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Hydrophobization and Bioconjugation for Enhanced Sirna Delivery and Targeting

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Hydrophobization and Bioconjugation for Enhanced Sirna Delivery and Targeting JOBNAME: RNA 13#4 2007 PAGE: 1 OUTPUT: Saturday February 24 04:28:17 2007 csh/RNA/131733/rna4598 Downloaded from rnajournal.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press REVIEW Hydrophobization and bioconjugation for enhanced siRNA delivery and targeting DANIEL DE PAULA,1,2 M. VITO´ RIA L.B. BENTLEY,1 and RAM I. MAHATO2 1Faculdade de Cieˆncias Farmaceˆuticas de Ribeira˜o Preto, Universidade de Sa˜o Paulo, Ribeira˜o Preto, SP, Brazil 2Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, Memphis, Tennessee 38163, USA ABSTRACT RNA interference (RNAi) is an evolutionarily conserved process by which double-stranded small interfering RNA (siRNA) induces sequence-specific, post-transcriptional gene silencing. Unlike other mRNA targeting strategies, RNAi takes advantage of the physiological gene silencing machinery. The potential use of siRNA as therapeutic agents has attracted great attention as a novel approach for treating severe and chronic diseases. RNAi can be achieved by either delivery of chemically synthesized siRNAs or endogenous expression of small hairpin RNA, siRNA, and microRNA (miRNA). However, the relatively high dose of siRNA required for gene silencing limits its therapeutic applications. This review discusses several strategies to improve therapeutic efficacy as well as to abrogate off-target effects and immunostimulation caused by siRNAs. There is an in-depth discussion on various issues related to the (1) mechanisms of RNAi, (2) methods of siRNA production, (3) barriers to RNAi-based therapies, (4) biodistribution, (5) design of siRNA molecules, (6) chemical modification and bioconjugation, (7) complex formation with lipids and polymers, (8) encapsulation into lipid particles, and (9) target specificity for enhanced therapeutic effectiveness. Keywords: RNA interference; small interfering RNA; bioconjugation; chemical modification; complex formation INTRODUCTION The revolutionary finding of RNAi resulted from the work of Andrew Fire and coworkers at the Carnegie RNA interference (RNAi) is an evolutionarily conserved Institute in Washington, D.C., who demonstrated in 1998 process by which double-stranded small interfering RNA that injection of dsRNA into Caenorhabditis elegans leads to (siRNA) induces sequence-specific, post-transcriptional efficient sequence-specific gene silencing (Fire et al. 1998). gene silencing (Hannon 2002). The process of RNAi At that time, the state of the art in gene silencing was the consists of an initiator step, in which long double-stranded use of antisense oligodeoxynucleotide (ODNs), which RNA (dsRNA) is cleaved into siRNA fragments, and an comprise single strands of short DNA or RNA comple- effector step, in which these fragments are incorporated mentary sequences that hybridize with the targeted mRNA into a protein complex, dissociated, and used as a guiding (Mahato et al. 2005). However, the dsRNA seemed to sequence to recognize homologous mRNA that is sub- induce silencing through a pathway distinct from classical sequently cleaved. RNAi is considered a self-defense mech- antisense therapies due to the catalytic nature of RNAi, in anism of eukaryotic cells to combat infection by RNA which one siRNA can be used over and over to guide the viruses and transposons. It is also assumed to tightly cleavage of many mRNA molecules (Dykxhoorn et al. regulate protein levels in response to various environmental 2003). Bertrand et al. (2002) have compared the effects of stimuli (Meister and Tuschl 2004). antisense ODNs and siRNAs targeting green fluorescent protein (GFP) in vitro and in vivo. siRNA was quantita- tively more efficient, and its effect lasted for a longer time Abbreviations: See list at end of paper. Reprint requests to: Ram I. Mahato, Department of Pharmaceutical in cell culture. In mice, siRNAs were able to silence gene Sciences, Health Science Center, University of Tennessee, 26 South Dunlap expression, whereas no effect was observed in the presence Street, Feurt Building, Room 413, Memphis, TN 38163, USA; e-mail: of antisense ODNs. [email protected]; fax: (901) 448-6092. Article published online ahead of print. Article and publication date are The first evidence that siRNAs can mediate sequence- at http://www.rnajournal.org/cgi/doi/10.1261/rna.459807. specific gene silencing in mammalian cells was provided in RNA (2007), 13:1–26. Published by Cold Spring Harbor Laboratory Press. Copyright Ó 2007 RNA Society. 1 rna4598 De Paula et al. REVIEW RA JOBNAME: RNA 13#4 2007 PAGE: 2 OUTPUT: Saturday February 24 04:28:18 2007 csh/RNA/131733/rna4598 Downloaded from rnajournal.cshlp.org on October 4, 2021 - Published by Cold Spring Harbor Laboratory Press De Paula et al. 2001 when the conversion of dsRNA into short RNA recognition by other RNAi components. Dicer is the fragments was shown to be bypassed by the transfection enzyme known to process dsRNA into siRNAs, and it of siRNA molecules into cells (Elbashir et al. 2001a). After contains dual catalytic domains and additional helicase and that, various in vivo effects of siRNA and short hairpin PAZ domains (Elbashir et al. 2001b; Zamore and Haley RNA (shRNA) have been reported (Lewis et al. 2002; 2005). Processed siRNA is incorporated into a protein McCaffrey et al. 2002; Xia et al. 2002; Song et al. 2003b). complex, termed RNA-induced silencing complex (RISC). For example, McCaffrey et al. (2002) showed that siRNA Dicer is also involved in the early steps of RISC formation and shRNA reduce luciferase (Luc) expression in the liver and may be required for siRNA entry into RISC (Lee et al. in a sequence-specific manner. Song et al. (2003b) found 2004). In mammalian cells, the protein Argonaute 2 (Ago2) that siRNA targeted to the Fas receptor protects mice from is the catalytic component of RISC that cleaves target liver fibrosis. mRNAs (Liu et al. 2004b; Meister et al. 2004). In the RISC Human clinical trials of RNAi-based drugs are currently assembly process, siRNAs are initially loaded into Ago2 as under way by Acuity Pharmaceuticals and Sirna Therapeu- duplexes, and then Ago2 cleaves the passenger strand, tics. Both companies are working on intravitreal adminis- thereby liberating the guide strand from the siRNA duplex tration of siRNA targeting vascular endothelial growth and producing active RISC capable of cleaving target factor (VEGF), whose overexpression is the primary cause mRNAs (Matranga et al. 2005; Leuschner et al. 2006). of age-related macular degeneration (AMD). The first The guide strand serves as a template for the recognition results are encouraging in terms of tolerability of siRNA of homologous mRNA, which upon binding to RISC is compounds. Other clinical trials involving siRNA for cleaved between bases 10 and 11 relative to the 59 base of treating chronic myeloid leukemia and respiratory syncytial the guide siRNA strand by the catalytic activity of Ago2 virus infection are also being carried out by the Hadassah (Elbashir et al. 2001c). The template siRNA is not affected Medical Organization and Alnylam Pharmaceutics, Inc., by this reaction, so the RISC can undergo numerous cycles respectively. of mRNA cleavage that comprise the high efficiency of Despite early excitement, siRNAs have shown to activate RNAi (Elbashir et al. 2001b; Meister and Tuschl 2004). The immune response in a sequence- and concentration-depen- mechanism by which passenger strands are cleaved by Ago2 dent manner, leading to nonspecific gene silencing (Jackson follows the same rules established for the siRNA-guided et al. 2003; Sioud 2005). In this respect, the introduction of cleavage of a target mRNA (Leuschner et al. 2006). chemical modifications and generation of designed siRNAs RNAi can also be induced by endogenous expression of have become essential for achieving high gene silencing small regulatory RNAs known as microRNAs (miRNAs) with a low degree of undesired effects. Chemical modifica- (Fig. 1). The generation of miRNAs occurs via sequential tions of siRNAs also appear to stabilize these molecules in processing and maturation of long primary transcripts (pri- serum and show enhanced gene silencing (Braasch et al. miRNA). Pre-microRNA (pre-miRNA) exits in the nucleus 2003; Chiu and Rana 2003; Layzer et al. 2004). A break- upon cleavage by Drosha and is recognized by the endo- through in this area has recently been achieved by linking nuclease Dicer, which processes the pre-miRNA into a cholesterol (Chol) to siRNAs (Soutschek et al. 2004), and 22-nt mature miRNA (Bartel 2004). The mature miRNA is by encapsulating them into stable nucleic acid lipid subsequently incorporated into the silencing complex particles (Zimmermann et al. 2006). Undoubtedly, effective (RISC), where it mediates translational repression. During in vivo delivery of siRNAs will be a key factor in turning RISC assembly, the Ago2-mediated cleavage of the passen- siRNA into a new class of therapeutics. In this review, we ger strand is blocked by mismatches between the guide discuss several strategies to abrogate undesired effects and and passenger strands of miRNAs. In this case, a slower improve the efficiency of siRNA, including chemical modifi- ‘‘backup’’ pathway dissociates and destroys the passenger cations, siRNA sequence design, bioconjugation, complex strand, liberating active RISC (Matranga et al. 2005; formation, and encapsulation into lipid particles. Leuschner et al. 2006). In contrast to siRNA, miRNAs medi- ate RNAi through a translational inhibition mechanism involving imperfect complementarity
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