Therapeutic siRNA: Principles, Challenges, and Strategies

RNA interference (RNAi) is a remarkable endogenous regulatory pathway that can bring about sequence-specific gene silencing. If harnessed effectively, RNAi could result in a potent targeted therapeutic modality with applications ranging from viral diseases to cancer. The major barrier to realizing the full medicinal potential of RNAi is the difficulty of delivering effector molecules, such as small interfering RNAs (siRNAs), in vivo. An effective delivery strategy for siRNAs must address limitations that include poor stability and non-targeted biodistribution, while protecting against the stimulation of an undesirable innate immune response. The design of such a system requires rigorous understanding of all mechanisms involved. This article reviews the mechanistic principles of RNA interference, its potential, the greatest challenges for use in biomedical applications, and some of the work that has been done toward engineering delivery systems that overcome some of the hurdles facing siRNA-based therapeutics.

[1]  Anil K Sood,et al.  Therapeutic EphA2 gene targeting in vivo using neutral liposomal small interfering RNA delivery. , 2005, Cancer research.

[2]  M. Zavolan,et al.  Strand-specific 5'-O-methylation of siRNA duplexes controls guide strand selection and targeting specificity. , 2007, RNA.

[3]  S. Akira,et al.  Sequence-specific potent induction of IFN-α by short interfering RNA in plasmacytoid dendritic cells through TLR7 , 2005, Nature Medicine.

[4]  M. Manoharan.  RNA interference and chemically modified small interfering RNAs. , 2004, Current opinion in chemical biology.

[5]  C. Noe,et al.  A proteomic study reveals unspecific apoptosis induction and reduction of glycolytic enzymes by the phosphorothioate antisense oligonucleotide oblimersen in human melanoma cells. , 2009, Journal of proteomics.

[6]  T. Allen.  Ligand-targeted therapeutics in anticancer therapy , 2002, Nature Reviews Cancer.

[7]  R. Schiffelers,et al.  Transporting silence: design of carriers for siRNA to angiogenic endothelium. , 2005, Journal of controlled release : official journal of the Controlled Release Society.

[8]  P. Zamore,et al.  ATP Requirements and Small Interfering RNA Structure in the RNA Interference Pathway , 2001, Cell.

[9]  Anastasia Khvorova,et al.  3′ UTR seed matches, but not overall identity, are associated with RNAi off-targets , 2006, Nature Methods.

[10]  B. Simon,et al.  Structure and nucleic-acid binding of the Drosophila Argonaute 2 PAZ domain , 2003, Nature.

[11]  A. Fire,et al.  Functional anatomy of a dsRNA trigger: differential requirement for the two trigger strands in RNA interference. , 2000, Molecular cell.

[12]  P. Opolon,et al.  Comparison of antisense oligonucleotides and siRNAs in cell culture and in vivo. , 2002, Biochemical and biophysical research communications.

[13]  M. Woodle,et al.  Harnessing in vivo siRNA delivery for drug discovery and therapeutic development , 2006, Drug Discovery Today.

[14]  K. Kataoka,et al.  Block copolymer micelles for drug delivery: design, characterization and biological significance. , 2001, Advanced drug delivery reviews.

[15]  Matthias John,et al.  Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs , 2004, Nature.

[16]  S. Hammond.  MicroRNA therapeutics: a new niche for antisense nucleic acids. , 2006, Trends in molecular medicine.

[17]  V. Torchilin,et al.  siRNA-containing liposomes modified with polyarginine effectively silence the targeted gene , 2006, Journal of Controlled Release.

[18]  Sha Jin,et al.  Nanoparticle-mediated gene delivery. , 2009, Methods in molecular biology.

[19]  Mark E. Davis,et al.  Sequence-specific knockdown of EWS-FLI1 by targeted, nonviral delivery of small interfering RNA inhibits tumor growth in a murine model of metastatic Ewing's sarcoma. , 2005, Cancer research.

[20]  F. Eckstein,et al.  Kinetic characterization of ribonuclease-resistant 2'-modified hammerhead ribozymes. , 1991, Science.

[21]  Anastasia Khvorova,et al.  Addendum: 3′ UTR seed matches, but not overall identity, are associated with RNAi off-targets , 2006 .

[22]  K. Ui-Tei,et al.  Functional dissection of siRNA sequence by systematic DNA substitution: modified siRNA with a DNA seed arm is a powerful tool for mammalian gene silencing with significantly reduced off-target effect , 2008, Nucleic acids research.

[23]  F. Szoka,et al.  Lipid-based Nanoparticles for Nucleic Acid Delivery , 2007, Pharmaceutical Research.

[24]  M. Cristea,et al.  Polymeric micelles for oral drug delivery: Why and how , 2004 .

[25]  T. Tuschl,et al.  Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate , 2001, The EMBO journal.

[26]  A. Reynolds,et al.  Rational siRNA design for RNA interference , 2004, Nature Biotechnology.

[27]  W. Filipowicz,et al.  The widespread regulation of microRNA biogenesis, function and decay , 2010, Nature Reviews Genetics.

[28]  Shan Jiang,et al.  Quantum-dot based nanoparticles for targeted silencing of HER2/neu gene via RNA interference. , 2007, Biomaterials.

[29]  S. Betigeri,et al.  Surface-modified and internally cationic polyamidoamine dendrimers for efficient siRNA delivery. , 2008, Bioconjugate chemistry.

[30]  W. Saltzman,et al.  Octa-functional PLGA nanoparticles for targeted and efficient siRNA delivery to tumors. , 2012, Biomaterials.

[31]  M. Behlke.  Progress towards in Vivo Use of siRNAs , 2006, Molecular Therapy.

[32]  Matthias John,et al.  Steroid and lipid conjugates of siRNAs to enhance cellular uptake and gene silencing in liver cells. , 2004, Bioorganic & medicinal chemistry letters.

[33]  K. Roy,et al.  Efficient gene silencing in lungs and liver using imidazole-modified chitosan as a nanocarrier for small interfering RNA. , 2010, Oligonucleotides.

[34]  Jay Z. Parrish,et al.  Functional genomic analysis of apoptotic DNA degradation in C. elegans. , 2003, Molecular cell.

[35]  T. Tuschl,et al.  RNA interference is mediated by 21- and 22-nucleotide RNAs. , 2001, Genes & development.

[36]  P. Sharp,et al.  Functional Delivery of siRNA in Mice Using Dendriworms , 2009, ACS nano.

[37]  A. Aigner,et al.  RNAi-mediated gene-targeting through systemic application of polyethylenimine (PEI)-complexed siRNA in vivo , 2005, Gene Therapy.

[38]  Anna Moore,et al.  In vivo imaging of siRNA delivery and silencing in tumors , 2007, Nature Medicine.

[39]  P. Guo,et al.  Construction of folate-conjugated pRNA of bacteriophage phi29 DNA packaging motor for delivery of chimeric siRNA to nasopharyngeal carcinoma cells , 2006, Gene Therapy.

[40]  Thomas Tuschl,et al.  Structural basis for 5′-end-specific recognition of guide RNA by the A. fulgidus Piwi protein , 2005, Nature.

[41]  H. Maeda.  The enhanced permeability and retention (EPR) effect in tumor vasculature: the key role of tumor-selective macromolecular drug targeting. , 2001, Advances in enzyme regulation.

[42]  K. Alexander,et al.  RNA interference using boranophosphate siRNAs: structure-activity relationships. , 2004, Nucleic acids research.

[43]  R. G. Anderson,et al.  Filipin-cholesterol complexes form in uncoated vesicle membrane derived from coated vesicles during receptor-mediated endocytosis of low density lipoprotein , 1983, The Journal of cell biology.

[44]  Hua Ai,et al.  N-Alkyl-PEI-functionalized iron oxide nanoclusters for efficient siRNA delivery. , 2011, Small.

[45]  Xiao-rong Li,et al.  Research progress on siRNA delivery with nonviral carriers , 2011, International journal of nanomedicine.

[46]  S. Hammond,et al.  An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells , 2000, Nature.

[47]  G. Hannon,et al.  Crystal Structure of Argonaute and Its Implications for RISC Slicer Activity , 2004, Science.

[48]  Christopher J. Cheng,et al.  Enhanced siRNA delivery into cells by exploiting the synergy between targeting ligands and cell-penetrating peptides. , 2011, Biomaterials.

[49]  H. Maeda,et al.  Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review. , 2000, Journal of controlled release : official journal of the Controlled Release Society.

[50]  Anton P. McCaffrey,et al.  Systemic administration of optimized aptamer-siRNA chimeras promotes regression of PSMA-expressing tumors , 2009, Nature Biotechnology.

[51]  Kenneth A Howard,et al.  RNA interference in vitro and in vivo using a novel chitosan/siRNA nanoparticle system. , 2006, Molecular therapy : the journal of the American Society of Gene Therapy.

[52]  J. Doudna,et al.  A three-dimensional view of the molecular machinery of RNA interference , 2009, Nature.

[53]  Robert Langer,et al.  A combinatorial library of lipid-like materials for delivery of RNAi therapeutics , 2008, Nature Biotechnology.

[54]  L. Lim,et al.  Widespread siRNA "off-target" transcript silencing mediated by seed region sequence complementarity. , 2006, RNA.

[55]  Daniel G. Anderson,et al.  Synergistic silencing: combinations of lipid-like materials for efficacious siRNA delivery. , 2011, Molecular therapy : the journal of the American Society of Gene Therapy.

[56]  S. Agrawal,et al.  Biodistribution and metabolism of a mixed backbone oligonucleotide (GEM 231) following single and multiple dose administration in mice. , 2000, Antisense & nucleic acid drug development.

[57]  T. Du,et al.  Asymmetry in the Assembly of the RNAi Enzyme Complex , 2003, Cell.

[58]  Mark E. Davis,et al.  Administration in non-human primates of escalating intravenous doses of targeted nanoparticles containing ribonucleotide reductase subunit M2 siRNA , 2007, Proceedings of the National Academy of Sciences.

[59]  B. Li,et al.  Expression profiling reveals off-target gene regulation by RNAi , 2003, Nature Biotechnology.

[60]  P. Low,et al.  Delivery of liposomes into cultured KB cells via folate receptor-mediated endocytosis. , 1994, The Journal of biological chemistry.

[61]  T. Minko,et al.  Surface-engineered targeted PPI dendrimer for efficient intracellular and intratumoral siRNA delivery. , 2009, Journal of controlled release : official journal of the Controlled Release Society.

[62]  M. Amarzguioui,et al.  An algorithm for selection of functional siRNA sequences. , 2004, Biochemical and biophysical research communications.

[63]  P. Fong,et al.  PEGylated PLGA nanoparticles for the improved delivery of doxorubicin. , 2009, Nanomedicine : nanotechnology, biology, and medicine.

[64]  H. Soifer,et al.  siRNA target site secondary structure predictions using local stable substructures , 2005, Nucleic acids research.

[65]  T. Tuschl,et al.  Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells , 2001, Nature.

[66]  P. Low,et al.  Tumor-selective radiopharmaceutical targeting via receptor-mediated endocytosis of gallium-67-deferoxamine-folate. , 1996, Journal of nuclear medicine : official publication, Society of Nuclear Medicine.

[67]  E. Sontheimer,et al.  Origins and Mechanisms of miRNAs and siRNAs , 2009, Cell.

[68]  J. Rossi,et al.  RNAi therapeutics: principles, prospects and challenges. , 2007, Advanced drug delivery reviews.

[69]  D. Barford,et al.  Structural insights into mRNA recognition from a PIWI domain–siRNA guide complex , 2005, Nature.

[70]  Ji-Joon Song,et al.  The crystal structure of the Argonaute2 PAZ domain reveals an RNA binding motif in RNAi effector complexes , 2003, Nature Structural Biology.

[71]  Peixuan Guo,et al.  Engineering RNA for Targeted siRNA Delivery and Medical Application , 2010, Advanced Drug Delivery Reviews.

[72]  Martin J. Simard,et al.  Argonaute proteins: key players in RNA silencing , 2008, Nature Reviews Molecular Cell Biology.

[73]  G. Sczakiel,et al.  Phosphorothioate‐stimulated uptake of short interfering RNA by human cells , 2005, EMBO reports.

[74]  Frank Baas,et al.  Evaluation of locked nucleic acid–modified small interfering RNA in vitro and in vivo , 2007, Molecular Cancer Therapeutics.

[75]  M. Carmell,et al.  Posttranscriptional Gene Silencing in Plants , 2006 .

[76]  M. D. Blanco,et al.  Targeted Nanoparticles for Cancer Therapy , 2012 .

[77]  Y. Maitani,et al.  Folate-linked lipid-based nanoparticles for synthetic siRNA delivery in KB tumor xenografts. , 2008, European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V.

[78]  C. Wahlestedt,et al.  Locked nucleic acid (LNA) mediated improvements in siRNA stability and functionality , 2005, Nucleic acids research.

[79]  F. Behm,et al.  Differentiation-independent retinoid induction of folate receptor type beta, a potential tumor target in myeloid leukemia. , 2000, Blood.

[80]  A. Pasquinelli,et al.  Regulation by let-7 and lin-4 miRNAs Results in Target mRNA Degradation , 2005, Cell.

[81]  T. Rana,et al.  RNAi in human cells: basic structural and functional features of small interfering RNA. , 2002, Molecular cell.

[82]  G. Rettig,et al.  Progress Toward In Vivo Use of siRNAs-II , 2006, Molecular Therapy.

[83]  W. Saltzman,et al.  High loading efficiency and tunable release of plasmid DNA encapsulated in submicron particles fabricated from PLGA conjugated with poly-L-lysine. , 2008, Journal of controlled release : official journal of the Controlled Release Society.

[84]  V. Patzel,et al.  Design of siRNAs producing unstructured guide-RNAs results in improved RNA interference efficiency , 2005, Nature Biotechnology.

[85]  Mark E. Davis,et al.  Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles , 2010, Nature.

[86]  Anna Moore,et al.  In Vivo Targeting of Underglycosylated MUC-1 Tumor Antigen Using a Multimodal Imaging Probe , 2004, Cancer Research.

[87]  P. D. Cook,et al.  Uniformly modified 2'-deoxy-2'-fluoro phosphorothioate oligonucleotides as nuclease-resistant antisense compounds with high affinity and specificity for RNA targets. , 1993, Journal of medicinal chemistry.

[88]  A. Caudy,et al.  Role for a bidentate ribonuclease in the initiation step of RNA interference , 2001 .

[89]  R. Shiekhattar,et al.  Human RISC Couples MicroRNA Biogenesis and Posttranscriptional Gene Silencing , 2005, Cell.

[90]  Anastasia Khvorova,et al.  Induction of the interferon response by siRNA is cell type- and duplex length-dependent. , 2006, RNA.

[91]  P. Sharp,et al.  RNAi Double-Stranded RNA Directs the ATP-Dependent Cleavage of mRNA at 21 to 23 Nucleotide Intervals , 2000, Cell.

[92]  Han-Oh Park,et al.  Chemical modification of siRNAs to improve serum stability without loss of efficacy. , 2006, Biochemical and biophysical research communications.

[93]  Judy Lieberman,et al.  Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors , 2005, Nature Biotechnology.

[94]  J. M. Thomson,et al.  Argonaute2 Is the Catalytic Engine of Mammalian RNAi , 2004, Science.

[95]  Sangdun Choi,et al.  Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy , 2005, Nature Biotechnology.

[96]  Huixin He,et al.  Multifunctional nanomedicine platform for cancer specific delivery of siRNA by superparamagnetic iron oxide nanoparticles-dendrimer complexes. , 2011, Current drug delivery.

[97]  Robert Langer,et al.  Tissue-specific gene delivery via nanoparticle coating. , 2010, Biomaterials.

[98]  Jennifer A. Doudna,et al.  In vitro reconstitution of the human RISC-loading complex , 2008, Proceedings of the National Academy of Sciences.

[99]  W. Mark Saltzman,et al.  Intravaginal gene silencing using biodegradable polymer nanoparticles densely loaded with small-interfering RNA , 2009, Nature materials.

[100]  A. Fire,et al.  Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans , 1998, Nature.

[101]  K. Howard.  Unlocking the money-making potential of RNAi , 2003, Nature Biotechnology.