Off-target effect In addition to siRNA desired silencing of gene, off-target effect led to unwanted toxicities. The siRNA therapeutics mainly associated with three types of off-target effects are: miRNA-like off-target effects; saturation of the endogenous RNAi machinery by exogenous siRNAs and inflammatory response by siRNAs and their cationic carriers. siRNAs share the same machinery to miRNAs in mRNA regulation process. siRNAs 5′ end of guide strand take the same effect to seed region of miRNA in mRNA recognition in the RISC. The siRNA related miRNA like off-target silencing is sequence-specific and originated from imperfect pairing of siRNA guide strand with sequence motifs of mRNAs [50]. Chemical modification of 2′-O-methyl ribosyl substitution at position 2 in siRNA guide strand [51] and use of a pool of multiple siRNA to the same target are strategies for reducing miRNA like offtarget effect [52]. An example is a pool of 10 strip siRNAs each at a concentration of 10 nM significantly reduced off-target effect when compared with any single strip at 100 nM while the target specificity was reserved [53]. The immune system recognizes a wide variety of agents, such as virus, parasitic worms, and exogenous particles and distinguishes them from the innate healthy tissue components. Mammalian immune cells recognize and response to exogenous materials via Toll-like receptors (TLRs) [54], siRNAs and cationic siRNA carriers are mainly recognized by TLR3, TLR7, TLR8 [55] and protein kinase receptors. Synthetic siRNA features like length and sequences are responsible for the elicitation of the immunological effect. Some specific sequence motifs in siRNA sequence activated the immune cells and induce the production of inflammatory cytokines [56]. siRNA sequences lacking these GU or AU rich motifs, or chemical modifications of the 2′ position of ribose could block the recognition by these TLRs [57]. Cationic siRNA carriers coated with neutral hydrophilic polymer effectively reduce their inflammatory response. Saturation of the endogenous RNAi machinery by exogenous siRNAs seldom occurs and can be avoided by reducing the siRNA application amount [58]. 3. Technologies for improved siRNA performance Different techniques were undertaken to improve siRNA therapeutic potency, including siRNA design and synthesis strategy, siRNA nucleotide and backbone chemical modification, and siRNA exploitation delivery vehicle development. These will prevent some of the barrier effects described before. 3.1. Chemical modification to siRNAs The performance of siRNA is significantly improved after chemical modification to siRNA strands, the sites include ribose, base, phosphorous acid, strand end or backbone of both sense and antisense strands [59,60]. Modification to ribose 2′-OH group with \CH3 group or fluoride atom within both sense strand and antisense strand increased their resistance to endonuclease degradation and the siRNA therapeutic potency [61]; chemical modification to 5′ end of sense strand and 3′ end overhang of antisense strand reduced sense strand selection by RISC,

subsequently increasing siRNA potency and specificity [62]; specially, asymmetry introduce 2 nt at a 3′ overhang only in the guide strand increased knockdown potency and decreased off-target effects [63] (see Fig. 3a). Conjugations of different functional groups to strand ends of siRNA with small molecules (such as folic acid and galactose), polymers (PEI, PEG, etc.), antibodies, peptide and aptamers improved their performance in vivo [60]. Intravenous injection of apolipoprotein B (ApoB) targeted cholesterol siRNA conjugates to transgenic mouse resulted in downregulation of plasma ApoB protein levels and total cholesterol reduction in the liver and jejunum [6,13]. For siRNA–PEG conjugates, after intravenous injection, the conjugates remained in active function, prolonged circulation time and reduced siRNA urine excretion [64]. Covalently conjugated cell penetrating peptides (CPPs) or protein transduction domains to siRNA also improved siRNA knockdown efficiency. For example, CPP– siRNA conjugates greatly decreased targeted gene expression for up to 7 days in two cell lines [65]. By linking siRNAs with complementary A5–8/T5–8 sequence at their 3′ overhangs, gene-like sticky siRNAs were constructed, and the sticky siRNAs increased complex stability hence RNase protection and gene silencing [66]. These chemical modifications protected siRNA from quick elimination and inactivation and improved siRNA therapeutic potency, but do not necessarily augment the efficacy of gene silencing in vivo.

3.2. Physical encapsulation to siRNAs 3.2.1. Cationic encapsulation Negatively charged siRNA cannot easily come across cell anionic bilayer membrane and reach the cytoplasm. Proper delivery vehicles nurse siRNA surface property and deliver siRNAs into the cells. Cationic carriers complex with siRNA to form positively charged NPs (see Fig. 3b and c), including cationic liposome [67], PEI [68], PLL [69], atelocollagen [70], chitosan [71], trans-membrane peptide [72], dendrimer [73], and cyclodextran polymers [74], and their exterior surface associate with siRNA to form a dense complex, which greatly reduces siRNA degradation by RNase and increases siRNA therapeutic potency in vitro and in vivo [75]. The common features of these positively charged polymers are their solubility in water and their high cationic charge density at physiological pH. Among the amino group rich polymers, synthetic polymer polyethyleinimine (PEI) was frequently used in siRNA delivery [76]. In PEI structure, amine groups occur in every third position. Under physiological pH, about twenty percent of the amine groups are protonated and this result in their high buffering capacity [77]. In vitro application of PEI/siRNA complexes successfully delivered siRNA into the cells, however, intravenously administered PEI complexes unspecifically bind to plasma opsonins and induced low efficiency and cytotoxicity. The PEI/siRNA complexes preferentially accumulated in the lung [78]. Dendrimers are highly branched, monodisperse synthetic polymers. Dendrimers have tunable molecular size and surface charge density. Increasing dendrimer generation (DG) resulted in their size, surface functional groups and surface charge density growth. Polycationic

Fig. 3. siRNA improved delivery: a) siRNA strand chemical modification, green spot represents modification to ribose, sugar, phosphate and nucleotide. R represents modification to siRNA strand end with molecules, SS: sense strand, AS: antisense strand; b) cationic complexation with siRNAs by polycationic polymers; c) siRNA encapsulation by cationic liposome; and d) siRNA stealth encapsulation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Please cite this article as: Y. Zhou, et al., Development of RNAi technology for targeted therapy — A track of siRNA based agents to RNAi therapeutics, J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.04.044

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dendrimers with defined size, structure homogeneity and charge density are beneficial for siRNA delivery. Polycationic dendrimers such as poly(amidoamine) (PAMAM) and poly(propylenimine) (PPI) with precisely controlled multivalent amine and surface amine groups protected siRNA from RNase degradation and delivered siRNAs into the cells [79]. siRNA preferentially bind to dendrimer surface protonated amine groups, and the dendrimer interior amine groups triggered proton sponge effect once in the cell endosome compartment and facilitated siRNA release into the cytoplasm [80]. PAMAM/siNA complexes protected siRNAs from enzymatic degradation and achieved substantial release of siRNA over extended time and efficient gene silencing. Moreover, higher generation of PAMAM dendrimers with more amine groups achieved higher gene silencing effect in cultured cells [81]. Polysaccharide polymer chitosans (CSs) are composed of D-glucosamines and N-acetyl-D-glucosamines, and their primary amines from de-acetylation of acetyl-D-glucosamines bind with siRNAs under acidic environment (pH 6.2–7.0). CSs show low toxicity, low immunogenicity as well as excellent biocompatibility. CSs are frequently used in pharmaceutics as wound therapy materials and dietary supplements, because of the electrostatic interaction of the CSs with the tissue anionic mucus layer [82]. CS deactylation degree is an important consideration when CSs are used as siRNA carrier, which determine the zeta potential and loading efficiency of siRNAs in CS/siRNA complexes. In the first reported CS/siRNA complex, siRNAs were transfected to CHO K1 and HEK 293 cell lines with high transfection efficiency in vitro [83]. Intraperitoneal administered CS/siRNA complexes to mice induced downregulation of local and systemic inflammation levels [84]. Some oligopeptides present the ability of translocation across cell membrane, and the translocation process is independent on cell surface receptors and is free of energy consumption. The Cell penetrating peptides (CPPs) are usually short peptides, rich in certain amino acids (such as lysine, histidine and arginine). The membrane translocation property presents new options for delivering therapeutics into the cells. CPPs delivered siRNAs via conjugation [65] or non-covalently complexation manners [85]. An amphiphilic CPP, CADY could form stable complexes with siRNAs, and this CPP combines aromatic tryptophan and hydrophilic arginine residues within its 20 amino acid residues. When the CADY/siRNA complex was transferred to cell membranes, its hydrophilic arginine residues and the aromatic tryptophan residues stretched to different directions, which increased both siRNA stability and the knockdown efficiency to the targeted gene at both mRNA and protein levels [86]. A typical class of lipid based molecule–lipidoid was identified recently in delivering siRNAs [87,88]. Lipidoids were synthesized by linear acrylic alkane esters or epoxide derivatives with amine compounds via additive reactions. Lipidoids could substitute cationic lipids as the positively charged materials in the construction of cationic liposomes or directly used as cationic materials for encapsulation of siRNAs. A C12-200 lipidoid–siRNA complex injected to mice at a dose below 0.01 mg/kg induced targeted gene silencing in the liver, and as a proof of its high siRNA delivery capacity, silencing of five genes in the liver with a single i.v. administration was achieved within the tissue tolerability range [88]. 3.2.2. Stealth encapsulation Even though cationic complexation greatly increased siRNA therapeutic potency, RNAi NPs need to resolve multiple environment barriers along the blood stream, such as renal clearance, blood opsonins and RES system, before they have a chance to interact with targeted tissue cells. Cationic RNAi NPs in the blood stream could be intensively affected by the specific blood opsonins via surface physical interaction. The opsonins include laminin, type I collagen, C-reactive protein, immunoglobulins, etc. [21]. The opsonized NPs quickly undergo RES filtration. RNAi NP surface engineered with low observable or stealth polymers effectively prolonged their circulation time and reduced elimination by the clearance systems. Hydrophilic polysaccharides chitosan, heparin and

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synthetic polymers polyvinyl, pyrrolidine and PEG were frequently used polymers to confer particles with stealth properties [21], these polymers formed dense, hydrophilic corona around cationic or hydrophobic particles and reduced their electrostatic or hydrophobic interaction with plasma components (see Fig. 3d). PEGs are electrically neutral, structure flexible polymers, plus their biocompatible and hydrophilic properties, PEGs, are frequently used to coat NPs. PEG coating reduced opsonin unspecific adhesion and macrophage recognition. For example, Doxil, the doxorubicin loaded PEGylated liposome suffered only small amount of opsonin proteins than free DOX, presented a biphasic circulation and prolonged half-life [21]. Opsonins adsorbed on PEG-coated NPs strongly depended on the molecular weight (Mw), length and contents of PEGs at the particle surface, as well as the surface charge, hydrophobicity and morphology of the particles [89]. PEG–PLA nanospheres with PEG MW 5000 absorbed the least opsonins and when at 2–5% weight ratio of PEG content, an optimal opsonin protein resistant was observed. “Stealth” NPs could be constructed by introducing PEG or other hydrophilic polymers to their surface via physical coating or chemical conjugation [90,91]. Physical coating often does not always lead to prolonged circulation, for example, the circulation time of poloxamine coated PLGA NPs [92] and liposomes [93] was not longer than the uncoated counterparts in mice, the cause was probably the coating layer quickly dissociated in the blood stream [18]. Chemical conjugation of PEG to cationic or hydrophobic polymers to form amphiphilic copolymers and conjugation of PEG to surface of NPs was prevailing manner in building stealth siRNA delivery vehicles. 3.2.2.1. PEGylated lipidic NPs. PEG–PE is a PEGylated phospholipid polymer. Due to the phospholipid structure similarity to lipidic components in liposomes, PEG–PE is frequently used as additive in construction stealth liposomes. PEG coated liposomes could be constructed by mixing PEG–PE ahead or after liposome formation, or chemical conjugation of PEG acibenzolar with reactive amine residues on liposome surface in aqueous phase [94]. PEG–PE assembles automatically into stable spherical micelles in aqueous phase. Their hydrophobic DSPE segment aggregated into the hydrophobic core and PEGs form the hydrophilic corona, and the phosphate part in the intermediate section plays an important role in the delivery of aromatic structured pharmaceutical agents [95]. PEG–PE micelles were used in the delivery of RNAi agent [96] and anticancer drugs [97,98]. The fact was that PEG–PE micelles encapsulated almost 100% free DOX in aqueous phase when mixed with PEG–PE micelles with DOX at a molar ratio of 2:1 [97,98]. Our laboratory constructed an R8–PEG–PE containing cationic liposome (R8–liposome) for active targeted delivery of siRNA. The arginine octamer (R8) oligopeptide was used as ligands for liposome targeted to cell membrane receptors. The complexation of human double minute gene 2 (HDM2) siRNA with R8–liposome (R8–liposome–siRNA) exhibited high stability in serum plasma and resistance to RNAse. R8–liposome transfected siRNA efficiently in the three tested lung cancer cell lines even in the presence of plasma proteins, however, the cytotoxicity was fairly low. The R8–liposome–siRNA complex significantly inhibited the growth of tumor cells in vitro, moreover, the inhibition mechanism induced by the complexed siRNAs was identified [99]. The degree of PEGylation to the cationic liposomes influenced their stability and biodistribution. Insufficient PEG coating resulted in their serum related aggregation and pulmonary capillary accumulation [100]. In addition to PEGylation to liposomes increasing their size, the enlarged liposome hydrodynamic radiuses contributed more to their lung deposition and biodistribution than the polycationic liposome [78]. Stable Nucleic Acid Lipid Particles (SNALPs) are PEG coated liposomes. SNALPs were comprised of cationic and fusogenic lipids. The PEG motif shielded and stabilized the vesicle, resisted recognition by opsonins and rapid clearance by the MPS system, the cationically charged lipid was favored for complexation with siRNAs, and the fusogenic lipids

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facilitated SNALP cellular uptake and siRNA endosome release [101]. SNALPs showed longer half life than traditional unPEGylated liposomes, and biodistribution and pharmacokinetic studies indicated SNALPs substantially accumulated in the liver and spleen in mice. In siRNA containing SNALPs, siRNA preferentially loaded into the lipid bilayers. Currently, SNALP based siRNA delivery agents accounted for multiple siRNA based RNAi therapeutics in clinical trial. 3.2.2.2. PEGylated cationic polymeric NPs. Introducing PEG to the surface of cationic NPs effectively shield their positive charge and endow them with stealth properties. Polylysines (PLLs) are biocompatible oligopeptides. The condensed amino groups from PLL side chain amino groups are favored for complexation of siRNAs, whereas PLLs caused adverse effect on cells due to their non-specific random interaction with cell membranes. Even after PLL complex with siRNAs, the strong cationic surface charge could still interact with the cell membrane and cause strong cytotoxicity. PEGylation to the backbone of PLLs or grafting of PEGs to PLL side chains significantly reduced their cytotoxicity, prolonged PLL circulation time in the blood stream and increased their tumoral accumulation [69]. Grafting of 40 kDa PLL with 10 kDa PEG at a ratio of 37% (PLL-g-PEG) prevented siRNA degradation by RNase even after preincubation with murine sera, and augmented PLL-g-PEG/siRNA NP lifetime in blood circulation and tumoral accumulation without loss of siRNA association activity [69]. Current PEI based siRNA delivery vehicles are quite toxic when administered intravenously and the siRNA releasing efficiency is quite low, even though linear PEIs exhibit less toxicity and induce weaker inflammatory effect than branched PEIs. PEGylation to PEI attenuates the toxicity generated from the cationic amine groups. In one study, both PEI and PEG grafted PEI effectively condensed siRNAs, their complex stability depends on the PEG grafting degree, and PEI/siRNA polyplex exhibited more stability than all the PEG–PEI/siRNA polyplexes, however, the release of siRNAs from PEG–PEI/siRNA polyplexes was more efficient. Pulmonary application of PEI/siRNA indicated that PEI was left in the lung and siRNAs were released and excreted, whereas PEG–PEI/ siRNA polyplexes showed higher pro-inflammatory potential, sustained siRNA release and no sign of histological abnormality [102]. In another study, introducing PEG to PEI also demonstrated advantages over PEIs. The PEG–PEI/siRNA complex showed much higher siRNA release (52%) than PEI/siRNA (b 5%). In vitro transfection demonstrated that the PEG–PEI/siGFP complexes showed much higher gene silencing effect (75%) than PEI/siRNA complex (19%) [103]. Chitosans (CSs) are highly recommended for siRNA delivery in clinical development because of their biodegradability in the human body. The limitations to CSs in siRNA delivery are their poor solubility in aqueous phase at physiological pH and low transfection efficiency in vivo [104]. PEGylation to CSs increases siRNA loading efficiency. CSs grafted with polyarginines (PLR) and PEGs for delivery of luciferase mRNA specific siRNAs, and the cellular GFP knockdown levels were highly reduced than CSs alone. Different from CS–PLR/siRNA complexes, the presence of PEG stabilized the complexes even in high concentration of serum content [105]. 4. Preclinical targeted delivery of siRNA based RNAi By optimization of the construct of siRNA NPs, their therapeutic potency was improved. Through targeted delivery siRNA local concentration was increased, and their nonspecific biodistribution and adverse effects were mitigated. 4.1. Local delivery Compared to systemic delivery, the benefits of local delivery are the relatively higher bioavailability, reduced adverse effects and less pharmaceutical concerns. siRNA based agents for topical diseases could be

administered via local approaches including intranasal, intraocular, intratumoral, intramuscular and intracerebral, etc. The applied formulations include, naked siRNAs, liposome encapsulated siRNAs, polymer complexed siRNAs, etc. [106]. Multiple local applications of siRNA based therapeutics in clinical trial are under investigation. Typically, some are applying naked siRNAs against molecular targets of ocular diseases, and these naked siRNAs are applied via intravitreal injection or topical application. In a study investigating the roles of VEGFR1 in ocular neovascularization (NV), naked siRNA Sirna-027 against vascular endothelial growth factor receptor 1 (VEGFR1) mRNA was identified, and Sirna-027 reduced VEGFR1 mRNA levels in mouse model of retinal and choroidal neovascularization (CNV). After intravitreous and periocular injection, significant reduction of VEGFR1 mRNA level to 57% and 40% was achieved, respectively [107]. Some local applied polyplexes demonstrated satisfying siRNA transfection result [108]. Intranasal administration of oligofectamine/siRNA complexes resulted in their effective delivery to the lung against the influenza virus infection [109]. Intrathecal delivery of PEI–siRNA conjugates targeted to the NR2B gene subunit not only decreased their mRNA and associated protein levels, but also abolished formalin induced pain in rat model [110]. Intratumoral injection of siRNA encapsulated cationic liposomes achieved targeted inhibition of invasive cutaneous melanomas [111].

4.2. Systemic delivery RNAi local application that only treated topical diseases is not manageable to most diseases related to the internal organs. Treatment to the internal organs needs to apply RNAi therapeutics intravenously for systemic delivery. Passive and active targeted systemic delivery strategies are applied to send RNAi therapeutics to the disease stricken internal tissues [112].

4.2.1. Passive delivery Taking advantage of the special anatomical and functional features of tumors, RNAi NPs could be passively delivered. In fact, passive delivery accounted for most tissue internalized RNAi NPs. The main internalization approach is through the EPR effect in solid tumor [113] and inflammatory tissues [114], where small molecule drugs diffuse in and out in concentration driven manner, whereas larger NPs (MW N 40 kDa) cannot easily diffuse back to the blood circulation once it passed through the abnormal angiogenesis vessels and finally accumulated in these tissues [27,115]. Furthermore, NP delivery can be influenced by the pre-mentioned systemic barriers and the features of RNAi delivery vehicles, such as particle size, surface charge, hydrophobicity, and stability [116]. Coating cationic or hydrophobic particles with hydrophilic polymer endow them with stealth properties, and reduce opsonization [18], subsequently accumulation in the abnormal angiogenesis vessels. Cationic liposomes for passive targeted delivery had a half life of about 20 min in the blood flow, however, PEGylated liposome extended to 5 days. SNALPs represent advanced liposome vehicles for passive siRNA delivery. SNALPs with a mean size of 100 nm, plus surface PEGylation endow them with much longer half-life in plasma when compared with traditional cationic liposomes. An apolipoprotein B (ApoB) targeted siRNA encapsulated SNALP confirmed the suitability of SNALPs for systemic delivery of siRNAs. By intravenous injection to cynomolgus monkeys in one dose at 2.5 mg/kg, a dose dependent silencing of ApoB mRNA level reached N 90%, the silencing effect occurred as a result of ApoB mRNA cleavage at the predicted site. Significant reduction in ApoB protein, serum cholesterol and low-density lipoprotein levels lasts for 11 days at the highest siRNA dose [117].

Please cite this article as: Y. Zhou, et al., Development of RNAi technology for targeted therapy — A track of siRNA based agents to RNAi therapeutics, J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.04.044

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4.2.2. Active, targeted delivery Followed by intravenous injection, RNAi agents entered the superficial layer of pathological tissue via specific or nonspecific binding, and most of the NPs still need to be explored in the subsurface or deeper layer before it can enter the cells. Theoretically, RNAi NPs decorated with active targeting ligands have more chance to enter deeper tissue sites and show greater treatment effect. The potential targets for ligand decorated NPs include tumor angiogenesis cells, tumor inner tissue cell and intracellular compartment endosome. Some ligand receptors were specifically over-expressed in diseased tissues, their ligands were chosen as targeting motif for ligand decorated RNAi NP targeted delivery (Fig. 4a), and these ligands include antibodies (Fig. 4b), peptides (Fig. 4c), aptamers (Fig. 4d) and small molecules (Fig. 4e). 4.2.2.1. Target to tumor angiogenesis cells. Transmembrane glycoprotein intergrins played an important role in cell proliferation. Most integrins are vital in cell survival, adhesion, signaling and migration, especially in cancer progression. Integrins bind to extracellular matrix proteins through the tripeptide Arg-Gly-Asp (RGD) motif. αvβ3 integrin is a crucial mediator in tumor angiogenesis, tumor neovascularization and tumor metastasis [28]. Their expression is upregulated on angiogenic endothelial cells in different cancer cell lines, and the inhibition of αvβ3 integrin suppresses tumor angiogenesis and retards tumor growth. The property of RGD peptide recognizing integrins has been applied for RNAi active targeting to tumor neovascularization cells. RGD conjugated NPs specifically bind with tumor neovasculature endothelium cells, deliver VEGF targeted siRNA into tumor endothelial cells, and inhibit the tumor neovascularization and metastasis [118]. RGD targeting effect was verified through a cRGD conjugated liposome (RGD–MEND). RGD–MEND could be co-localized with tumor endothelial cells (TECs), comparatively, YSK–MEND and PEG–MEND diffused over the entire tumor tissue, and they neither accumulated in TECs nor inhibited TEC proliferation [29]. In another study, intravenously administered nanoplex of RGD–PEG–PEI (RPP) polymer with FITC–siRNA

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produced appreciable FITC fluorescence in the tumor, but poor in the liver and the lung, indicating that the RGD motif of RPP complex preferentially accumulated in the tumor tissue through ligand binding. In the in vivo transfection, simultaneously administered luciferase plasmid RPP polyplexes and the plasmid targeted siRNA–RPP polyplexes inhibited plasmid expression by 90%. Moreover, when RPP delivered siRNAs against vascular endothelial growth factor (VEGF) R2 to tumor bearing mice, strong inhibition effect to tumor growth rate was identified [119]. 4.2.2.2. Target to tumor inner tissue cells. Some RNAi NPs enter the inner sites of tumor by diffusion or convection, and ligand decorated RNAi NPs specifically bind to tumor cell surface which are over-expressing relative receptors or proteins and subsequently increase their uptake. Antibodies (such as monoclonal antibodies (mAbs) [120], fragment antibodies (Fabs) [121], single chain fragment variable antibodies (scFvs) [122]), peptide and proteins (such as RGD, RVG [123], and transferrin (TF) [124]), small molecules (such as folic acid [125] and carbohydrates [126]) and aptamer [127] were the most frequently studied ligands. Antibodies (Abs) bind to their relative antigens with high affinity and specificity, Engineered antibody fragment such as Fabs and scFvs with only their recognition motif and reduced size shows advantages over the whole antibodies, and is more efficiently adopted with NPs for cell type specific drug delivery. Her2 [128], erbB2 [121] and HIV-1 gp160 [129], etc. are examples of antibodies adopted for active targeted delivery. Song et al. developed a protamine–antibody fusion protein (F105-P) to deliver siRNA into HIV infected or HIV envelope transfected cells. In vitro transfection showed complexes of the HIV-1 capsid gag gene targeted siRNAs and F105-P induced gene silence only in cells expressing HIV-1 envelope. Intratumoral or intravenous administration F105-P/FITC–siRNA complexes into mice which was bearing HIV infected B16 melanoma cells, FITC–siRNA only enriched in gp160(+) tumor cells, not in adjacent tissue. In intratumoral or intravenous injection of complexes of F105-P with pooled siRNAs targeting c-myc, MDM2 and VEGF genes, the gp160-B16 expressing tumor growth was significantly

Fig. 4. Popular ligands involved in systemic active targeted siRNA delivery: a) overview of ligand conjugated RNAi NPs; b) antibodies (mAb, Fab, scFv, etc.); c) peptides (RGD, polyarginines, transferrin, etc.); d) aptamer and e) small molecules (folic acid, cholesterol, carbohydrate, etc.).

Please cite this article as: Y. Zhou, et al., Development of RNAi technology for targeted therapy — A track of siRNA based agents to RNAi therapeutics, J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.04.044

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retarded in mice compared to those treated with free siRNA alone [121]. Yan-dan Yao et al. reported a fusion of proteins of ScFvs and protamine for active targeted delivery siRNAs to tumor [128]. Polo-like kinase 1 (PLK1) was selected as the siRNA target because it is a highly conserved kinase in diverse cancer types, PLK1 promotes cell division and correlates with aggressive behavior, and PLK1 inhibition leads to cancer cell cycle arrest, apoptosis and “mitotic catastrophe”. PLK1 targeted siRNAs complexed with a Her2(+)–ScFv–protamine peptide fusion protein (F5-P) to form PLK1–siRNAs/F5-P complexes. Intravenously injected this complexes concentrated into Her2(+) breast cancer xenografts, and their distribution in irrelevant normal tissues was neglectable. The complexes retarded Her2(+) breast tumor growth, reduced tumor metastasis and prolonged mice survival [128]. Transferrin (TF) is a blood plasma glycoprotein for iron delivery, however, transferrin receptors are over-expressed on various fast growing endogenous cancer cell surfaces. The intracellular routing of TF receptor mediated cell endocytosis has been fully characterized; therefore, TF is another type of prevailing ligand for active targeted drug delivery. TF conjugated NPs were frequently used as siRNA delivery vehicles for tumor targeted gene silencing in vivo. In a representative study, siRNA and TF were crosslinked to form stable NPs (psi-tTF NPs). After systemic injection, fluorescent dye labeled psi-tTF NPs showed an increasing fluorescence intensity from 3 h post-injection to 24 h in tumor tissue, and the tumor was clearly delineated from normal tissues, suggesting the psi-tF NPs more preferentially accumulated in the TF receptor over-expressing tumor tissues. In contrast, NIR signal of naked siRNA markedly increased throughout the body and disappeared after 24 h. In RFP/B16F10 bearing mouse model, RFP gene was significantly knocked down by the intravenously administered antiRFP siRNA containing psi-tTF NPs [124]. The first clinical trial agent CALAA-01 was also utilizing TF as its targeting ligand. CALAA-01 is mainly composed of TF conjugated PEG, TF and adamantine conjugated PEG, cyclodextran (CD) polymers and RRM2 gene targeted siRNA sequences. Early data indicated that systemic injected CALAA-01 NPs mainly localized in tumor cells of melanoma in a dose-dependent manner [130], and CALAA-01 was well tolerated after multiple dosing in non-human primates [131]. Aptamers are synthetic, single strand short oligonucleic acid molecules, and are capable of specifically binding with different molecular ligands with the secondary or tertiary structure [132]. After two decades of development, aptamers are enabled as small molecule inhibitors, diagnostic tools and therapeutics. Their low immunogenicity in vivo and ease of chemical modification as well as the ability of recognizing and binding with specific protein or cell surface receptors have been adopted as ligands for targeted siRNA delivery [133]. Aptamers and siRNA could be readily linked via covalent or non-covalent linkages, or fabricated into a nanoplexes. An aptamer–siRNA chimera (PSMA– Plk1) with aptamer segment selectively binding with PSMA (prostate specific membrane antigen) delivered PSMA–Plk1 into prostate cancer cells in vitro. Intra-peritoneally administered PSMA–Plk1 chimera to prostate tumor bearing athymic mice completely achieved regression of 70% of tumors at the end of treatment. The RNAi specificity by the chimera siRNAs was also identified on tumor bearing animals treated with the PSMA–Plk1 chimeras [134]. Small molecule ligands are also effective chemicals in active targeted siRNA delivery. Cholesterol [135], folic acid [136] and carbohydrate [137] etc. are typical representative small targeting ligands. Modification of oligonucleotides with cholesterol increased their stability and cellular uptake and reduced their interaction with low density lipoproteins (LDLs) and high density lipoproteins (HDLs). Intravenously injected apolipoprotein B (ApoB) targeted siRNA cholesterol conjugates (Chol–siRNAs) silenced ApoB in mice liver [13]. Folate receptors (FRs) were overexpressed on some cancer cell surface, whereas limited on normal cell. FA or FA derivative modified liposomes efficiently deliver ODN to KB cells that over-express FRs. Uptake of FA modified liposomes by KB cells is about 8–10 fold than those without folate or free ODN [138].

Cell penetrating peptides (CPPs) with cell membrane penetration property are cationic oligopeptides derived from natural protein sequences of viral, insect or mammalian proteins [139]. Covalent conjugation and electrostatic interaction are manners for CPPs to aid the delivery of drugs. RNAi NPs linked with CPPs increase their cellular uptake, and CPPs assist their internalization via increasing cellular plasma membrane permeability. Most CPPs lack specificity in cellular targeting, and are frequently used as subsidiaries in assisting NP cellular internalization. HIV–TAT family, Penetratin and chimeric peptide Transportan and Oligoarginines are the typical representative CPPs [140]. The repeating unit of arginines in the Oligoarginines sequence endows them with strong cationic nature. A chimeric peptide from conjugation of nona-arginine peptide with RVG peptide (RVG-9R) for siRNA delivery. Intravenous administration of the RVG-9R/siRNA complexes led to specific gene silencing in the central nervous system in mice [123]. 4.2.2.3. Target to cell endosome. RNAi particles internalized via clathrin mediated endocytic pathway entrapped in endosomes might be degraded in the lysosome where acid and active enzymes exist. This led to limited delivery of siRNAs into the cytoplasm and eventually low gene knockdown efficiency. Several approaches facilitate RNAi agent escape from early endosomes. The identified approaches include endosome membrane pore formation and endosome membrane disruption. pH sensitive peptide, pH buffering polymers, fusogenic lipids, and chemical molecules were agents used to promote RNAi particle escape from endosome. Cationic polymers PEI, poly(L-histidine) and synthetic pH-sensitive cationic polymer with highly protonable amino groups [42] induced proton sponge effect at endosomal pH led to osmotic pressure increasing inside the endosome which finally disrupted the endosome membrane [141]. Some photo-sensitizers are useful for RNAi NP escape from endosome. Upon exposure to light, photo-sensitizers [142] in the endosome/lysosome membrane generated singlet oxygen which destroyed the endosomal/lysosomal membrane, followed by releasing their content to the cytosol [143]. Endosome membrane could also be destabilized when fused with pH sensitive peptides. When in the endosome membrane, the pH sensitive peptides undergo conformational changes upon pH variation, allowing the peptides to fuse with the endosome lipid bilayer. Some protein fragments, such as GALA [144] and diINF-7 [49], originating from virus single integral membrane proteins were used for RNAi NP endosome escape. GALA is a pH sensitive, amphiphilic peptide with 30 amino acids and glutamic acid–alanine–leucine–alanine (GALA) sequence repeats. This peptide adopts a random coil conformation in neutral pH, while α-helix in the endosomal pH, thereby promoting GALA binding to endosome membrane and resulting in endosome membrane disruption [145]. A multifunctional siRNA delivery envelope-type nanodevice (MEND) achieved eradication of the tumor lung metastasis. The key to success was attributed greatly to the surface GALA peptide. Tail vein and earlobe blood vessel injected GALA–MEND/Cy3-siRNA complexes gradually accumulated along the lung capillary. The negative GALA peptide shielded the cationic liposome from aggregation with blood opsonins and acted as positively charged endosome destabilizer [144]. 5. RNAi in clinical trials Since the first siRNA based RNAi agent entered clinical trial in 2004, there were more than 20 siRNA based RNAi therapeutics that entered clinical trial for local or systemic disease treatment [146]. With the development of siRNA delivery vehicles, there was an increasing number of siRNA agents that entered the systemic clinical testing. The siRNA agents in their clinical trials at different clinical trial stages showed inspiring therapeutic effect and were on their way to further identification (see Table 1). Most of the naked siRNAs were locally

Please cite this article as: Y. Zhou, et al., Development of RNAi technology for targeted therapy — A track of siRNA based agents to RNAi therapeutics, J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.04.044

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administered to treat topical diseases (including AMD, DME and NAION) or virus infections (such as RSV and HCV). A notable naked siRNA therapeutic I5NP in its phase I/II trial targeting prophylaxis of delayed graft function in kidney transplantation was administered intravenously. Several siRNA agents terminated their clinical trial due to low efficacy, off-target effect or capital issues. Up to date, the terminated siRNA agents in clinical trials were naked delivered Bevasiranib, AGN-745, and PF-655; a SNALP based delivery agent TKM–ApoB and the first active targeted delivery agent CALAA-01. Bevasiranib was terminated because of low efficacy at its phase III clinical trial on therapy of AMD/DME. AGN-745 was terminated in the treatment of AMD due to off-target effect at its phase II clinical testing. Whereas, TKM–ApoB was terminated because of only transient reduction of cholesterol level was observed at its phase I clinical study. A common adverse effect of these agents was activation of TLRs. Even though most terminated RNAi agents were naked siRNAs, the local administered naked siRNAs still have chances to enter further testing as long as they show appropriate safety and efficacy. The only active targeted therapeutic CALAA-01 was also terminated recently, the actual reason was not announced, but the observed phenomena with fast clearance and an acute inflammation cytokine elevation in the preclinical trials might show some clues [147]. Preliminary study indicated that liposome based delivery system was generally safe and well tolerated. SNALP based siRNA delivery platform was accounted for multiple siRNA agents in clinical trial, and most of the SNALP based agents were in their phase I stage testing. Typically, 50% of patients with hepatocellular carcinoma showed anti-VEGF effect after treatment with ALN-VSP02, which encapsulated VEGF and kinesin spindle protein (KSP) targeted siRNAs. The SNALP based ALN-TTR01 exhibited statistically significant serum transthyretin (TTR) protein level reduction in both wide-type and mutant TTR proteins in ATTR patients after a single dose. Another ATTR siRNA agent ALN-TTR02 led to robust knockdown of serum TTR protein levels up to 94% after just a single injection [148]. In another representative RNAi agent, Atu027 was applied a complex of siRNA and cationic liposome AtuPLEX, in its phase I clinical trial for evaluating toxicity, and no drug related side effect was observed from 27 out of the 33 patients who were with advanced solid tumor. A phase Ib/IIa trial was under way with some selected chemotherapeutical regimens to study the pharmacological activity. Some other RNAi agent like CALAA01 utilized cyclodextran polymers as its positively charged polymer, and siG12D LODER utilized a microdevice for siRNAs locally release.

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6. RNAi future prospect Despite that some RNAi agents are terminated even at the advanced clinical trial stage, experiences from these and on-going trials could direct the future study of RNAi, and new attempts from the scientific research community are booming and on its way of creating more practical RNAi therapeutics. Avoiding multiple barriers from the blood circulation system, RNAi agents only require naked siRNAs or structure simplified vehicles for local application should have more chance to enter clinical trials earlier. Chemical modification or chemical stabilization to siRNAs enhances their bioavailability and improves their efficiency. The combinations of several types of chemical modifications synergistically effectively improve siRNA performance and generation of superior siRNAs. Carriers of siRNA are critical issues that need to be resolved for successful systemic delivery. Development of multi-functional non-viral carriers is currently the best option. The roles of the carriers playing in siRNA transportation process should cover these areas: endow siRNAs with long circulation time, protect siRNAs from RNase degradation and renal clearance, rectify and guide siRNA targeted distribution and their escape from endosome. Size, charge and morphology control are critical issues that need to be considered in the construction of RNAi NPs. To fulfill these requirements, targeting ligands, stealth polymer, cationically charged materials and membrane disruption components are necessary requisites in constructing the multi-functional siRNA carriers. AtuPLEX and SNALPs are optimized liposomes, which represent the most advanced siRNA carriers presently. Especially, SNALPs contain almost all the necessary component required for optimal siRNA carriers, and their current systemic disease targets were almost exclusively in the RES tissues. The clear liver physiologic structure sheds light on RNAi therapeutics to target liver related diseases, meanwhile this poses a challenge to develop RNAi therapeutics targeting sites out of the RES tissues. In this perspective, stealth spherical NPs with controlled size, such as less than 100 nm or much less in diameter, should be in favor of their distribution to the other organs. Collectively, improvement on siRNAs and their carriers is the leading consideration for designing more precisely targeted and potent RNAi therapeutics. 7. Conclusions In summary, the gene target specificity and diseases target universality endowed siRNA agent substantial potential in the treatment of

Table 1 Representative RNAi agents in clinical trials.a CTA

Candidate

Disease target

Delivery route

Vehicle

Phase stage

Status

Company

2004 2005 2007 2008 2009 2009 2009 2011 2007 2008 2008 2009 2009 2009 2010 2011 2008

Sirna-027 Bevasiranib PF-4523655 TD101 SPC3649 QPI-1007 SYL040012 SYL1001 ALN-RSV01 I5NP ALN-VSP02 TKM-ApoB ALN-TTR01 Atu027 siG12D LODER ALN-PCS02 CALAA01

AMD AMD/DME AMD/DME PC HCV NAION Intraocular pressure Dry eye/ocular pain RSV AKI Solid tumor High cholesterol ATTR Advanced solid tumor Advanced pancreatic cancer LDL-C Solid tumor

Intravitreal Intravitreal Intravitreal Intralesional Subcutaneous Intravitreal Eye drop Eye drop Nebulization i.v. i.v. i.v. i.v. i.v. Intratumoral injection i.v. i.v.

Free siRNA Free siRNA Free siRNA Free siRNA LNA siRNA Free siRNA Free siRNA Free siRNA Free siRNA Free siRNA SNALP SNALP SNALP Lipoplex LODER polymer SNALP Polyplex

II III II Ib IIa I II I IIb II I I I I I I I

Terminated Terminated Completed Completed Active Active Completed Active Completed Active Completed Terminated Completed Completed Active Completed Terminated

Allergan/Sirna Opko Health Quark/Pfizer TransDerm/IPCC Santaris Quark Pharma Sylentis Sylentis Alnylam/Cubist Quark Pharma Alnylan/Tekmira Tekmira Alnylam Silence Therapeutics Silenceed Ltd. Alnylan Calando Pharma

Abbreviations: clinical trial approval (CTA); age-related macular edema (AMD); diabetic macular edema (DME); Pachyonychia Congenital (PC); Hepatitis C Viral (HCV); transthyretin (TTR) mediated amyloidosis (ATTR); Acute Non-Arteritic Anterior Ischemic Optic Neuropathy (NAION); respiratory syncytial virus (RSV); Acute Kidney Injury (AKI); low density lipoprotein-cholesterol (LDL-C). a Cited from http://www.clinicaltrials.gov/.

Please cite this article as: Y. Zhou, et al., Development of RNAi technology for targeted therapy — A track of siRNA based agents to RNAi therapeutics, J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.04.044

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diseases in humans. Application of new technologies greatly altered siRNA behaviors, increased their therapeutic potency and reduced offtarget effect. The great progress made in engineered RNAi agents in the treatment of cancer, virus and other genetic disorders paved a way for the conversion of siRNA agents to siRNA based RNAi therapeutics.

Acknowledgement This work was financially supported by grants from the National Natural Science Foundation of China (nos. 51203179 and 81173633).

References [1] A. Fire, S. Xu, M.K. Montgomery, S.A. Kostas, S.E. Driver, C.C. Mello, Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans, Nature 391 (1998) 806–811. [2] C.A. Sledz, B.R. Williams, RNA interference in biology and disease, Blood 106 (2005) 787–794. [3] L.P. Ford, M.M. Toloue, Delivery of RNAi mediators, Wiley interdisciplinary reviews, RNA 1 (2010) 341–350. [4] N. Manjunath, D.M. Dykxhoorn, Advances in synthetic siRNA delivery, Discov. Med. 9 (2010) 418–430. [5] S.L. Ameres, J. Martinez, R. Schroeder, Molecular basis for target RNA recognition and cleavage by human RISC, Cell 130 (2007) 101–112. [6] M. DiFiglia, M. Sena-Esteves, K. Chase, E. Sapp, E. Pfister, M. Sass, J. Yoder, P. Reeves, R.K. Pandey, K.G. Rajeev, M. Manoharan, D.W. Sah, P.D. Zamore, N. Aronin, Therapeutic silencing of mutant huntingtin with siRNA attenuates striatal and cortical neuropathology and behavioral deficits, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 17204–17209. [7] J. DeVincenzo, R. Lambkin-Williams, T. Wilkinson, J. Cehelsky, S. Nochur, E. Walsh, R. Meyers, J. Gollob, A. Vaishnaw, A randomized, double-blind, placebo-controlled study of an RNAi-based therapy directed against respiratory syncytial virus, Proc. Natl. Acad. Sci. U. S. A. 107 (2010) 8800–8805. [8] S.I. Pai, Y.Y. Lin, B. Macaes, A. Meneshian, C.F. Hung, T.C. Wu, Prospects of RNA interference therapy for cancer, Gene Ther. 13 (2006) 464–477. [9] F.T. Vicentini, L.N. Borgheti-Cardoso, L.V. Depieri, D. de Macedo Mano, T.F. Abelha, R. Petrilli, M.V. Bentley, Delivery systems and local administration routes for therapeutic siRNA, Pharm. Res. 30 (2013) 915–931. [10] J. Whelan, First clinical data on RNAi, Drug Discov. Today 10 (2005) 1014–1015. [11] K. Bruno, Using drug–excipient interactions for siRNA delivery, Adv. Drug Deliv. Rev. 63 (2011) 1210–1226. [12] K.A. Whitehead, R. Langer, D.G. Anderson, Knocking down barriers: advances in siRNA delivery, Nat. Rev. Drug Discov. 8 (2009) 129–138. [13] J. Soutschek, A. Akinc, B. Bramlage, K. Charisse, R. Constien, M. Donoghue, S. Elbashir, A. Geick, P. Hadwiger, J. Harborth, M. John, V. Kesavan, G. Lavine, R.K. Pandey, T. Racie, K.G. Rajeev, I. Rohl, I. Toudjarska, G. Wang, S. Wuschko, D. Bumcrot, V. Koteliansky, S. Limmer, M. Manoharan, H.P. Vornlocher, Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs, Nature 432 (2004) 173–178. [14] D.M. Dykxhoorn, D. Palliser, J. Lieberman, The silent treatment: siRNAs as small molecule drugs, Gene Ther. 13 (2006) 541–552. [15] D.W. Bartlett, M.E. Davis, Effect of siRNA nuclease stability on the in vitro and in vivo kinetics of siRNA-mediated gene silencing, Biotechnol. Bioeng. 97 (2007) 909–921. [16] M. Longmire, P.L. Choyke, H. Kobayashi, Clearance properties of nano-sized particles and molecules as imaging agents: considerations and caveats, Nanomedicine 3 (2008) 703–717. [17] A. Sato, S.W. Choi, M. Hirai, A. Yamayoshi, R. Moriyama, T. Yamano, M. Takagi, A. Kano, A. Shimamoto, A. Maruyama, Polymer brush-stabilized polyplex for a siRNA carrier with long circulatory half-life, J. Control. Release 122 (2007) 209–216. [18] D.E. Owens, N.A. Peppas, Opsonization, biodistribution, and pharmacokinetics of polymeric nanoparticles, Int. J. Pharm. 307 (2006) 93–102. [19] S. Dokka, D. Toledo, X.G. Shi, V. Castranova, Y. Rojanasakul, Oxygen radicalmediated pulmonary toxicity induced by some cationic liposomes, Pharm. Res. 17 (2000) 521–525. [20] D.T. Auguste, K. Furman, A. Wong, J. Fuller, S.P. Armes, T.J. Deming, R. Langer, Triggered release of siRNA from poly(ethylene glycol)-protected, pH-dependent liposomes, J. Control. Release 130 (2008) 266–274. [21] S. Salmaso, P. Caliceti, Stealth properties to improve therapeutic efficacy of drug nanocarriers, J. Drug Deliv. 2013 (2013) (article ID: 374252). [22] D. Peer, J. Lieberman, Special delivery: targeted therapy with small RNAs, Gene Ther. 18 (2011) 1127–1133. [23] J. Heyes, L. Palmer, K. Bremner, I. MacLachlan, Cationic lipid saturation influences intracellular delivery of encapsulated nucleic acids, J. Control. Release 107 (2005) 276–287. [24] R. Juliano, M.R. Alam, V. Dixit, H. Kang, Mechanisms and strategies for effective delivery of antisense and siRNA oligonucleotides, Nucleic Acids Res. 36 (2008) 4158–4171.

[25] Y. Matsumura, H. Maeda, A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs, Cancer Res. 46 (1986) 6387–6392. [26] H. Maeda, J. Wu, T. Sawa, Y. Matsumura, K. Hori, Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review, J. Control. Release 65 (2000) 271–284. [27] G.J. Villares, M. Zigler, H. Wang, V.O. Melnikova, H. Wu, R. Friedman, M.C. Leslie, P.E. Vivas-Mejia, G. Lopez-Berestein, A.K. Sood, M. Bar-Eli, Targeting melanoma growth and metastasis with systemic delivery of liposome-incorporated proteaseactivated receptor-1 small interfering RNA, Cancer Res. 68 (2008) 9078–9086. [28] J.S. Desgrosellier, D.A. Cheresh, Integrins in cancer: biological implications and therapeutic opportunities, nature reviews, Cancer 10 (2010) 9–22. [29] Y. Sakurai, H. Hatakeyama, Y. Sato, M. Hyodo, H. Akita, N. Ohga, K. Hida, H. Harashima, RNAi-mediated gene knockdown and anti-angiogenic therapy of RCCs using a cyclic RGD-modified liposomal–siRNA system, J. Control. Release 173 (2014) 110–118. [30] H. Hillaireau, P. Couvreur, Nanocarriers' entry into the cell: relevance to drug delivery, Cell. Mol. Life Sci. 66 (2009) 2873–2896. [31] J. Nguyen, F.C. Szoka, Nucleic acid delivery: the missing pieces of the puzzle? Acc. Chem. Res. 45 (2012) 1153–1162. [32] D. Vercauteren, J. Rejman, T.F. Martens, J. Demeester, S.C. De Smedt, K. Braeckmans, On the cellular processing of non-viral nanomedicines for nucleic acid delivery: mechanisms and methods, J. Control. Release 161 (2012) 566–581. [33] S. Kumari, M.G. Swetha, S. Mayor, Endocytosis unplugged: multiple ways to enter the cell, Cell Res. 20 (2010) 256–275. [34] L.M. Bareford, P.W. Swaan, Endocytic mechanisms for targeted drug delivery, Adv. Drug Deliv. Rev. 59 (2007) 748–758. [35] J. Rejman, V. Oberle, I.S. Zuhorn, D. Hoekstra, Size-dependent internalization of particles via the pathways of clathrin- and caveolae-mediated endocytosis, Biochem. J. 377 (2004) 159–169. [36] S. Mao, O. Germershaus, D. Fischer, T. Linn, R. Schnepf, T. Kissel, Uptake and transport of PEG-graft-trimethyl-chitosan copolymer-insulin nanocomplexes by epithelial cells, Pharm. Res. 22 (2005) 2058–2068. [37] O. Harush-Frenkel, N. Debotton, S. Benita, Y. Altschuler, Targeting of nanoparticles to the clathrin-mediated endocytic pathway, Biochem. Biophys. Res. Commun. 353 (2007) 26–32. [38] H.R. Kim, K. Andrieux, S. Gil, M. Taverna, H. Chacun, D. Desmaele, F. Taran, D. Georgin, P. Couvreur, Translocation of poly(ethylene glycol-co-hexadecyl)cyanoacrylate nanoparticles into rat brain endothelial cells: role of apolipoproteins in receptor-mediated endocytosis, Biomacromolecules 8 (2007) 793–799. [39] M. Ogris, P. Steinlein, S. Carotta, S. Brunner, E. Wagner, DNA/polyethylenimine transfection particles: influence of ligands, polymer size, and PEGylation on internalization and gene expression, AAPS PharmSci 3 (2001) E21. [40] C. Foerg, U. Ziegler, J. Fernandez-Carneado, E. Giralt, R. Rennert, A.G. BeckSickinger, H.P. Merkle, Decoding the entry of two novel cell-penetrating peptides in HeLa cells: lipid raft-mediated endocytosis and endosomal escape, Biochemistry 44 (2005) 72–81. [41] C. Moreira, H. Oliveira, L.R. Pires, S. Simoes, M.A. Barbosa, A.P. Pego, Improving chitosan-mediated gene transfer by the introduction of intracellular buffering moieties into the chitosan backbone, Acta Biomater. 5 (2009) 2995–3006. [42] H. Yu, Y. Zou, Y. Wang, X. Huang, G. Huang, B.D. Sumer, D.A. Boothman, J. Gao, Overcoming endosomal barrier by amphotericin B-loaded dual pH-responsive PDMA-b-PDPA micelleplexes for siRNA delivery, ACS Nano 5 (2011) 9246–9255. [43] S. Kakimoto, T. Hamada, Y. Komatsu, M. Takagi, T. Tanabe, H. Azuma, S. Shinkai, T. Nagasaki, The conjugation of diphtheria toxin T domain to poly(ethylenimine) based vectors for enhanced endosomal escape during gene transfection, Biomaterials 30 (2009) 402–408. [44] H. Jenssen, P. Hamill, R.E. Hancock, Peptide antimicrobial agents, Clin. Microbiol. Rev. 19 (2006) 491–511. [45] T. Endoh, T. Ohtsuki, Cellular siRNA delivery using cell-penetrating peptides modified for endosomal escape, Adv. Drug Deliv. Rev. 61 (2009) 704–709. [46] S.C. Semple, A. Akinc, J. Chen, A.P. Sandhu, B.L. Mui, C.K. Cho, D.W. Sah, D. Stebbing, E.J. Crosley, E. Yaworski, I.M. Hafez, J.R. Dorkin, J. Qin, K. Lam, K.G. Rajeev, K.F. Wong, L.B. Jeffs, L. Nechev, M.L. Eisenhardt, M. Jayaraman, M. Kazem, M.A. Maier, M. Srinivasulu, M.J. Weinstein, Q. Chen, R. Alvarez, S.A. Barros, S. De, S.K. Klimuk, T. Borland, V. Kosovrasti, W.L. Cantley, Y.K. Tam, M. Manoharan, M.A. Ciufolini, M.A. Tracy, A. de Fougerolles, I. MacLachlan, P.R. Cullis, T.D. Madden, M.J. Hope, Rational design of cationic lipids for siRNA delivery, Nat. Biotechnol. 28 (2010) 172–176. [47] N.D. Sonawane, F.C. Szoka, A.S. Verkman, Chloride accumulation and swelling in endosomes enhances DNA transfer by polyamine–DNA polyplexes, J. Biol. Chem. 278 (2003) 44826–44831. [48] A. Kichler, A.J. Mason, A. Marquette, B. Bechinger, Histidine-rich cationic amphipathic peptides for plasmid DNA and siRNA delivery, Methods Mol. Biol. 948 (2013) 85–103. [49] A.K. Varkouhi, M. Scholte, G. Storm, H.J. Haisma, Endosomal escape pathways for delivery of biologicals, J. Control. Release 151 (2011) 220–228. [50] A.L. Jackson, S.R. Bartz, J. Schelter, S.V. Kobayashi, J. Burchard, M. Mao, B. Li, G. Cavet, P.S. Linsley, Expression profiling reveals off-target gene regulation by RNAi, Nat. Biotechnol. 21 (2003) 635–637. [51] A.L. Jackson, J. Burchard, D. Leake, A. Reynolds, J. Schelter, J. Guo, J.M. Johnson, L. Lim, J. Karpilow, K. Nichols, W. Marshall, A. Khvorova, P.S. Linsley, Positionspecific chemical modification of siRNAs reduces “off-target” transcript silencing, RNA 12 (2006) 1197–1205. [52] E. Iorns, C.J. Lord, N. Turner, A. Ashworth, Utilizing RNA interference to enhance cancer drug discovery, Nat. Rev. Drug Discov. 6 (2007) 556–568.

Please cite this article as: Y. Zhou, et al., Development of RNAi technology for targeted therapy — A track of siRNA based agents to RNAi therapeutics, J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.04.044

Y. Zhou et al. / Journal of Controlled Release xxx (2014) xxx–xxx [53] R. Kittler, V. Surendranath, A.K. Heninger, M. Slabicki, M. Theis, G. Putz, K. Franke, A. Caldarelli, H. Grabner, K. Kozak, J. Wagner, E. Rees, B. Korn, C. Frenzel, C. Sachse, B. Sonnichsen, J. Guo, J. Schelter, J. Burchard, P.S. Linsley, A.L. Jackson, B. Habermann, F. Buchholz, Genome-wide resources of endoribonuclease-prepared short interfering RNAs for specific loss-of-function studies, Nat. Methods 4 (2007) 337–344. [54] M. Schlee, V. Hornung, G. Hartmann, siRNA and isRNA: two edges of one sword, Mol. Ther. 14 (2006) 463–470. [55] A.L. Jackson, P.S. Linsley, Recognizing and avoiding siRNA off-target effects for target identification and therapeutic application, Nat. Rev. Drug Discov. 9 (2010) 57–67. [56] V. Hornung, M. Guenthner-Biller, C. Bourquin, A. Ablasser, M. Schlee, S. Uematsu, A. Noronha, M. Manoharan, S. Akira, A. de Fougerolles, S. Endres, G. Hartmann, Sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7, Nat. Med. 11 (2005) 263–270. [57] A. Judge, I. MacLachlan, Overcoming the innate immune response to small interfering RNA, Hum. Gene Ther. 19 (2008) 111–124. [58] A.A. Khan, D. Betel, M.L. Miller, C. Sander, C.S. Leslie, D.S. Marks, Transfection of small RNAs globally perturbs gene regulation by endogenous microRNAs, Nat. Biotechnol. 27 (2009) 549–555. [59] S. Shukla, C.S. Sumaria, P.I. Pradeepkumar, Exploring chemical modifications for siRNA therapeutics: a structural and functional outlook, ChemMedChem 5 (2010) 328–349. [60] J.B. Bramsen, J. Kjems, Chemical modification of small interfering RNA, Methods Mol. Biol. 721 (2011) 77–103. [61] S. Choung, Y.J. Kim, S. Kim, H.O. Park, Y.C. Choi, Chemical modification of siRNAs to improve serum stability without loss of efficacy, Biochem. Biophys. Res. Commun. 342 (2006) 919–927. [62] J.B. Bramsen, M.B. Laursen, A.F. Nielsen, T.B. Hansen, C. Bus, N. Langkjaer, B.R. Babu, T. Hojland, M. Abramov, A. Van Aerschot, D. Odadzic, R. Smicius, J. Haas, C. Andree, J. Barman, M. Wenska, P. Srivastava, C. Zhou, D. Honcharenko, S. Hess, E. Muller, G.V. Bobkov, S.N. Mikhailov, E. Fava, T.F. Meyer, J. Chattopadhyaya, M. Zerial, J.W. Engels, P. Herdewijn, J. Wengel, J. Kjems, A large-scale chemical modification screen identifies design rules to generate siRNAs with high activity, high stability and low toxicity, Nucleic Acids Res. 37 (2009) 2867–2881. [63] M. Amarzguioui, J.J. Rossi, Principles of Dicer substrate (D-siRNA) design and function, Methods Mol. Biol. 442 (2008) 3–10. [64] F. Iversen, C.X. Yang, F. Dagnaes-Hansen, D.H. Schaffert, J. Kjems, S. Gao, Optimized siRNA–PEG conjugates for extended blood circulation and reduced urine excretion in mice, Theranostics 3 (2013) 201–209. [65] B.R. Meade, S.F. Dowdy, Exogenous siRNA delivery using peptide transduction domains/cell penetrating peptides, Adv. Drug Deliv. Rev. 59 (2007) 134–140. [66] A.L. Bolcato-Bellemin, M.E. Bonnet, G. Creusat, P. Erbacher, J.P. Behr, Sticky overhangs enhance siRNA-mediated gene silencing, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 16050–16055. [67] A. Schroeder, C.G. Levins, C. Cortez, R. Langer, D.G. Anderson, Lipid-based nanotherapeutics for siRNA delivery, J. Intern. Med. 267 (2010) 9–21. [68] S. Hobel, A. Aigner, Polyethylenimine (PEI)/siRNA-mediated gene knockdown in vitro and in vivo, Methods Mol. Biol. 623 (2010) 283–297. [69] A. Kano, K. Moriyama, T. Yamano, I. Nakamura, N. Shimada, A. Maruyama, Grafting of poly(ethylene glycol) to poly-lysine augments its lifetime in blood circulation and accumulation in tumors without loss of the ability to associate with siRNA, J. Control. Release 149 (2011) 2–7. [70] E. Kawakami, N. Kinouchi, T. Adachi, Y. Ohsawa, N. Ishimaru, H. Ohuchi, Y. Sunada, Y. Hayashi, E. Tanaka, S. Noji, Atelocollagen-mediated systemic administration of myostatin-targeting siRNA improves muscular atrophy in caveolin-3-deficient mice, Dev. Growth Differ. 53 (2011) 48–54. [71] M. Chen, S. Gao, M. Dong, J. Song, C. Yang, K.A. Howard, J. Kjems, F. Besenbacher, Chitosan/siRNA nanoparticles encapsulated in PLGA nanofibers for siRNA delivery, ACS Nano 6 (2012) 4835–4844. [72] U.l. Langel, Cell-penetrating Peptides: Methods and Protocols, Humana Press, New York, NY, 2011. [73] X.X. Liu, P. Rocchi, L. Peng, Dendrimers as non-viral vectors for siRNA delivery, N. J. Chem. 36 (2012) 256–263. [74] J.D. Heidel, T. Schluep, Cyclodextrin-containing polymers: versatile platforms of drug delivery materials, J. Drug Deliv. 2012 (2012) (Article ID 262731). [75] A.W. Tong, C.M. Jay, N. Senzer, P.B. Maples, J. Nemunaitis, Systemic therapeutic gene delivery for cancer: crafting Paris' arrow, Curr. Gene Ther. 9 (2009) 45–60. [76] Y. Wang, Z.G. Li, Y. Han, L.H. Liang, A.M. Ji, Nanoparticle-based delivery system for application of siRNA in vivo, Curr. Drug Metab. 11 (2010) 182–196. [77] S. Hobel, I. Koburger, M. John, F. Czubayko, P. Hadwiger, H.P. Vornlocher, A. Aigner, Polyethylenimine/small interfering RNA-mediated knockdown of vascular endothelial growth factor in vivo exerts anti-tumor effects synergistically with Bevacizumab, J. Gene Med. 12 (2010) 287–300. [78] S. Gao, F. Dagnaes-Hansen, E.J.B. Nielsen, J. Wengel, F. Besenbacher, K.A. Howard, J. Kjems, The effect of chemical modification and nanoparticle formulation on stability and biodistribution of siRNA in mice, Mol. Ther. 17 (2009) 1225–1233. [79] J. Zhou, K.T. Shum, J.C. Burnett, J.J. Rossi, Nanoparticle-based delivery of RNAi therapeutics: progress and challenges, Pharmaceuticals (Basel) 6 (2013) 85–107. [80] X. Liu, J. Wu, M. Yammine, J. Zhou, P. Posocco, S. Viel, C. Liu, F. Ziarelli, M. Fermeglia, S. Pricl, G. Victorero, C. Nguyen, P. Erbacher, J.P. Behr, L. Peng, Structurally flexible triethanolamine core PAMAM dendrimers are effective nanovectors for DNA transfection in vitro and in vivo to the mouse thymus, Bioconjug. Chem. 22 (2011) 2461–2473.

11

[81] J.H. Zhou, J.Y. Wu, N. Hafdi, J.P. Behr, P. Erbacher, L. Peng, PAMAM dendrimers for efficient siRNA delivery and potent gene silencing, Chem. Commun. (2006) 2362–2364. [82] W.E. Rudzinski, T.M. Aminabhavi, Chitosan as a carrier for targeted delivery of small interfering RNA, Int. J. Pharm. 399 (2010) 1–11. [83] H. Katas, H.O. Alpar, Development and characterisation of chitosan nanoparticles for siRNA delivery, J. Control. Release 115 (2006) 216–225. [84] K.A. Howard, S.R. Paludan, M.A. Behlke, F. Besenbacher, B. Deleuran, J. Kjems, Chitosan/siRNA nanoparticle-mediated TNF-alpha knockdown in peritoneal macrophages for anti-inflammatory treatment in a murine arthritis model, Mol. Ther. 17 (2009) 162–168. [85] F. Heitz, M.C. Morris, G. Divita, Twenty years of cell-penetrating peptides: from molecular mechanisms to therapeutics, Br. J. Pharmacol. 157 (2009) 195–206. [86] L. Crombez, G. Aldrian-Herrada, K. Konate, Q.N. Nguyen, G.K. McMaster, R. Brasseur, F. Heitz, G. Divita, A new potent secondary amphipathic cell-penetrating peptide for siRNA delivery into mammalian cells, Mol. Ther. 17 (2009) 95–103. [87] A. Akinc, A. Zumbuehl, M. Goldberg, E.S. Leshchiner, V. Busini, N. Hossain, S.A. Bacallado, D.N. Nguyen, J. Fuller, R. Alvarez, A. Borodovsky, T. Borland, R. Constien, A. de Fougerolles, J.R. Dorkin, K.N. Jayaprakash, M. Jayaraman, M. John, V. Koteliansky, M. Manoharan, L. Nechev, J. Qin, T. Racie, D. Raitcheva, K.G. Rajeev, D.W.Y. Sah, J. Soutschek, I. Toudjarska, H.P. Vornlocher, T.S. Zimmermann, R. Langer, D.G. Anderson, A combinatorial library of lipid-like materials for delivery of RNAi therapeutics, Nat. Biotechnol. 26 (2008) 561–569. [88] K.T. Love, K.P. Mahon, C.G. Levins, K.A. Whitehead, W. Querbes, J.R. Dorkin, J. Qin, W. Cantley, L.L. Qin, T. Racie, M. Frank-Kamenetsky, K.N. Yip, R. Alvarez, D.W.Y. Sah, A. de Fougerolles, K. Fitzgerald, V. Koteliansky, A. Akinc, R. Langer, D.G. Anderson, Lipid-like materials for low-dose, in vivo gene silencing, Proc. Natl. Acad. Sci. U. S. A. 107 (2010) 1864–1869. [89] R. Gref, M. Luck, P. Quellec, M. Marchand, E. Dellacherie, S. Harnisch, T. Blunk, R.H. Muller, ‘Stealth’ corona-core nanoparticles surface modified by polyethylene glycol (PEG): influences of the corona (PEG chain length and surface density) and of the core composition on phagocytic uptake and plasma protein adsorption, Colloids Surf. B Biointerfaces 18 (2000) 301–313. [90] X. Yan, G.L. Scherphof, J.A. Kamps, Liposome opsonization, J. Liposome Res. 15 (2005) 109–139. [91] J.G. Li, D. Cheng, T.H. Yin, W.C. Chen, Y.J. Lin, J.F. Chen, R.T. Li, X.T. Shuai, Copolymer of poly(ethylene glycol) and poly((L)-lysine) grafting polyethylenimine through a reducible disulfide linkage for siRNA delivery, Nanoscale 6 (2014) 1732–1740. [92] S. Stolnik, S.E. Dunn, M.C. Garnett, M.C. Davies, A.G. Coombes, D.C. Taylor, M.P. Irving, S.C. Purkiss, T.F. Tadros, S.S. Davis, et al., Surface modification of poly(lactide-co-glycolide) nanospheres by biodegradable poly(lactide)-poly(ethylene glycol) copolymers, Pharm. Res. 11 (1994) 1800–1808. [93] K. Kostarelos, T.F. Tadros, P.F. Luckham, Physical conjugation of (tri-) block copolymers to liposomes toward the construction of sterically stabilized vesicle systems, Langmuir 15 (1999) 369–376. [94] K. Kostarelos, A.D. Miller, Synthetic, self-assembly ABCD nanoparticles; a structural paradigm for viable synthetic non-viral vectors, Chem. Soc. Rev. 34 (2005) 970–994. [95] J. Wang, Y. Wang, W. Liang, Delivery of drugs to cell membranes by encapsulation in PEG–PE micelles, J. Control. Release 160 (2012) 637–651. [96] T. Musacchio, O. Vaze, G. D'Souza, V.P. Torchilin, Effective stabilization and delivery of siRNA: reversible siRNA–phospholipid conjugate in nanosized mixed polymeric micelles, Bioconjug. Chem. 21 (2010) 1530–1536. [97] N. Tang, G. Du, N. Wang, C. Liu, H. Hang, W. Liang, Improving penetration in tumors with nanoassemblies of phospholipids and doxorubicin, J. Natl. Cancer Inst. 99 (2007) 1004–1015. [98] L. Qin, F. Zhang, X. Lu, X. Wei, J. Wang, X. Fang, D. Si, Y. Wang, C. Zhang, R. Yang, C. Liu, W. Liang, Polymeric micelles for enhanced lymphatic drug delivery to treat metastatic tumors, J. Control. Release 171 (2013) 133–142. [99] C. Zhang, N. Tang, X. Liu, W. Liang, W. Xu, V.P. Torchilin, siRNA-containing liposomes modified with polyarginine effectively silence the targeted gene, J. Control. Release 112 (2006) 229–239. [100] A. Aigner, Nonviral in vivo delivery of therapeutic small interfering RNAs, Curr. Opin. Mol. Ther. 9 (2007) 345–352. [101] J.J. Rossi, RNAi therapeutics: SNALPing siRNAs in vivo, Gene Ther. 13 (2006) 583–584. [102] O.M. Merkel, A. Beyerle, D. Librizzi, A. Pfestroff, T.M. Behr, B. Sproat, P.J. Barth, T. Kissel, Nonviral siRNA delivery to the lung: investigation of PEG–PEI polyplexes and their in vivo performance, Mol. Pharm. 6 (2009) 1246–1260. [103] L.R. Tsai, M.H. Chen, C.T. Chien, M.K. Chen, F.S. Lin, K.M. Lin, Y.K. Hwu, C.S. Yang, S.Y. Lin, A single-monomer derived linear-like PEI-co-PEG for siRNA delivery and silencing, Biomaterials 32 (2011) 3647–3653. [104] S.R. Mao, W. Sun, T. Kissel, Chitosan-based formulations for delivery of DNA and siRNA, Adv. Drug Deliv. Rev. 62 (2010) 12–27. [105] M. Ravina, E. Cubillo, D. Olmeda, R. Novoa-Carballal, E. Fernandez-Megia, R. Riguera, A. Sanchez, A. Cano, M.J. Alonso, Hyaluronic acid/chitosan-g-poly(ethylene glycol) nanoparticles for gene therapy: an application for pDNA and siRNA delivery, Pharm. Res. 27 (2010) 2544–2555. [106] A. Daka, D. Peer, RNAi-based nanomedicines for targeted personalized therapy, Adv. Drug Deliv. Rev. 64 (2012) 1508–1521. [107] J. Shen, R. Samul, R.L. Silva, H. Akiyama, H. Liu, Y. Saishin, S.F. Hackett, S. Zinnen, K. Kossen, K. Fosnaugh, C. Vargeese, A. Gomez, K. Bouhana, R. Aitchison, P. Pavco, P.A. Campochiaro, Suppression of ocular neovascularization with siRNA targeting VEGF receptor 1, Gene Ther. 13 (2006) 225–234. [108] X.Y. Niu, Z.L. Peng, W.Q. Duan, H. Wang, P. Wang, Inhibition of HPV 16 E6 oncogene expression by RNA interference in vitro and in vivo, Int. J. Gynecol. Cancer 16 (2006) 743–751.

Please cite this article as: Y. Zhou, et al., Development of RNAi technology for targeted therapy — A track of siRNA based agents to RNAi therapeutics, J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.04.044

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[109] S.M. Tompkins, C.Y. Lo, T.M. Tumpey, S.L. Epstein, Protection against lethal influenza virus challenge by RNA interference in vivo, Proc. Natl. Acad. Sci. U. S. A. 101 (2004) 8682–8686. [110] P.H. Tan, L.C. Yang, H.C. Shih, K.C. Lan, J.T. Cheng, Gene knockdown with intrathecal siRNA of NMDA receptor NR2B subunit reduces formalin-induced nociception in the rat, Gene Ther. 12 (2005) 59–66. [111] M.A. Tran, R. Gowda, A. Sharma, E.J. Park, J. Adair, M. Kester, N.B. Smith, G.P. Robertson, Targeting B-V600E-Raf and AW using nanoliposomal-small interfering RNA inhibits cutaneous melanocytic lesion development, Cancer Res. 68 (2008) 7638–7649. [112] S. David, B. Pitard, J.P. Benoit, C. Passirani, Non-viral nanosystems for systemic siRNA delivery, Pharmacol. Res. 62 (2010) 100–114. [113] A.K. Iyer, G. Khaled, J. Fang, H. Maeda, Exploiting the enhanced permeability and retention effect for tumor targeting, Drug Discov. Today 11 (2006) 812–818. [114] T. Ishimoto, Y. Takei, Y. Yuzawa, K. Hanai, S. Nagahara, Y. Tarumi, S. Matsuo, K. Kadomatsu, Downregulation of monocyte chemoattractant protein-1 involving short interfering RNA attenuates hapten-induced contact hypersensitivity, Mol. Ther. 16 (2008) 387–395. [115] E. Kawata, E. Ashihara, S. Kimura, K. Takenaka, K. Sato, R. Tanaka, A. Yokota, Y. Kamitsuji, M. Takeuchi, J. Kuroda, F. Tanaka, T. Yoshikawa, T. Maekawa, Administration of PLK-1 small interfering RNA with atelocollagen prevents the growth of liver metastases of lung cancer, Mol. Cancer Ther. 7 (2008) 2904–2912. [116] T.M. Allen, P.R. Cullis, Liposomal drug delivery systems: from concept to clinical applications, Adv. Drug Deliv. Rev. 65 (2013) 36–48. [117] T.S. Zimmermann, A.C. Lee, A. Akinc, B. Bramlage, D. Bumcrot, M.N. Fedoruk, J. Harborth, J.A. Heyes, L.B. Jeffs, M. John, A.D. Judge, K. Lam, K. McClintock, L.V. Nechev, L.R. Palmer, T. Racie, I. Rohl, S. Seiffert, S. Shanmugam, V. Sood, J. Soutschek, I. Toudjarska, A.J. Wheat, E. Yaworski, W. Zedalis, V. Koteliansky, M. Manoharan, H.P. Vornlocher, I. MacLachlan, RNAi-mediated gene silencing in non-human primates, Nature 441 (2006) 111–114. [118] R.O. Hynes, A reevaluation of integrins as regulators of angiogenesis, Nat. Med. 8 (2002) 918–921. [119] R.M. Schiffelers, A. Ansari, J. Xu, Q. Zhou, Q. Tang, G. Storm, G. Molema, P.Y. Lu, P.V. Scaria, M.C. Woodle, Cancer siRNA therapy by tumor selective delivery with ligand-targeted sterically stabilized nanoparticle, Nucleic Acids Res. 32 (2004) e149. [120] X.F. Zheng, C. Vladau, X.S. Zhang, M. Suzuki, T.E. Ichim, Z.X. Zhang, M. Li, E. Carrier, B. Garcia, A.M. Jevnikar, W.P. Min, A novel in vivo siRNA delivery system specifically targeting dendritic cells and silencing CD40 genes for immunomodulation, Blood 113 (2009) 2646–2654. [121] E.W. Song, P.C. Zhu, S.K. Lee, D. Chowdhury, S. Kussman, D.M. Dykxhoorn, Y. Feng, D. Palliser, D.B. Weiner, P. Shankar, W.A. Marasco, J. Lieberman, Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors, Nat. Biotechnol. 23 (2005) 709–717. [122] P. Kumar, H.S. Ban, S.S. Kim, H.Q. Wu, T. Pearson, D.L. Greiner, A. Laouar, J.H. Yao, V. Haridas, K. Habiro, Y.G. Yang, J.H. Jeong, K.Y. Lee, Y.H. Kim, S.W. Kim, M. Peipp, G.H. Fey, N. Manjunath, L.D. Shultz, S.K. Lee, P. Shankar, T cell-specific siRNA delivery suppresses HIV-1 infection in humanized mice, Cell 134 (2008) 577–586. [123] P. Kumar, H.Q. Wu, J.L. McBride, K.E. Jung, M.H. Kim, B.L. Davidson, S.K. Lee, P. Shankar, N. Manjunath, Transvascular delivery of small interfering RNA to the central nervous system, Nature 448 (2007) 39–43. [124] J.Y. Yhee, S.J. Lee, S. Lee, S. Song, H.S. Min, S.W. Kang, S. Son, S.Y. Jeong, I.C. Kwon, S.H. Kim, K. Kim, Tumor-targeting transferrin nanoparticles for systemic polymerized siRNA delivery in tumor-bearing mice, Bioconjug. Chem. 24 (2013) 1850–1860. [125] G.Y. Xiang, J. Wu, Y.H. Lu, Z.L. Liu, R.J. Lee, Synthesis and evaluation of a novel ligand for folate-mediated targeting liposomes, Int. J. Pharm. 356 (2008) 29–36. [126] D. Peer, J.M. Karp, S. Hong, O.C. FaroKHzad, R. Margalit, R. Langer, Nanocarriers as an emerging platform for cancer therapy, Nat. Nanotechnol. 2 (2007) 751–760. [127] X. Li, Q.H. Zhao, L.Y. Qiu, Smart ligand: aptamer-mediated targeted delivery of chemotherapeutic drugs and siRNA for cancer therapy, J. Control. Release 171 (2013) 152–162. [128] Y.D. Yao, T.M. Sun, S.Y. Huang, S. Dou, L. Lin, J.N. Chen, J.B. Ruan, C.Q. Mao, F.Y. Yu, M.S. Zeng, J.Y. Zang, Q. Liu, F.X. Su, P. Zhang, J. Lieberman, J. Wang, E. Song, Targeted

[129] [130]

[131]

[132] [133] [134]

[135] [136]

[137]

[138]

[139] [140] [141] [142]

[143]

[144]

[145]

[146] [147]

[148]

delivery of PLK1–siRNA by ScFv suppresses Her2+ breast cancer growth and metastasis, Sci. Transl. Med. 4 (2012) 130ra148. S.S. Kim, S. Subramanya, D. Peer, M. Shimaoka, P. Shankar, Antibody-mediated delivery of siRNAs for anti-HIV therapy, Methods Mol. Biol. 721 (2011) 339–353. M.E. Davis, J.E. Zuckerman, C.H. Choi, D. Seligson, A. Tolcher, C.A. Alabi, Y. Yen, J.D. Heidel, A. Ribas, Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles, Nature 464 (2010) 1067–1070. J.D. Heidel, Z. Yu, J.Y. Liu, S.M. Rele, Y. Liang, R.K. Zeidan, D.J. Kornbrust, M.E. Davis, Administration in non-human primates of escalating intravenous doses of targeted nanoparticles containing ribonucleotide reductase subunit M2 siRNA, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 5715–5721. A.C. Yan, M. Levy, Aptamers and aptamer targeted delivery, RNA Biol. 6 (2009) 316–320. A. Daka, D. Peer, RNAi-based nanomedicines for targeted personalized therapy, Adv. Drug Deliv. Rev. 64 (2012) 1508–1521. J.P. Dassie, X.Y. Liu, G.S. Thomas, R.M. Whitaker, K.W. Thiel, K.R. Stockdale, D.K. Meyerholz, A.P. McCaffrey, J.O. McNamara II, P.H. Giangrande, Systemic administration of optimized aptamer–siRNA chimeras promotes regression of PSMAexpressing tumors, Nat. Biotechnol. 27 (2009) 839–849. D. Hymel, B.R. Peterson, Synthetic cell surface receptors for delivery of therapeutics and probes, Adv. Drug Deliv. Rev. 64 (2012) 797–810. H. Arima, A. Yoshimatsu, H. Ikeda, A. Ohyama, K. Motoyama, T. Higashi, A. Tsuchiya, T. Niidome, Y. Katayama, K. Hattori, T. Takeuchi, Folate-PEG-appended dendrimer conjugate with alpha-cyclodextrin as a novel cancer cell-selective siRNA delivery carrier, Mol. Pharm. 9 (2012) 2591–2604. M. Oishi, Y. Nagasaki, N. Nishiyama, K. Itaka, M. Takagi, A. Shimamoto, Y. Furuichi, K. Kataoka, Enhanced growth inhibition of hepatic multicellular tumor spheroids by lactosylated poly(ethylene glycol)-siRNA conjugate formulated in PEGylated polyplexes, ChemMedChem 2 (2007) 1290–1297. W. Zhou, X. Yuan, A. Wilson, L. Yang, M. Mokotoff, B. Pitt, S. Li, Efficient intracellular delivery of oligonucleotides formulated in folate receptor-targeted lipid vesicles, Bioconjug. Chem. 13 (2002) 1220–1225. A.T. Jones, E.J. Sayers, Cell entry of cell penetrating peptides: tales of tails wagging dogs, J. Control. Release 161 (2012) 582–591. B. Gupta, V.P. Torchilin, Transactivating transcriptional activator-mediated drug delivery, Expert Opin. Drug Deliv. 3 (2006) 177–190. C. Lin, J.F. Engbersen, Effect of chemical functionalities in poly(amido amine)s for non-viral gene transfection, J. Control. Release 132 (2008) 267–272. N. Nishiyama, Arnida, W.D. Jang, K. Date, K. Miyata, K. Kataoka, Photochemical enhancement of transgene expression by polymeric micelles incorporating plasmid DNA and dendrimer-based photosensitizer, J. Drug Target. 14 (2006) 413–424. P.J. Lou, P.S. Lai, M.J. Shieh, A.J. Macrobert, K. Berg, S.G. Bown, Reversal of doxorubicin resistance in breast cancer cells by photochemical internalization, Int. J. Cancer 119 (2006) 2692–2698. K. Sasaki, K. Kogure, S. Chaki, Y. Nakamura, R. Moriguchi, H. Hamada, R. Danev, K. Nagayama, S. Futaki, H. Harashima, An artificial virus-like nano carrier system: enhanced endosomal escape of nanoparticles via synergistic action of pH-sensitive fusogenic peptide derivatives, Anal. Bioanal. Chem. 391 (2008) 2717–2727. H. Hatakeyama, E. Ito, H. Akita, M. Oishi, Y. Nagasaki, S. Futaki, H. Harashima, A pHsensitive fusogenic peptide facilitates endosomal escape and greatly enhances the gene silencing of siRNA-containing nanoparticles in vitro and in vivo, J. Control. Release 139 (2009) 127–132. J.C. Burnett, J.J. Rossi, K. Tiemann, Current progress of siRNA/shRNA therapeutics in clinical trials, Biotechnol. J. 6 (2011) 1130–1146. D.L. Stirland, J.W. Nichols, S. Miura, Y.H. Bae, Mind the gap: a survey of how cancer drug carriers are susceptible to the gap between research and practice, J. Control. Release 172 (2013) 1045–1064. T. Coelho, D. Adams, A. Silva, P. Lozeron, P.N. Hawkins, T. Mant, J. Perez, J. Chiesa, S. Warrington, E. Tranter, M. Munisamy, R. Falzone, J. Harrop, J. Cehelsky, B.R. Bettencourt, M. Geissler, J.S. Butler, A. Sehgal, R.E. Meyers, Q.M. Chen, T. Borland, R.M. Hutabarat, V.A. Clausen, R. Alvarez, K. Fitzgerald, C. Gamba-Vitalo, S.V. Nochur, A.K. Vaishnaw, D.W.Y. Sah, J.A. Gollob, O.B. Suhr, Safety and efficacy of RNAi therapy for transthyretin amyloidosis, N. Engl. J. Med. 369 (2013) 819–829.

Please cite this article as: Y. Zhou, et al., Development of RNAi technology for targeted therapy — A track of siRNA based agents to RNAi therapeutics, J. Control. Release (2014), http://dx.doi.org/10.1016/j.jconrel.2014.04.044