Journal Home > Volume 11 , Issue 10

Ribonucleic acids (RNAs) possess great therapeutic potential and can be used to treat a variety of diseases. The unique biophysical properties of RNAs, such as high molecular weight, negative charge, hydrophilicity, low stability, and potential immunogenicity, require chemical modification and development of carriers to enable intracellular delivery of RNAs for clinical use. A variety of nanomaterials have been developed for the effective in vivo delivery of short/ small RNAs, messenger RNAs, and RNAs required for gene editing technologies including clustered regularly interspaced palindromic repeat (CRISPR)/Cas. This review outlines the challenges of delivering RNA therapeutics, explores the chemical synthesis of RNA modifications and carriers, and describes the efforts to design nanomaterials that can be used for a variety of clinical indications.


menu
Abstract
Full text
Outline
About this article

Design of synthetic materials for intracellular delivery of RNAs: From siRNA-mediated gene silencing to CRISPR/Cas gene editing

Show Author's information Jason B. Miller1,2Daniel J. Siegwart1,2( )
Simmons Comprehensive Cancer CenterUniversity of Texas Southwestern Medical CenterDallasTX75390USA
Department of BiochemistryUniversity of Texas Southwestern Medical CenterDallasTX75390USA

Abstract

Ribonucleic acids (RNAs) possess great therapeutic potential and can be used to treat a variety of diseases. The unique biophysical properties of RNAs, such as high molecular weight, negative charge, hydrophilicity, low stability, and potential immunogenicity, require chemical modification and development of carriers to enable intracellular delivery of RNAs for clinical use. A variety of nanomaterials have been developed for the effective in vivo delivery of short/ small RNAs, messenger RNAs, and RNAs required for gene editing technologies including clustered regularly interspaced palindromic repeat (CRISPR)/Cas. This review outlines the challenges of delivering RNA therapeutics, explores the chemical synthesis of RNA modifications and carriers, and describes the efforts to design nanomaterials that can be used for a variety of clinical indications.

Keywords: nanoparticles, nucleic acid therapeutics, synthetic nanomaterials, RNA interference (RNAi), messenger RNA (mRNA), clustered regularly interspaced palindromic repeat (CRISPR)/Cas

References(229)

1

Wu, S. Y.; Lopez–Berestein, G.; Calin, G. A.; Sood, A. K. RNAi therapies: Drugging the undruggable. Sci. Transl. Med. 2014, 6, 240ps7.

2

Cox, A. D.; Fesik, S. W.; Kimmelman, A. C.; Luo, J.; Der, C. J. Drugging the undruggable RAS: Mission possible? Nat. Rev. Drug Discovery 2014, 13, 828–851.

3

Zhou, K. J.; Nguyen, L. H.; Miller, J. B.; Yan, Y. F.; Kos, P.; Xiong, H.; Li, L.; Hao, J.; Minnig, J. T.; Zhu, H. et al. Modular degradable dendrimers enable small RNAs to extend survival in an aggressive liver cancer model. Proc. Natl. Acad. Sci. USA 2016, 113, 520–525.

4

Daige, C. L.; Wiggins, J. F.; Priddy, L.; Nelligan–Davis, T.; Zhao, J.; Brown, D. Systemic delivery of a miR34a mimic as a potential therapeutic for liver cancer. Mol. Cancer Ther. 2014, 13, 2352–2360.

5

Rupaimoole, R.; Slack, F. J. MicroRNA therapeutics: Towards a new era for the management of cancer and other diseases. Nat. Rev. Drug Discovery 2017, 16, 203–222.

6

Cutting, G. R. Cystic fibrosis genetics: From molecular understanding to clinical application. Nat. Rev. Genet. 2015, 16, 45–56.

7

Tabebordbar, M.; Zhu, K. X.; Cheng, J. K. W.; Chew, W. L.; Widrick, J. J.; Yan, W. X.; Maesner, C.; Wu, E. Y.; Xiao, R.; Ran, F. A. et al. In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science 2016, 351, 407–411.

8

Nelson, C. E.; Hakim, C. H.; Ousterout, D. G.; Thakore, P. I.; Moreb, E. A.; Rivera, R. M. C.; Madhavan, S.; Pan, X. F.; Ran, F. A.; Yan, W. X. et al. In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science 2016, 351, 403–407.

9

Long, C. Z.; Amoasii, L.; Mireault, A. A.; McAnally, J. R.; Li, H.; Sanchez–Ortiz, E.; Bhattacharyya, S.; Shelton, J. M.; Bassel–Duby, R.; Olson, E. N. Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy. Science 2016, 351, 400–403.

10

Sánchez–Rivera, F. J.; Papagiannakopoulos, T.; Romero, R.; Tammela, T.; Bauer, M. R.; Bhutkar, A.; Joshi, N. S.; Subbaraj, L.; Bronson, R. T.; Xue, W. et al. Rapid modelling of cooperating genetic events in cancer through somatic genome editing. Nature 2014, 516, 428–431.

11

Verdine, G. L.; Walensky, L. D. The challenge of drugging undruggable targets in cancer: Lessons learned from targeting BCL–2 family members. Clin. Cancer Res. 2007, 13, 7264–7270.

12

Aagaard, L.; Rossi, J. J. RNAi therapeutics: Principles, prospects and challenges. Adv. Drug Deliver. Rev. 2007, 59, 75–86.

13

Bobbin, M. L.; Rossi, J. J. RNA interference (RNAi)–based therapeutics: Delivering on the promise? Ann. Rev. Pharm. Tox. 2016, 56, 103–122.

14

Akinc, A.; Zumbuehl, A.; Goldberg, M.; Leshchiner, E. S.; Busini, V.; Hossain, N.; Bacallado, S. A.; Nguyen, D. N.; Fuller, J.; Alvarez, R. et al. A combinatorial library of lipidlike materials for delivery of RNAi therapeutics. Nat. Biotechnol. 2008, 26, 561–569.

15

Kanasty, R.; Dorkin, J. R.; Vegas, A.; Anderson, D. Delivery materials for siRNA therapeutics. Nat. Mater. 2013, 12, 967–977.

16

Whitehead, K.; Langer, R.; Anderson, D. G. Knocking down barriers: Advances in siRNA delivery. Nat. Rev. Drug Discovery 2009, 8, 129–138.

17

Fire, A.; Xu, S. Q.; Montgomery, M. K.; Kostas, S. A.; Driver, S. E.; Mello, C. C. Potent and specific genetic interference by double–stranded RNA in Caenorhabditis elegans. Nature 1998, 391, 806–811.

18

Hammond, S. M.; Bernstein, E.; Beach, D.; Hannon, G. J. An RNA–directed nuclease mediates post–transcriptional gene silencing in Drosophila cells. Nature 2000, 404, 293–296.

19

Elbashir, S. M.; Harborth, J.; Lendeckel, W.; Yalcin, A.; Weber, K.; Tuschl, T. Duplexes of 21–nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001, 411, 494–498.

20

Haussecker, D. The business of RNAi therapeutics. Hum. Gene Ther. 2008, 19, 451–462.

21

Haussecker, D. The business of RNAi therapeutics in 2012. Mol. Ther. Nucl. Acids 2012, 1, e8.

22

Pack, D. W.; Hoffman, A. S.; Pun, S. Z.; Stayton, P. S. Design and development of polymers for gene delivery. Nat. Rev. Drug Discovery 2005, 4, 581–593.

23

Kim, H.; Kim, J. S. A guide to genome engineering with programmable nucleases. Nat. Rev. Genet. 2014, 15, 321–334.

24

Geall, A. J.; Verma, A.; Otten, G. R.; Shaw, C. A.; Hekele, A.; Banerjee, K.; Cu, Y.; Beard, C. W.; Brito, L. A.; Krucker, T. et al. Nonviral delivery of self–amplifying RNA vaccines. Proc. Natl. Acad. Sci. USA 2012, 109, 14604–14609.

25

Kowalczyk, A.; Doener, F.; Zanzinger, K.; Noth, J.; Baumhof, P.; Fotin–Mleczek, M.; Heidenreich, R. Self–adjuvanted mRNA vaccines induce local innate immune responses that lead to a potent and boostable adaptive immunity. Vaccine 2016, 34, 3882–3893.

26

Li, M.; Zhao, M. N.; Fu, Y.; Li, Y.; Gong, T.; Zhang, Z. R.; Sun, X. Enhanced intranasal delivery of mRNA vaccine by overcoming the nasal epithelial barrier via intra–and paracellular pathways. J. Control. Release 2016, 228, 9–19.

27

Pardi, N.; Hogan, M. J.; Porter, F. W.; Weissman, D. mRNA vaccines — a new era in vaccinology. Nat. Rev. Drug Discovery 2018, 17, 261–279.

28

Petsch, B.; Schnee, M.; Vogel, A. B.; Lange, E.; Hoffmann, B.; Voss, D.; Schlake, T.; Thess, A.; Kallen, K. J.; Stitz, L. et al. Protective efficacy of in vitro synthesized, specific mRNA vaccines against influenza A virus infection. Nat. Biotechnol. 2012, 30, 1210–1216.

29

Richner, J. M.; Himansu, S.; Dowd, K. A.; Butler, S. L.; Salazar, V.; Fox, J. M.; Julander, J. G.; Tang, W. W.; Shresta, S.; Pierson, T. C. et al. Modified mRNA vaccines protect against zika virus infection. Cell 2017, 168, 1114–1125. e10.

30

Richner, J. M.; Jagger, B. W.; Shan, C.; Fontes, C. R.; Dowd, K. A.; Cao, B.; Himansu, S.; Caine, E. A.; Nunes, B. T. D.; Medeiros, D. B. A. et al. Vaccine mediated protection against zika virus–induced congenital disease. Cell 2017, 170, 273–283. e12.

31

Sahin, U.; Karikó, K.; Türeci, Ö. mRNA–based therapeutics — developing a new class of drugs. Nat. Rev. Drug Discovery 2014, 13, 759–780.

32

Thran, M.; Mukherjee, J.; Pönisch, M.; Fiedler, K.; Thess, A.; Mui, B. L.; Hope, M. J.; Tam, Y. K.; Horscroft, N.; Heidenreich, R. et al. mRNA mediates passive vaccination against infectious agents, toxins, and tumors. EMBO Mol. Med. 2017, 9, 1434–1447.

33

Kranz, L. M.; Diken, M.; Haas, H.; Kreiter, S.; Loquai, C.; Reuter, K. C.; Meng, M.; Fritz, D.; Vascotto, F.; Hefesha, H. et al. Systemic RNA delivery to dendritic cells exploits antiviral defence for cancer immunotherapy. Nature 2016, 534, 396–401.

34

Kübler, H.; Scheel, B.; Gnad–Vogt, U.; Miller, K.; Schultze–Seemann, W.; vom Dorp, F.; Parmiani, G.; Hampel, C.; Wedel, S.; Trojan, L. et al. Self–adjuvanted mRNA vaccination in advanced prostate cancer patients: A first–in–man phase I/IIa study. J. Immunotherapy Can. 2015, 3, 26.

35

Scheel, B.; Aulwurm, S.; Probst, J.; Stitz, L.; Hoerr, I.; Rammensee, H. G.; Weller, M.; Pascolo, S. Therapeutic anti–tumor immunity triggered by injections of immunostimulating single–stranded RNA. Eur. J. Immun. 2006, 36, 2807–2816.

36

Stadler, C. R.; Bähr–Mahmud, H.; Celik, L.; Hebich, B.; Roth, A. S.; Roth, R. P.; Karikó, K.; Türeci, Ö.; Sahin, U. Elimination of large tumors in mice by mRNA–encoded bispecific antibodies. Nat. Med. 2017, 23, 815–817.

37

Hajj, K. A.; Whitehead, K. A. Tools for translation: Non–viral materials for therapeutic mRNA delivery. Nat. Rev. Mater. 2017, 2, 17056.

38

An, D.; Schneller, J. L.; Frassetto, A.; Liang, S.; Zhu, X. L.; Park, J. S.; Theisen, M.; Hong, S. J.; Zhou, J.; Rajendran, R. et al. Systemic messenger RNA therapy as a treatment for methylmalonic acidemia. Cell Rep. 2017, 21, 3548–3558.

39

DeRosa, F.; Guild, B.; Karve, S.; Smith, L.; Love, K.; Dorkin, J. R.; Kauffman, K. J.; Zhang, J.; Yahalom, B.; Anderson, D. G. et al. Therapeutic efficacy in a hemophilia B model using a biosynthetic mRNA liver depot system. Gene Ther. 2016, 23, 699–707.

40

Karikó, K.; Muramatsu, H.; Keller, J. M.; Weissman, D. Increased erythropoiesis in mice injected with submicrogram quantities of pseudouridine–containing mrna encoding erythropoietin. Mol. Ther. 2012, 20, 948–953.

41

Kormann, M. S. D.; Hasenpusch, G.; Aneja, M. K.; Nica, G.; Flemmer, A. W.; Herber–Jonat, S.; Huppmann, M.; Mays, L. E.; Illenyi, M.; Schams, A. et al. Expression of therapeutic proteins after delivery of chemically modified mRNA in mice. Nat. Biotechnol. 2011, 29, 154–157.

42

Ramaswamy, S.; Tonnu, N.; Tachikawa, K.; Limphong, P.; Vega, J. B.; Karmali, P. P.; Chivukula, P.; Verma, I. M. Systemic delivery of factor IX messenger RNA for protein replacement therapy. Proc. Natl. Acad. Sci. USA 2017, 114, E1941–E1950.

43

Ziller, A.; Nogueira, S. S.; Hühn, E.; Funari, S. S.; Brezesinski, G.; Hartmann, H.; Sahin, U.; Haas, H.; Langguth, P. Incorporation of mRNA in lamellar lipid matrices for parenteral administration. Mol. Pharmaceut. 2018, 15, 642–651.

44

Song, M. The CRISPR/Cas9 system: Their delivery, in vivo and ex vivo applications and clinical development by startups. Biotechnol. Progr. 2017, 33, 1035–1045.

45

Chen, J.; Guo, Z. P.; Tian, H. Y.; Chen, X. S. Production and clinical development of nanoparticles for gene delivery. Mol. Ther. –Methods Clin. Dev. 2016, 3, 16023.

46

Liu, F.; Huang, L. Development of non–viral vectors for systemic gene delivery. J. Control. Release 2002, 78, 259–266.

47

Zhi, D. F.; Zhang, S. B.; Cui, S. H.; Zhao, Y. A.; Wang, Y. H.; Zhao, D. F. The headgroup evolution of cationic lipids for gene delivery. Bioconjugate Chem. 2013, 24, 487–519.

48

Zhi, D. F.; Zhang, S. B.; Wang, B.; Zhao, Y. N.; Yang, B. L.; Yu, S. J. Transfection efficiency of cationic lipids with different hydrophobic domains in gene delivery. Bioconjugate Chem. 2010, 21, 563–577.

49

Kim, H. J.; Kim, A.; Miyata, K.; Kataoka, K. Recent progress in development of siRNA delivery vehicles for cancer therapy. Adv. Drug Deliver. Rev. 2016, 104, 61–77.

50

Sarett, S. M.; Nelson, C. E.; Duvall, C. L. Technologies for controlled, local delivery of siRNA. J. Control. Release 2015, 218, 94–113.

51

Zuckerman, J. E.; Davis, M. E. Clinical experiences with systemically administered siRNA–based therapeutics in cancer. Nat. Rev. Drug Discovery 2015, 14, 843–856.

52

Granot, Y.; Peer, D. Delivering the right message: Challenges and opportunities in lipid nanoparticles–mediated modified mRNA therapeutics—An innate immune system standpoint. Semin. Immunol. 2017, 34, 68–77.

53

Guan, S.; Rosenecker, J. Nanotechnologies in delivery of mRNA therapeutics using nonviral vector–based delivery systems. Gene Ther. 2017, 24, 133–143.

54

Kauffman, K. J.; Webber, M. J.; Anderson, D. G. Materials for non–viral intracellular delivery of messenger RNA therapeutics. J. Control. Release 2016, 240, 227–234.

55

Wang, H. X.; Li, M. Q.; Lee, C. M.; Chakraborty, S.; Kim, H. W.; Bao, G.; Leong, K. W. CRISPR/Cas9–based genome editing for disease modeling and therapy: Challenges and opportunities for nonviral delivery. Chem. Rev. 2017, 117, 9874–9906.

56

Sander, J. D.; Joung, J. K. CRISPR–Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 2014, 32, 347–355.

57

Cullis, P. R.; Hope, M. J. Lipid nanoparticle systems for enabling gene therapies. Mol. Ther. 2017, 25, 1467–1475.

58

Whitehead, K. A.; Matthews, J.; Chang, P. H.; Niroui, F.; Dorkin, J. R.; Severgnini, M.; Anderson, D. G. In vitro–in vivo translation of lipid nanoparticles for hepatocellular siRNA delivery. ACS Nano 2012, 6, 6922–6929.

59

Zatsepin, T. S.; Kotelevtsev, Y. V.; Koteliansky, V. Lipid nanoparticles for targeted siRNA delivery–going from bench to bedside. Int. J. Nanomed. 2016, 11, 3077–3086.

60

Gary, D. J.; Puri, N.; Won, Y. Y. Polymer–based siRNA delivery: Perspectives on the fundamental and phenomenological distinctions from polymer–based DNA delivery. J. Control. Release 2007, 121, 64–73.

61

Guillot–Nieckowski, M.; Eisler, S.; Diederich, F. Dendritic vectors for gene transfection. New J. Chem. 2007, 31, 1111–1127.

62

Svenson, S.; Tomalia, D. A. Dendrimers in biomedical applications–reflections on the field. Adv. Drug Deliver. Rev. 2005, 57, 2106–2129.

63

Chen, D. Q.; Dougherty, C. A.; Zhu, K. C.; Hong, H. Theranostic applications of carbon nanomaterials in cancer: Focus on imaging and cargo delivery. J. Control. Release 2015, 210, 230–245.

64

Kam, N. W. S.; Liu, Z.; Dai, H. J. Functionalization of carbon nanotubes via cleavable disulfide bonds for efficient intracellular delivery of siRNA and potent gene silencing. J. Am. Chem. Soc. 2005, 127, 12492–12493.

65

Prato, M.; Kostarelos, K.; Bianco, A. Functionalized carbon nanotubes in drug design and discovery. Acc. Chem. Res. 2008, 41, 60–68.

66

Ghosh, P. S.; Kim, C. K.; Han, G.; Forbes, N. S.; Rotello, V. M. Efficient gene delivery vectors by tuning the surface charge density of amino acid–functionalized gold nanoparticles. ACS Nano 2008, 2, 2213–2218.

67

Loh, X. J.; Lee, T. C.; Dou, Q. Q.; Deen, G. R. Utilising inorganic nanocarriers for gene delivery. Biomat. Sci. 2016, 4, 70–86.

68

Sokolova, V.; Epple, M. Inorganic nanoparticles as carriers of nucleic acids into cells. Angew. Chem., Int. Ed. 2008, 47, 1382–1395.

69

Xu, Z. P.; Zeng, Q. H.; Lu, G. Q.; Yu, A. B. Inorganic nanoparticles as carriers for efficient cellular delivery. Chem. Eng. Sci. 2006, 61, 1027–1040.

70

Yan, Y. F.; Zhou, K. J.; Xiong, H.; Miller, J. B.; Motea, E. A.; Boothman, D. A.; Liu, L.; Siegwart, D. J. Aerosol delivery of stabilized polyester–siRNA nanoparticles to silence gene expression in orthotopic lung tumors. Biomaterials 2017, 118, 84–93.

71

Fehring, V.; Schaeper, U.; Ahrens, K.; Santel, A.; Keil, O.; Eisermann, M.; Giese, K.; Kaufmann, J. Delivery of therapeutic siRNA to the lung endothelium via novel lipoplex formulation DACC. Mol. Ther. 2014, 22, 811–820.

72

Ishiwata, H.; Suzuki, N.; Ando, S.; Kikuchi, H.; Kitagawa, T. Characteristics and biodistribution of cationic liposomes and their DNA complexes. J. Control. Release 2000, 69, 139–148.

73

Love, K. T.; Mahon, K. P.; Levins, C. G.; Whitehead, K. A.; Querbes, W.; Dorkin, J. R.; Qin, J.; Cantley, W.; Qin, L. L.; Racie, T. et al. Lipid–like materials for low–dose, in vivo gene silencing. Proc. Natl. Acad. Sci. USA 2010, 107, 1864–1869.

74

Semple, S. C.; Akinc, A.; Chen, J. X.; Sandhu, A. P.; Mui, B. L.; Cho, C. K.; Sah, D. W. Y.; Stebbing, D.; Crosley, E. J.; Yaworski, E. et al. Rational design of cationic lipids for siRNA delivery. Nat. Biotechnol. 2010, 28, 172–176.

75

Dong, Y. Z.; Love, K. T.; Dorkin, J. R.; Sirirungruang, S.; Zhang, Y. L.; Chen, D. L.; Bogorad, R. L.; Yin, H.; Chen, Y.; Vegas, A. J. et al. Lipopeptide nanoparticles for potent and selective siRNA delivery in rodents and nonhuman primates. Proc. Natl. Acad. Sci. USA 2014, 111, 3955–3960.

76

Jayaraman, M.; Ansell, S. M.; Mui, B. L.; Tam, Y. K.; Chen, J. X.; Du, X. Y.; Butler, D.; Eltepu, L.; Matsuda, S.; Narayanannair, J. K. et al. Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo. Angew. Chem., Int. Ed. 2012, 51, 8529–8533.

77

Maier, M. A.; Jayaraman, M.; Matsuda, S.; Liu, J.; Barros, S.; Querbes, W.; Tam, Y. K.; Ansell, S. M.; Kumar, V.; Qin, J. et al. Biodegradable lipids enabling rapidly eliminated lipid nanoparticles for systemic delivery of RNAi therapeutics. Mol. Ther. 2013, 21, 1570–1578.

78

Wittrup, A.; Ai, A.; Liu, X.; Hamar, P.; Trifonova, R.; Charisse, K.; Manoharan, M.; Kirchhausen, T.; Lieberman, J. Visualizing lipid–formulated siRNA release from endosomes and target gene knockdown. Nat. Biotechnol. 2015, 33, 870–976.

79

Sahay, G.; Querbes, W.; Alabi, C.; Eltoukhy, A.; Sarkar, S.; Zurenko, C.; Karagiannis, E.; Love, K.; Chen, D. L.; Zoncu, R. et al. Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling. Nat. Biotechnol. 2013, 31, 653–658.

80

Gilleron, J.; Querbes, W.; Zeigerer, A.; Borodovsky, A.; Marsico, G.; Schubert, U.; Manygoats, K.; Seifert, S.; Andree, C.; Stoter, M. et al. Image–based analysis of lipid nanoparticle–mediated siRNA delivery, intracellular trafficking and endosomal escape. Nat. Biotechnol. 2013, 31, 638–646.

81

Sahay, G.; Alakhova, D. Y.; Kabanov, A. V. Endocytosis of nanomedicines. J. Control. Release 2010, 145, 182–195.

82

Rehman, Z.; Zuhorn, I. S.; Hoekstra, D. How cationic lipids transfer nucleic acids into cells and across cellular membranes: Recent advances. J. Control. Release 2013, 166, 46–56.

83

Layzer, J. M.; McCaffrey, A. P.; Tanner, A. K.; Huang, Z.; Kay, M. A.; Sullenger, B. A. In vivo activity of nucleaseresistant siRNAs. RNA 2004, 10, 766–771.

84

Khvorova, A.; Watts, J. K. The chemical evolution of oligonucleotide therapies of clinical utility. Nat. Biotechnol. 2017, 35, 238–248.

85

Behlke, M. A. Chemical modification of siRNAs for in vivo use. Oligonucleotides 2008, 18, 305–320.

86

Patra, A.; Paolillo, M.; Charisse, K.; Manoharan, M.; Rozners, E.; Egli, M. 2'–fluoro RNA shows increased watson–crick H–bonding strength and stacking relative to RNA: Evidence from NMR and thermodynamic data. Angew. Chem., Int. Ed. 2012, 51, 11863–11866.

87

Kalota, A.; Karabon, L.; Swider, C. R.; Viazovkina, E.; Elzagheid, M.; Damha, M. J.; Gewirtz, A. M. 2'–Deoxy–2'–fluoro–β–d–arabinonucleic acid (2'F–ANA) modified oligonucleotides (ON) effect highly efficient, and persistent, gene silencing. Nucleic Acids Res. 2006, 34, 451–461.

88

Braasch, D. A.; Paroo, Z.; Constantinescu, A.; Ren, G.; Öz, O. K.; Mason, R. P.; Corey, D. R. Biodistribution of phosphodiester and phosphorothioate siRNA. Bioorg. Med. Chem. Lett. 2004, 14, 1139–1143.

89

Corey, D. R. Nusinersen, an antisense oligonucleotide drug for spinal muscular atrophy. Nat. Neurosci. 2017, 20, 497–499.

90

Meade, B. R.; Gogoi, K.; Hamil, A. S.; Palm–Apergi, C.; van den Berg, A.; Hagopian, J. C.; Springer, A. D.; Eguchi, A.; Kacsinta, A. D.; Dowdy, C. F. et al. Efficient delivery of RNAi prodrugs containing reversible charge–neutralizing phosphotriester backbone modifications. Nat. Biotechnol. 2014, 32, 1256–1261.

91

Kole, R.; Krainer, A. R.; Altman, S. RNA therapeutics: Beyond RNA interference and antisense oligonucleotides. Nat. Rev. Drug Discovery 2012, 11, 125–140.

92

Ray, A.; Nordén, B. Peptide nucleic acid (PNA): Its medical and biotechnical applications and promise for the future. FASEB J. 2000, 14, 1041–1060.

93

Almeida, M. I.; Reis, R. M.; Calin, G. A. MicroRNA history: Discovery, recent applications, and next frontiers. Mutat. Res. /Fund. Mol. Mechan. Mutag. 2011, 717, 1–8.

94

Ling, H.; Fabbri, M.; Calin, G. A. MicroRNAs and other non–coding RNAs as targets for anticancer drug development. Nat. Rev. Drug Discovery 2013, 12, 847–865.

95

Matsui, M.; Corey, D. R. Non–coding RNAs as drug targets. Nat. Rev. Drug Discovery 2017, 16, 167–179.

96

Youngblood, D. S.; Hatlevig, S. A.; Hassinger, J. N.; Iversen, P. L.; Moulton, H. M. Stability of cell–penetrating peptide–morpholino oligomer conjugates in human serum and in cells. Bioconjugate Chem. 2007, 18, 50–60.

97

Echigoya, Y.; Nakamura, A.; Nagata, T.; Urasawa, N.; Lim, K. R. Q.; Trieu, N.; Panesar, D.; Kuraoka, M.; Moulton, H. M.; Saito, T. et al. Effects of systemic multiexon skipping with peptide–conjugated morpholinos in the heart of a dog model of Duchenne muscular dystrophy. Proc. Natl. Acad. Sci. USA 2017, 114, 4213–4218.

98

Lim, K. R. Q.; Maruyama, R.; Yokota, T. Eteplirsen in the treatment of Duchenne muscular dystrophy. Drug Design Dev. Ther. 2017, 11, 533–545.

99

Lu–Nguyen, N.; Malerba, A.; Popplewell, L.; Schnell, F.; Hanson, G.; Dickson, G. Systemic antisense therapeutics for dystrophin and myostatin exon splice modulation improve muscle pathology of adult mdx mice. Mol. Ther. Nucl. Acids 2017, 6, 15–28.

100

Vaish, N.; Chen, F.; Seth, S.; Fosnaugh, K.; Liu, Y.; Adami, R.; Brown, T.; Chen, Y.; Harvie, P.; Johns, R. et al. Improved specificity of gene silencing by siRNAs containing unlocked nucleobase analogs. Nucleic Acids Res. 2011, 39, 1823–1832.

101

Campbell, M. A.; Wengel, J. Locked vs. unlocked nucleic acids (LNA vs. UNA): Contrasting structures work towards common therapeutic goals. Chem. Soc. Rev. 2011, 40, 5680–5689.

102

Yanagi, T.; Tachikawa, K.; Wilkie–Grantham, R.; Hishiki, A.; Nagai, K.; Toyonaga, E.; Chivukula, P.; Matsuzawa, S. I. Lipid nanoparticle–mediated siRNA transfer against PCTAIRE1/PCTK1/Cdk16 inhibits in vivo cancer growth. Mol. Ther. –Nucl. Acids 2016, 5, e327.

103

Yaghi, N. K.; Wei, J.; Hashimoto, Y.; Kong, L. Y.; Gabrusiewicz, K.; Nduom, E. K.; Ling, X.; Huang, N.; Zhou, S.; Kerrigan, B. C. P. et al. Immune modulatory nanoparticle therapeutics for intracerebral glioma. Neuro Oncol. 2017, 19, 372–382.

104

Aleku, M.; Schulz, P.; Keil, O.; Santel, A.; Schaeper, U.; Dieckhoff, B.; Janke, O.; Endruschat, J.; Durieux, B.; Röder, N. et al. Atu027, a liposomal small interfering RNA formulation targeting protein kinase N3, inhibits cancer progression. Cancer Res. 2008, 68, 9788–9798.

105

Santel, A.; Aleku, M.; Röder, N.; Möpert, K.; Durieux, B.; Janke, O.; Keil, O.; Endruschat, J.; Dames, S.; Lange, C. et al. Atu027 prevents pulmonary metastasis in experimental and spontaneous mouse metastasis models. Clin. Cancer Res. 2010, 16, 5469–5480.

106

Schultheis, B.; Strumberg, D.; Santel, A.; Vank, C.; Gebhardt, F.; Keil, O.; Lange, C.; Giese, K.; Kaufmann, J.; Khan, M. et al. First–in–human phase I study of the liposomal RNA interference therapeutic Atu027 in patients with advanced solid tumors. J. Clin. Oncol. 2014, 32, 4141–4148.

107

Dudek, H.; Wong, D. H.; Arvan, R.; Shah, A.; Wortham, K.; Ying, B.; Diwanji, R.; Zhou, W.; Holmes, B.; Yang, H. L. et al. Knockdown of β–catenin with dicer–substrate siRNAs reduces liver tumor burden in vivo. Mol. Ther. 2014, 22, 92–101.

108

Ganesh, S.; Koser, M. L.; Cyr, W. A.; Chopda, G. R.; Tao, J. Y.; Shui, X.; Ying, B.; Chen, D. Y.; Pandya, P.; Chipumuro, E. et al. Direct pharmacological inhibition of β–catenin by RNA interference in tumors of diverse origin. Mol. Cancer Ther. 2016, 15, 2143–2154.

109

Lee, S. H.; Kang, Y. Y.; Jang, H. E.; Mok, H. Current preclinical small interfering RNA (siRNA)–based conjugate systems for RNA therapeutics. Adv. Drug Deliver. Rev. 2016, 104, 78–92.

110

Dohmen, C.; Fröhlich, T.; Lächelt, U.; Röhl, I.; Vornlocher, H. P.; Hadwiger, P.; Wagner, E. Defined folate–PEG–siRNA conjugates for receptor–specific gene silencing. Mol. Ther. –Nucl. Acids 2012, 1, e7.

111

Zwicke, G. L.; Mansoori, G. A.; Jeffery, C. J. Utilizing the folate receptor for active targeting of cancer nanotherapeutics. Nano Rev. 2012, 3, 18496.

112

Alam, M. R.; Ming, X.; Fisher, M.; Lackey, J.; Rajeev, K. G.; Manoharan, M.; Juliano, R. Multivalent cyclic RGD conjugates for targeted delivery of siRNA. Bioconjugate Chem. 2011, 22, 1673–1681.

113

Danhier, F.; Le Breton, A.; Préat, V. RGD–based strategies to target alpha(v) beta(3) integrin in cancer therapy and diagnosis. Mol. Pharmaceutics 2012, 9, 2961–2973.

114

Alterman, J. F.; Hall, L. M.; Coles, A. H.; Hassler, M. R.; Didiot, M. C.; Chase, K.; Abraham, J.; Sottosanti, E.; Johnson, E.; Sapp, E. et al. Hydrophobically modified siRNAs silence huntingtin mRNA in primary neurons and mouse brain. Mol. Ther. –Nucl. Acids 2015, 4, e266.

115

Nikan, M.; Osborn, M. F.; Coles, A. H.; Godinho, B. M. D. C.; Hall, L. M.; Haraszti, R. A.; Hassler, M. R.; Echeverria, D.; Aronin, N.; Khvorova, A. Docosahexaenoic acid conjugation enhances distribution and safety of siRNA upon local administration in mouse brain. Mol. Ther. –Nucl. Acids 2016, 5, e344.

116

Nair, J. K.; Willoughby, J. L. S.; Chan, A.; Charisse, K.; Alam, M. R.; Wang, Q. F.; Hoekstra, M.; Kandasamy, P.; Kel'in, A. V.; Milstein, S. et al. Multivalent N–acetylgalactosamine–conjugated siRNA localizes in hepatocytes and elicits robust RNAi–mediated gene silencing. J. Am. Chem. Soc. 2014, 136, 16958–16961.

117

Ramanathan, A.; Robb, G. B.; Chan, S. H. mRNA capping: Biological functions and applications. Nucleic Acids Res. 2016, 44, 7511–7526.

118

Grudzien–Nogalska, E.; Jemielity, J.; Kowalska, J.; Darzynkiewicz, E.; Rhoads, R. E. Phosphorothioate cap analogs stabilize mRNA and increase translational efficiency in mammalian cells. RNA 2007, 13, 1745–1755.

119

Kowalska, J.; Lewdorowicz, M.; Zuberek, J.; Grudzien–Nogalska, E.; Bojarska, E.; Stepinski, J.; Rhoads, R. E.; Darzynkiewicz, E.; Davis, R. E.; Jemielity, J. Synthesis and characterization of mRNA cap analogs containing phosphorothioate substitutions that bind tightly to eIF4E and are resistant to the decapping pyrophosphatase DcpS. RNA 2008, 14, 1119–1131.

120

Kowalska, J.; Wypijewska del Nogal, A.; Darzynkiewicz, Z. M.; Buck, J.; Nicola, C.; Kuhn, A. N.; Lukaszewicz, M.; Zuberek, J.; Strenkowska, M.; Ziemniak, M. et al. Synthesis, properties, and biological activity of boranophosphate analogs of the mRNA cap: Versatile tools for manipulation of therapeutically relevant cap–dependent processes. Nucleic Acids Res. 2014, 42, 10245–10264.

121

Karikó, K.; Muramatsu, H.; Welsh, F. A.; Ludwig, J.; Kato, H.; Akira, S.; Weissman, D. Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Mol. Ther. 2008, 16, 1833–1840.

122

Kauffman, K. J.; Mir, F. F.; Jhunjhunwala, S.; Kaczmarek, J. C.; Hurtado, J. E.; Yang, J. H.; Webber, M. J.; Kowalski, P. S.; Heartlein, M. W.; DeRosa, F. et al. Efficacy and immunogenicity of unmodified and pseudouridine–modified mRNA delivered systemically with lipid nanoparticles in vivo. Biomaterials 2016, 109, 78–87.

123

Li, B.; Luo, X.; Dong, Y. Z. Effects of chemically modified messenger RNA on protein expression. Bioconjugate Chem. 2016, 27, 849–853.

124

Svitkin, Y. V.; Cheng, Y. M.; Chakraborty, T.; Presnyak, V.; John, M.; Sonenberg, N. N1–methyl–pseudouridine in mRNA enhances translation through eIF2α–dependent and independent mechanisms by increasing ribosome density. Nucleic Acids Res. 2017, 45, 6023–6036.

125

Anderson, B. R.; Muramatsu, H.; Nallagatla, S. R.; Bevilacqua, P. C.; Sansing, L. H.; Weissman, D.; Karikó, K. Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation. Nucleic Acids Res. 2010, 38, 5884–5892.

126

Mauro, V. P.; Chappell, S. A. A critical analysis of codon optimization in human therapeutics. Trends Mol. Med. 2014, 20, 604–613.

127

Thess, A.; Grund, S.; Mui, B. L.; Hope, M. J.; Baumhof, P.; Fotin–Mleczek, M.; Schlake, T. Sequence–engineered mRNA without chemical nucleoside modifications enables an effective protein therapy in large animals. Mol. Ther. 2015, 23, 1456–1464.

128

Balmayor, E. R.; Geiger, J. P.; Aneja, M. K.; Berezhanskyy, T.; Utzinger, M.; Mykhaylyk, O.; Rudolph, C.; Plank, C. Chemically modified RNA induces osteogenesis of stem cells and human tissue explants as well as accelerates bone healing in rats. Biomaterials 2016, 87, 131–146.

129

Cheng, X. W.; Lee, R. J. The role of helper lipids in lipid nanoparticles (LNPs) designed for oligonucleotide delivery. Adv. Drug Deliver. Rev. 2016, 99, 129–137.

130

Maurer, N.; Wong, K. F.; Stark, H.; Louie, L.; McIntosh, D.; Wong, T.; Scherrer, P.; Semple, S. C.; Cullis, P. R. Spontaneous entrapment of polynucleotides upon electrostatic interaction with ethanol–destabilized cationic liposomes. Biophys. J. 2001, 80, 2310–2326.

131

Jeffs, L. B.; Palmer, L. R.; Ambegia, E. G.; Giesbrecht, C.; Ewanick, S.; MacLachlan, I. A scalable, extrusion–free method for efficient liposomal encapsulation of plasmid DNA. Pharm. Res. 2005, 22, 362–372.

132

Belliveau, N. M.; Huft, J.; Lin, P. J. C.; Chen, S.; Leung, A. K. K.; Leaver, T. J.; Wild, A. W.; Lee, J. B.; Taylor, R. J.; Tam, Y. K. et al. Microfluidic synthesis of highly potent limit–size lipid nanoparticles for in vivo delivery of siRNA. Mol. Ther. –Nucl. Acids 2012, 1, e37.

133

Leung, A. K. K.; Hafez, I. M.; Baoukina, S.; Belliveau, N. M.; Zhigaltsev, I. V.; Afshinmanesh, E.; Tieleman, D. P.; Hansen, C. L.; Hope, M. J.; Cullis, P. R. Correction to "Lipid nanoparticles containing siRNA synthesized by microfluidic mixing exhibit an electron–dense nanostructured core". J. Phys. Chem. C 2012, 116, 22104.

134

Leung, A. K. K.; Tam, Y. Y. C.; Chen, S.; Hafez, I. M.; Cullis, P. R. Microfluidic mixing: A general method for encapsulating macromolecules in lipid nanoparticle systems. J. Phys. Chem. B 2015, 119, 8698–8706.

135

van Meer, G.; Voelker, D. R.; Feigenson, G. W. Membrane lipids: Where they are and how they behave. Nat. Rev. Mol. Cell Biol. 2008, 9, 112–124.

136

Miller, J. B.; Zhang, S. Y.; Kos, P.; Xiong, H.; Zhou, K. J.; Perelman, S. S.; Zhu, H.; Siegwart, D. J. Non–viral CRISPR/Cas gene editing in vitro and in vivo enabled by synthetic nanoparticle co–delivery of Cas9 mRNA and sgRNA. Angew. Chem., Int. Ed. 2017, 56, 1059–1063.

137

Alabi, C. A.; Love, K. T.; Sahay, G.; Yin, H.; Luly, K. M.; Langer, R.; Anderson, D. G. Multiparametric approach for the evaluation of lipid nanoparticles for siRNA delivery. Proc. Natl. Acad. Sci. USA 2013, 110, 12881–12886.

138

Paunovska, K.; Sago, C. D.; Monaco, C. M.; Hudson, W. H.; Castro, M. G.; Rudoltz, T. G.; Kalathoor, S.; Vanover, D. A.; Santangelo, P. J.; Ahmed, R. et al. A direct comparison of in vitro and in vivo nucleic acid delivery mediated by hundreds of nanoparticles reveals a weak correlation. Nano Lett. 2018, 18, 2148–2157.

139

Roberts, L. R. Sorafenib in liver cancer — Just the beginning. New Engl. J. Med. 2008, 359, 420–422.

140

Scudellari, M. Drug development: Try and try again. Nature 2014, 516, S4–S6.

141

Tousignant, J. D.; Gates, A. L.; Ingram, L. A.; Johnson, C. L.; Nietupski, J. B.; Cheng, S. H.; Eastman, S. J.; Scheule, R. K. Comprehensive analysis of the acute toxicities induced by systemic administration of cationic lipid: Plasmid DNA complexes in mice. Hum. Gene Ther. 2000, 11, 2493–2513.

142

Lv, H. T.; Zhang, S. B.; Wang, B.; Cui, S. H.; Yan, J. Toxicity of cationic lipids and cationic polymers in gene delivery. J. Control. Release 2006, 114, 100–109.

143

Zhang, S. Y.; Zhou, K. J.; Luo, X.; Li, L.; Tu, H. C.; Sehgal, A.; Nguyen, L. H.; Zhang, Y.; Gopal, P.; Tarlow, B. D. et al. The polyploid state plays a tumor–suppressive role in the liver. Dev Cell 2018, 44, 447–459. e5.

144

Zhang, S. Y.; Nguyen, L. H.; Zhou, K. J.; Tu, H. C.; Sehgal, A.; Nassour, I.; Li, L.; Gopal, P.; Goodman, J.; Singal, A. G. et al. Knockdown of anillin actin binding protein blocks cytokinesis in hepatocytes and reduces liver tumor development in mice without affecting regeneration. Gastroenterology 2018, 154, 1421–1434.

145

Whitehead, K. A.; Dorkin, J. R.; Vegas, A. J.; Chang, P. H.; Veiseh, O.; Matthews, J.; Fenton, O. S.; Zhang, Y. L.; Olejnik, K. T.; Yesilyurt, V. et al. Degradable lipid nanoparticles with predictable in vivo siRNA delivery activity. Nat. Commun. 2014, 5, 4277.

146

Akinc, A.; Querbes, W.; De, S.; Qin, J.; Frank–Kamenetsky, M.; Jayaprakash, K. N.; Jayaraman, M.; Rajeev, K. G.; Cantley, W. L.; Dorkin, J. R. et al. Targeted delivery of RNAi therapeutics with endogenous and exogenous ligand–based mechanisms. Mol. Ther. 2010, 18, 1357–1364.

147

Adams, D.; Suhr, O. B.; Dyck, P. J.; Litchy, W. J.; Leahy, R. G.; Chen, J. H.; Gollob, J.; Coelho, T. Trial design and rationale for APOLLO, a Phase 3, placebo–controlled study of patisiran in patients with hereditary ATTR amyloidosis with polyneuropathy. BMC Neurol. 2017, 17, 181.

148

Coelho, T.; Adams, D.; Silva, A.; Lozeron, P.; Hawkins, P. N.; Mant, T.; Perez, J.; Chiesa, J.; Warrington, S.; Tranter, E. et al. Safety and efficacy of RNAi therapy for transthyretin amyloidosis. New Engl. J. Med. 2013, 369, 819–829.

149

Heyes, J.; Palmer, L.; Bremner, K.; MacLachlan, I. Cationic lipid saturation influences intracellular delivery of encapsulated nucleic acids. J. Control. Release 2005, 107, 276–287.

150

Santel, A.; Aleku, M.; Keil, O.; Endruschat, J.; Esche, V.; Fisch, G.; Dames, S.; Löffler, K.; Fechtner, M.; Arnold, W. et al. A novel siRNA–lipoplex technology for RNA interference in the mouse vascular endothelium. Gene Ther. 2006, 13, 1222–1234.

151

Adami, R. C.; Seth, S.; Harvie, P.; Johns, R.; Fam, R.; Fosnaugh, K.; Zhu, T. Y.; Farber, K.; McCutcheon, M.; Goodman, T. T. et al. An amino acid–based amphoteric liposomal delivery system for systemic administration of siRNA. Mol. Ther. 2011, 19, 1141–1151.

152

Bader, A. G. miR–34–a microRNA replacement therapy is headed to the clinic. Front. Genet. 2012, 3, 120.

153

Sato, Y.; Murase, K.; Kato, J.; Kobune, M.; Sato, T.; Kawano, Y.; Takimoto, R.; Takada, K.; Miyanishi, K.; Matsunaga, T. et al. Resolution of liver cirrhosis using vitamin A–coupled liposomes to deliver siRNA against a collagen–specific chaperone. Nat. Biotechnol. 2008, 26, 431–442.

154

Kohli, A. G.; Kierstead, P. H.; Venditto, V. J.; Walsh, C. L.; Szoka, F. C. Designer lipids for drug delivery: From heads to tails. J. Control. Release 2014, 190, 274–287.

155

Miller, J. B.; Kos, P.; Tieu, V.; Zhou, K. J.; Siegwart, D. J. Development of cationic quaternary ammonium sulfonamide amino lipids for nucleic acid delivery. ACS Appl. Mater. Interfaces 2018, 10, 2302–2311.

156

Bohdanowicz, M.; Grinstein, S. Role of phospholipids in endocytosis, phagocytosis, and macropinocytosis. Physiol. Rev. 2013, 93, 69–106.

157

Shao, Q.; Jiang, S. Y. Molecular understanding and design of zwitterionic materials. Adv. Mater. 2015, 27, 15–26.

158

Kim, S. T.; Saha, K.; Kim, C.; Rotello, V. M. The role of surface functionality in determining nanoparticle cytotoxicity. Acc. Chem. Res. 2013, 46, 681–691.

159

Kim, G.; Park, S.; Jung, J.; Heo, K.; Yoon, J.; Kim, H.; Kim, I. J.; Kim, J. R.; Lee, J. I.; Ree, M. Novel brush polymers with phosphorylcholine bristle ends: Synthesis, structure, properties, and biocompatibility. Adv. Funct. Mater. 2009, 19, 1631–1644.

160

Venditto, V. J.; Dolor, A.; Kohli, A.; Salentinig, S.; Boyd, B. J.; Szoka, F. C. Sulfated quaternary amine lipids: A new class of inverse charge zwitterlipids. Chem. Commun. 2014, 50, 9109–9111.

161

Walsh, C. L.; Nguyen, J.; Szoka, F. C. Synthesis and characterization of novel zwitterionic lipids with pHresponsive biophysical properties. Chem. Commun. 2012, 48, 5575–5577.

162

Lorenzer, C.; Dirin, M.; Winkler, A. M.; Baumann, V.; Winkler, J. Going beyond the liver: Progress and challenges of targeted delivery of siRNA therapeutics. J. Control. Release 2015, 203, 1–15.

163

Dahlman, J. E.; Barnes, C.; Khan, O. F.; Thiriot, A.; Jhunjunwala, S.; Shaw, T. E.; Xing, Y. P.; Sager, H. B.; Sahay, G.; Speciner, L. et al. In vivo endothelial siRNA delivery using polymeric nanoparticles with low molecular weight. Nat. Nanotechnol. 2014, 9, 648–655.

164

Choi, H. S.; Ashitate, Y.; Lee, J. H.; Kim, S. H.; Matsui, A.; Insin, N.; Bawendi, M. G.; Semmler–Behnke, M.; Frangioni, J. V.; Tsuda, A. Rapid translocation of nanoparticles from the lung airspaces to the body. Nat. Biotechnol. 2010, 28, 1300–1303.

165

Davis, M. E.; Zuckerman, J. E.; Choi, C. H. J.; Seligson, D.; Tolcher, A.; Alabi, C. A.; Yen, Y.; Heidel, J. D.; Ribas, A. Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles. Nature 2010, 464, 1067–1070.

166

Zuckerman, J. E.; Gritli, I.; Tolcher, A.; Heidel, J. D.; Lim, D.; Morgan, R.; Chmielowski, B.; Ribas, A.; Davis, M. E.; Yen, Y. Correlating animal and human phase Ia/Ib clinical data with CALAA–01, a targeted, polymer–based nanoparticle containing siRNA. Proc. Natl. Acad. Sci. USA 2014, 111, 11449–11454.

167

Rozema, D. B.; Lewis, D. L.; Wakefield, D. H.; Wong, S. C.; Klein, J. J.; Roesch, P. L.; Bertin, S. L.; Reppen, T. W.; Chu, Q. L.; Blokhin, A. V. et al. Dynamic polyconjugates for targeted in vivo delivery of siRNA to hepatocytes. Proc. Natl. Acad. Sci. USA 2007, 104, 12982–12987.

168

Wakefield, D. H.; Klein, J. J.; Wolff, J. A.; Rozema, D. B. Membrane activity and transfection ability of amphipathic polycations as a function of alkyl group size. Bioconjugate Chem. 2005, 16, 1204–1208.

169

Parmar, R. G.; Busuek, M.; Walsh, E. S.; Leander, K. R.; Howell, B. J.; Sepp–Lorenzino, L.; Kemp, E.; Crocker, L. S.; Leone, A.; Kochansky, C. J. et al. Endosomolytic bioreducible poly(amido amine disulfide) polymer conjugates for the in vivo systemic delivery of siRNA therapeutics. Bioconjugate Chem. 2013, 24, 640–647.

170

Parmar, R. G.; Poslusney, M.; Busuek, M.; Williams, J. M.; Garbaccio, R.; Leander, K.; Walsh, E.; Howell, B.; Sepp–Lorenzino, L.; Riley, S. et al. Novel endosomolytic poly(amido amine) polymer conjugates for systemic delivery of siRNA to hepatocytes in rodents and nonhuman primates. Bioconjugate Chem. 2014, 25, 896–906.

171

Wooddell, C. I.; Yuen, M. F.; Chan, H. L. Y.; Gish, R. G.; Locarnini, S. A.; Chavez, D.; Ferrari, C.; Given, B. D.; Hamilton, J.; Kanner, S. B. et al. RNAi–based treatment of chronically infected patients and chimpanzees reveals that integrated hepatitis B virus DNA is a source of HBsAg. Sci. Transl. Med. 2017, 9, eaan0241.

172

McKinlay, C. J.; Vargas, J. R.; Blake, T. R.; Hardy, J. W.; Kanada, M.; Contag, C. H.; Wender, P. A.; Waymouth, R. M. Charge–altering releasable transporters (CARTs) for the delivery and release of mRNA in living animals. Proc. Natl. Acad. Sci. USA 2017, 114, E448–E456.

173

Zorde Khvalevsky, E.; Gabai, R.; Rachmut, I. H.; Horwitz, E.; Brunschwig, Z.; Orbach, A.; Shemi, A.; Golan, T.; Domb, A. J.; Yavin, E. et al. Mutant KRAS is a druggable target for pancreatic cancer. Proc. Natl. Acad. Sci. USA 2013, 110, 20723–20728.

174

Dong, Y. Z.; Dorkin, J. R.; Wang, W. H.; Chang, P. H.; Webber, M. J.; Tang, B. C.; Yang, J.; Abutbul–Ionita, I.; Danino, D.; DeRosa, F. et al. Poly(glycoamidoamine) brushes formulated nanomaterials for systemic siRNA and mRNA delivery in vivo. Nano Lett. 2016, 16, 842–848.

175

Yang, X. Z.; Dou, S.; Sun, T. M.; Mao, C. Q.; Wang, H. X.; Wang, J. Systemic delivery of siRNA with cationic lipid assisted PEG–PLA nanoparticles for cancer therapy. J. Control. Release 2011, 156, 203–211.

176

Yang, X. Z.; Dou, S.; Wang, Y. C.; Long, H. Y.; Xiong, M. H.; Mao, C. Q.; Yao, Y. D.; Wang, J. Single–step assembly of cationic lipid–polymer hybrid nanoparticles for systemic delivery of siRNA. ACS Nano 2012, 6, 4955–4965.

177

Lv, S. J.; Wang, J.; Dou, S.; Yang, X. Z.; Ni, X.; Sun, R.; Tian, Z. G.; Wei, H. M. Nanoparticles encapsulating hepatitis B virus cytosine–phosphate–guanosine induce therapeutic immunity against HBV infection. Hepatology 2014, 59, 385–394.

178

Luo, Y. L.; Xu, C. F.; Li, H. J.; Cao, Z. T.; Liu, J.; Wang, J. L.; Du, X. J.; Yang, X. Z.; Gu, Z.; Wang, J. Macrophagespecific in vivo gene editing using cationic lipid–assisted polymeric nanoparticles. ACS Nano 2018, 12, 994–1005.

179

Shi, J. J.; Xiao, Z. Y.; Votruba Alexander, R.; Vilos, C.; Farokhzad Omid, C. Differentially charged hollow core/shell lipid–polymer–lipid hybrid nanoparticles for small interfering RNA delivery. Angew. Chem., Int. Ed. 2011, 50, 7027–7031.

180

Xu, X. Y.; Xie, K.; Zhang, X. Q.; Pridgen, E. M.; Park, G. Y.; Cui, D. S.; Shi, J. J.; Wu, J.; Kantoff, P. W.; Lippard, S. J. et al. Enhancing tumor cell response to chemotherapy through nanoparticle–mediated codelivery of siRNA and cisplatin prodrug. Proc. Natl. Acad. Sci. USA 2013, 110, 18638–18643.

181

Lynn, D. M.; Anderson, D. G.; Putnam, D.; Langer, R. Accelerated discovery of synthetic transfection vectors: Parallel synthesis and screening of a degradable polymer library. J. Am. Chem. Soc. 2001, 123, 8155–8156.

182

Green, J. J.; Langer, R.; Anderson, D. G. A combinatorial polymer library approach yields insight into nonviral gene delivery. Acc. Chem. Res. 2008, 41, 749–759.

183

Kozielski, K. L.; Tzeng, S. Y.; Green, J. J. A bioreducible linear poly(β–amino ester) for siRNA delivery. Chem. Commun. 2013, 49, 5319–5321.

184

Kozielski, K. L.; Tzeng, S. Y.; Hurtado De Mendoza, B. A.; Green, J. J. Bioreducible cationic polymer–based nanoparticles for efficient and environmentally triggered cytoplasmic siRNA delivery to primary human brain cancer cells. ACS Nano 2014, 8, 3232–3241.

185

Kaczmarek, J. C.; Patel, A. K.; Kauffman, K. J.; Fenton, O. S.; Webber, M. J.; Heartlein, M. W.; DeRosa, F.; Anderson, D. G. Polymer–lipid nanoparticles for systemic delivery of mRNA to the lungs. Angew. Chem., Int. Ed. 2016, 55, 13808–13812.

186

Su, X. F.; Fricke, J.; Kavanagh, D. G.; Irvine, D. J. In vitro and in vivo mRNA delivery using lipid–enveloped pH–responsive polymer nanoparticles. Mol. Pharmaceutics 2011, 8, 774–787.

187

Hao, J.; Elkassih, S.; Siegwart, D. J. Progress towards the synthesis of amino polyesters via ring–opening polymerization (ROP) of functional lactones. Synlett 2016, 27, 2285–2292.

188

Hao, J.; Kos, P.; Zhou, K. J.; Miller, J. B.; Xue, L.; Yan, Y. F.; Xiong, H.; Elkassih, S.; Siegwart, D. J. Rapid synthesis of a lipocationic polyester library via ring–opening polymerization of functional valerolactones for efficacious siRNA delivery. J. Am. Chem. Soc. 2015, 137, 9206–9209.

189

Yan, Y. F.; Liu, L.; Xiong, H.; Miller, J. B.; Zhou, K. J.; Kos, P.; Huffman, K. E.; Elkassih, S.; Norman, J. W.; Carstens, R. et al. Functional polyesters enable selective siRNA delivery to lung cancer over matched normal cells. Proc. Natl. Acad. Sci. USA 2016, 113, E5702–E5710.

190

Yan, Y. F.; Xue, L.; Miller, J. B.; Zhou, K. J.; Kos, P.; Elkassih, S.; Liu, L.; Nagai, A.; Xiong, H.; Siegwart, D. J. One–pot synthesis of functional poly(amino ester sulfide)s and utility in delivering pDNA and siRNA. Polymer 2015, 72, 271–280.

191

Yan, Y. F.; Siegwart, D. J. Scalable synthesis and derivation of functional polyesters bearing ene and epoxide side chains. Polym. Chem. 2014, 5, 1362–1371.

192

Yan, Y. F.; Xiong, H.; Zhang, X. Y.; Cheng, Q.; Siegwart, D. J. Systemic mRNA delivery to the lungs by functional polyester–based carriers. Biomacromolecules 2017, 18, 4307–4315.

193

Zangi, L.; Lui, K. O.; von Gise, A.; Ma, Q.; Ebina, W.; Ptaszek, L. M.; Später, D.; Xu, H. S.; Tabebordbar, M.; Gorbatov, R. et al. Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction. Nat. Biotechnol. 2013, 31, 898–907.

194

Kauffman, K. J.; Dorkin, J. R.; Yang, J. H.; Heartlein, M. W.; DeRosa, F.; Mir, F. F.; Fenton, O. S.; Anderson, D. G. Optimization of lipid nanoparticle formulations for mRNA delivery in vivo with fractional factorial and definitive screening designs. Nano Lett. 2015, 15, 7300–7306.

195

Yin, H.; Song, C. –Q.; Dorkin, J. R.; Zhu, L. J.; Li, Y. X.; Wu, Q. Q.; Park, A.; Yang, J.; Suresh, S.; Bizhanova, A. et al. Therapeutic genome editing by combined viral and non–viral delivery of CRISPR system components in vivo. Nat. Biotechnol. 2016, 34, 328–333.

196

Yin, H.; Song, C. –Q.; Suresh, S.; Wu, Q. Q.; Walsh, S.; Rhym, L. H.; Mintzer, E.; Bolukbasi, M. F.; Zhu, L. J.; Kauffman, K. et al. Structure–guided chemical modification of guide RNA enables potent non–viral in vivo genome editing. Nat. Biotechnol. 2017, 35, 1179–1187.

197

Li, B.; Luo, X.; Deng, B. B.; Wang, J. F.; McComb, D. W.; Shi, Y. M.; Gaensler, K. M. L.; Tan, X.; Dunn, A. L.; Kerlin, B. A. et al. An orthogonal array optimization of lipid–like nanoparticles for mRNA delivery in vivo. Nano Lett. 2015, 15, 8099–8107.

198

Fenton, O. S.; Kauffman, K. J.; McClellan, R. L.; Appel, E. A.; Dorkin, J. R.; Tibbitt, M. W.; Heartlein, M. W.; DeRosa, F.; Langer, R.; Anderson, D. G. Bioinspired alkenyl amino alcohol ionizable lipid materials for highly potent in vivo mRNA delivery. Adv. Mater. 2016, 28, 2939–2943.

199

Fenton, O. S.; Kauffman, K. J.; Kaczmarek, J. C.; McClellan, R. L.; Jhunjhunwala, S.; Tibbitt, M. W.; Zeng, M. D.; Appel, E. A.; Dorkin, J. R.; Mir, F. F. et al. Synthesis and biological evaluation of ionizable lipid materials for the in vivo delivery of messenger RNA to B lymphocytes. Adv. Mater. 2017, 29, 1606944.

200

Jiang, C.; Mei, M.; Li, B.; Zhu, X. R.; Zu, W. H.; Tian, Y. J.; Wang, Q. N.; Guo, Y.; Dong, Y. Z.; Tan, X. A non–viral CRISPR/Cas9 delivery system for therapeutically targeting HBV DNA and pcsk9 in vivo. Cell Res. 2017, 27, 440–443.

201

Li, B.; Luo, X.; Deng, B. B.; Giancola, J. B.; McComb, D. W.; Schmittgen, T. D.; Dong, Y. Z. Effects of local structural transformation of lipid–like compounds on delivery of messenger RNA. Sci. Rep. 2016, 6, 22137.

202

Dong, Y. Z.; Eltoukhy, A. A.; Alabi, C. A.; Khan, O. F.; Veiseh, O.; Dorkin, J. R.; Sirirungruang, S.; Yin, H.; Tang, B. C.; Pelet, J. M. et al. Lipid–like nanomaterials for simultaneous gene expression and silencing in vivo. Adv. Healthcare Mater. 2014, 3, 1392–1397.

203

Jarzębińska, A.; Pasewald, T.; Lambrecht, J.; Mykhaylyk, O.; Kümmerling, L.; Beck, P.; Hasenpusch, G.; Rudolph, C.; Plank, C.; Dohmen, C. A single methylene group in oligoalkylamine–based cationic polymers and lipids promotes enhanced mRNA delivery. Angew. Chem., Int. Ed. 2016, 55, 9591–9595.

204

Benenato, K. E.; Kumarasinghe, E. S.; Cornebise, M. Compounds and compositions for intracellular delivery of therapeutic agents: US Patent 20170210697. 2017–07–27.

205

Jinek, M.; Chylinski, K.; Fonfara, I.; Hauer, M.; Doudna, J. A.; Charpentier, E. A programmable Dual–RNA–Guided DNA endonuclease in adaptive bacterial immunity. Science 2012, 337, 816–821.

206

Cong, L.; Ran, F. A.; Cox, D.; Lin, S. L.; Barretto, R.; Habib, N.; Hsu, P. D.; Wu, X. B.; Jiang, W. Y.; Marraffini, L. A. et al. Multiplex genome engineering using CRISPR/cas systems. Science 2013, 339, 819–823.

207

Mali, P.; Yang, L. H.; Esvelt, K. M.; Aach, J.; Guell, M.; DiCarlo, J. E.; Norville, J. E.; Church, G. M. RNA–guided human genome engineering via Cas9. Science 2013, 339, 823–826.

208

Sanchez–Rivera, F. J.; Jacks, T. Applications of the CRISPR–Cas9 system in cancer biology. Nat. Rev. Cancer 2015, 15, 387–395.

209

Doudna, J. A.; Charpentier, E. The new frontier of genome engineering with CRISPR–Cas9. Science 2014, 346, 1258096.

210

Ran, F. A.; Hsu, P. D.; Wright, J.; Agarwala, V.; Scott, D. A.; Zhang, F. Genome engineering using the CRISPR–Cas9 system. Nat. Protoc. 2013, 8, 2281–2308.

211

Sternberg, S. H.; Redding, S.; Jinek, M.; Greene, E. C.; Doudna, J. A. DNA interrogation by the CRISPR RNAguided endonuclease Cas9. Nature 2014, 507, 62–67.

212

Davis, A. J.; Chen, D. J. DNA double strand break repair via non–homologous end–joining. Trans. Cancer Res. 2013, 2, 130–143.

213

Richardson, C. D.; Ray, G. J.; DeWitt, M. A.; Curie, G. L.; Corn, J. E. Enhancing homology–directed genome editing by catalytically active and inactive CRISPR–Cas9 using asymmetric donor DNA. Nat. Biotechnol. 2016, 34, 339–344.

214

Platt, R. J.; Chen, S. D.; Zhou, Y.; Yim, M. J.; Swiech, L.; Kempton, H. R.; Dahlman, J. E.; Parnas, O.; Eisenhaure, T. M.; Jovanovic, M. et al. CRISPR–Cas9 knockin mice for genome editing and cancer modeling. Cell 2014, 159, 440–455.

215

Xue, W.; Chen, S. D.; Yin, H.; Tammela, T.; Papagiannakopoulos, T.; Joshi, N. S.; Cai, W. X.; Yang, G.; Bronson, R.; Crowley, D. G. et al. CRISPR–mediated direct mutation of cancer genes in the mouse liver. Nature 2014, 514, 380–384.

216

Yin, H.; Xue, W.; Chen, S. D.; Bogorad, R. L.; Benedetti, E.; Grompe, M.; Koteliansky, V.; Sharp, P. A.; Jacks, T.; Anderson, D. G. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat. Biotechnol. 2014, 32, 551–553.

217

Chen, S. D.; Sanjana, N. E.; Zheng, K. J.; Shalem, O.; Lee, K.; Shi, X.; Scott, D. A.; Song, J.; Pan, J. Q.; Weissleder, R. et al. Genome–wide CRISPR screen in a mouse model of tumor growth and metastasis. Cell 2015, 160, 1246–1260.

218

Zetsche, B.; Gootenberg, J. S.; Abudayyeh, O. O.; Slaymaker, I. M.; Makarova, K. S.; Essletzbichler, P.; Volz, S. E.; Joung, J.; van der Oost, J.; Regev, A. et al. Cpf1 is a single RNA–guided endonuclease of a class 2 CRISPR–cas system. Cell 2015, 163, 759–771.

219

Komor, A. C.; Kim, Y. B.; Packer, M. S.; Zuris, J. A.; Liu, D. R. Programmable editing of a target base in genomic DNA without double–stranded DNA cleavage. Nature 2016, 533, 420–424.

220

Komor, A. C.; Zhao, K. T.; Packer, M. S.; Gaudelli, N. M.; Waterbury, A. L.; Koblan, L. W.; Kim, Y. B.; Badran, A. H.; Liu, D. R. Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C: G–to–T: A base editors with higher efficiency and product purity. Sci. Adv. 2017, 3, eaan4774.

221

Hu, J. H.; Miller, S. M.; Geurts, M. H.; Tang, W. X.; Chen, L. W.; Sun, N.; Zeina, C. M.; Gao, X.; Rees, H. A.; Lin, Z. et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature 2018, 556, 57–63.

222

Han, X.; Liu, Z. B.; Jo, M. C.; Zhang, K.; Li, Y.; Zeng, Z. H.; Li, N.; Zu, Y. L.; Qin, L. D. CRISPR–Cas9 delivery to hard–to–transfect cells via membrane deformation. Sci. Adv. 2015, 1, e1500454.

223

Zuris, J. A.; Thompson, D. B.; Shu, Y. L.; Guilinger, J. P.; Bessen, J. L.; Hu, J. H.; Maeder, M. L.; Joung, J. K.; Chen, Z. Y.; Liu, D. R. Cationic lipid–mediated delivery of proteins enables efficient protein–based genome editing in vitro and in vivo. Nat. Biotechnol. 2015, 33, 73–80.

224

Wang, M.; Zuris, J. A.; Meng, F. T.; Rees, H.; Sun, S.; Deng, P.; Han, Y.; Gao, X.; Pouli, D.; Wu, Q. et al. Efficient delivery of genome–editing proteins using bioreducible lipid nanoparticles. Proc. Natl. Acad. Sci. USA 2016, 113, 2868–2873.

225

Sun, W. J.; Ji, W. Y.; Hall, J. M.; Hu, Q. Y.; Wang, C.; Beisel, C. L.; Gu, Z. Self–assembled DNA nanoclews for the efficient delivery of CRISPR–Cas9 for genome editing. Angew. Chem., Int. Ed. 2015, 54, 12029–12033.

226

Wang, M.; Glass, Z. A.; Xu, Q. Non–viral delivery of genome–editing nucleases for gene therapy. Gene Ther. 2017, 24, 144–150.

227

Nishimasu, H.; Ran, F. A.; Hsu, P. D.; Konermann, S.; Shehata, S. I.; Dohmae, N.; Ishitani, R.; Zhang, F.; Nureki, O. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 2014, 156, 935–949.

228

Morrissey, D. V.; Patel, M. C.; Finn, J. D.; Smith, A. M. R.; Shaw, L. J.; Dombrowski, C.; Shah, R. R. Lipid nanoparticle formulations for CRISPR/Cas components: US Patent Application PCT/US2017/024973. 2017–03–30.

229

Finn, J. D.; Smith, A. R.; Patel, M. C.; Shaw, L.; Youniss, M. R.; van Heteren, J.; Dirstine, T.; Ciullo, C.; Lescarbeau, R.; Seitzer, J. et al. A single administration of CRISPR/Cas9 lipid nanoparticles achieves robust and persistent in vivo genome editing. Cell Rep. 2018, 22, 2227–2235.

Publication history
Copyright
Acknowledgements

Publication history

Received: 21 March 2018
Revised: 15 May 2018
Accepted: 18 May 2018
Published: 07 August 2018
Issue date: October 2018

Copyright

© Tsinghua University Press and Springer-Verlag GmbH Germany, part of Springer Nature 2018

Acknowledgements

Acknowledgements

D. J. S. acknowledges financial support from the Welch Foundation (I-1855), American Cancer Society (RSG-17-012-01), Department of Defense (CA150245P3), and Cancer Prevention and Research Institute of Texas (CPRIT) (R1212).

Return