Peer

Dan PeerEditor

Nanotechnologyfor the Delivery of

Therapeutic Nucleic Acids

9-78981-4411042ISBN 978-981-4411-04-2

V335

Nanotechnology for the D

elivery of Therapeutic Nucleic Acids

“Nucleic acid–based therapy would have revolutionized medicine many times if the problem of delivery had been solved, at least in small part. However, that is proving to be as challenging a problem as any in the sciences—and of the few with truly transformational implications for the health of all. Thus, I welcome with enthusiasm this book, edited by my good friend and extraordinarily distinguished colleague Dan Peer. The topics featured in the various chapters offer a very sound review of the major problem areas, and some of the most promising strategies for addressing them.”

Prof. Mauro FerrariThe Methodist Hospital Research Institute, USA

“This excellent book comes at a critical time to the field of delivery of therapeutic nucleic acids. I wish I had a book with a similar approach and design in the field of drug delivery when we started to develop Doxil®. I intend to use this book to cover nucleic acid delivery in my graduate course on drug delivery systems.”

Prof. Yechezkel (Chezy) BarenholzHebrew University of Jerusalem, Israel

“This timely and nicely arranged book represents an excellent collection of cutting-edge studies and approaches for the delivery of nucleic acid–based therapeutics, including siRNA. The editor has built a volume in which leading scientists cover the broad variety of nucleic acid delivery platforms, such as lipid- and polymer-based systems, aptamers, and chemical conjugates, as well as biological properties of these systems.”

Prof. Vladimir TorchilinNortheastern University, USA

Nucleic acid (NA) therapeutics has been extensively studied both in the academia and in the pharmaceutical industry and is still considered the promise for new therapeutic modalities, especially in personalized medicine. The only hurdle that limits the translation of NA therapeutics from an academic idea to the new therapeutic modality is the lack of efficient and safe delivery strategies. In this book, written by world experts in the field of nanotechnology for NA delivery, the contributing authors bring together the state of the art in delivery strategies with strong emphasis on aspects that are of essence to the pharmaceutical industry, such as stability, general toxicity, immune-toxicity, pharmacokinetics, efficacy, and validation of new drug targets using unique approaches based on exquisite nanotechnology strategies.

Dan Peer is an associate professor and head of the laboratory of NanoMedicine at Tel Aviv University. His research was one of the first to demonstrate the systemic delivery of RNAi using targeted nanocarriers to the immune system and the first to demonstrate the in vivo validation of new drug targets using RNAi in the immune system. Prof. Peer has authored and edited several books on biomaterials and nanomedicine. He is on the editorial board of several journals, including Nanotechnology, Journal of

Controlled Release, Journal of Biomedical Nanotechnology, Biomedical Microdevices, and Cancer Letters.

Pan StanfordSeries on

BiomedicalNanotechnology

Volume 4

Nanotechnologyfor the Delivery of

Therapeutic Nucleic Acids

Titles in the Series

Vol. 1Handbook of Materials for NanomedicineVladimir Torchilin and Monsoor Amiji, eds. 2010

978-981-4267-55-7 (Hardcover)978-981-4267-58-8 (eBook)

Vol. 2NanoimagingBeth A. Goins and William T. Phillips, eds. 2011

978-981-4267-09-0 (Hardcover)978-981-4267-91-5 (eBook)

Vol. 3Biomedical NanosensorsJoseph Irudayraj, ed. 2013

978-981-4303-03-3 (Hardcover)978-981-4303-04-0 (eBook)

Vol. 4Nanotechnology for the Delivery of Therapeutic Nucleic AcidsDan Peer, ed. 2013

978-981-4411-04-2 (Hardcover)978-981-4411-05-9 (eBook)

Vol. 5Inorganic NanomedicineBhupinder Singh Sekhon, ed. 2014

Vol. 6Nanotechnology for CancerJulia Ljubimova, ed. 2014

Vol. 7Nanotechnology for Delivery of DNA and Related MaterialsBengt Fadeel, ed. 2015

Vol. 8Translation Industrial NanotechnologyThomas Redelmeier, ed. 2015

Pan Stanford Series on Biomedical Nanotechnology

Series Editors

Vladimir Torchilin and Monsoor Amiji

Nanotechnologyfor the Delivery of

Therapeutic Nucleic Acids

Dan PeerEditor

Published by

Pan Stanford Publishing Pte. Ltd.Penthouse Level, Suntec Tower 3 8 Temasek Boulevard Singapore 038988 Email: editorial@panstanford.com Web: www.panstanford.com

British Library Cataloguing-in-Publication DataA catalogue record for this book is available from the British Library.

Nanotechnology for the Delivery of Therapeutic Nucleic Acids

Copyright © 2013 Pan Stanford Publishing Pte. Ltd.All rights reserved. This book, or parts thereof, may not be reproduced in any form or by any means, electronic or mechanical, including photocopying, recording or any information storage and retrieval system now known or to be invented, without written permission from the publisher.

For photocopying of material in this volume, please pay a copying fee through the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA. In this case permission to photocopy is not required from the publisher.

ISBN 978-981-4411-04-2 (Hardcover)ISBN 978-981-4411-05-9 (eBook)

Printed in the USA

Contents

Preface xi

1. Lipoplexes and Polyplexes: From Gene Delivery to Gene Expression 1

Gerardo Byk, Mirit Cohen-Ohana, and Fiana Mirkin

1.1 Introduction 1 1.2 Lipopolyamines 4 1.3 Lipopolyamine Co-formulation with DNA

Complexing Peptides 9 1.4 Lipopolyaminoguanidines 10 1.4.1 Biodegradable Lipoplexes: Reduction-Sensitive

Lipopolyamines 12 1.4.2 Biodegradable Polyplexes: Reduction-Sensitive

Dendrimers 15 1.5 Towards Non-Electrostatic DNA Complexing

Agents 18 1.6 Site-Specific Chemical Ligation of Targeting

Peptides to Plasmid DNA 20 1.7 Concluding Remarks and Future Directions 20

2. Cationic Polymers for the Delivery of Therapeutic Nucleotides 27

Wahid Khan, Saravanan Muthupandian, and Abraham J. Domb

2.1 Introduction 28 2.2 Cationic Polymer Targeted Delivery of Nucleotides 29 2.3 Major Cationic Polymers Used for Delivery of

Nucleotides 31 2.3.1 Polyethylenimine 31 2.3.2 Poly(L-lysine) 35 2.3.3 Cationic Polysaccharides 37

vi Contents

2.3.3.1 Chitosan 38 2.3.3.2 Cyclodextrins 39 2.3.3.3 Dextran, dextran-spermine 40

2.3.4 Dendrimers 41 2.3.5 Other Cationic Polymers 42

2.3.5.1 Cationic polyesters 42 2.3.5.2 Poly(amino ester)s 44 2.3.5.3 Poly(amido amine)s 45

2.4 Factors Influencing Cationic Polymer Mediated Nucleotides Delivery 46

2.5 Biomedical Applications 47 2.5.1 Tumor Therapy 47 2.5.2 siRNA Delivery 48 2.5.3 DNA Vaccination 49 2.5.4 Lung and Liver Delivery 49 2.5.5 Brain Delivery 50 2.6 Conclusion 50

3. Membrane/Core Nanoparticles for Delivery of Therapeutic Nucleic Acid 57

Younjee Chung and Leaf Huang

3.1 Introduction 58 3.2 Challenges in Nanocarrier Systems 60 3.3 Current Non-Viral Carrier Systems 62 3.4 Membrane/Core NPs 65 3.4.1 LPD 67

3.4.1.1 Formulation of LPD 67 3.4.1.2 The effect of surface modification

of LPD 69 3.4.1.3 Therapeutic applications of LPD 71 3.4.1.4 Modified LPD formulations 73

3.4.2 LCP 76 3.4.2.1 Physicochemical characteristic

of LCP 78 3.4.2.2 Potential therapeutic effect of LCP 78

3.5 Conclusion 80

viiContents

4. Delivery of Single siRNA Molecules 93

Caroline Palm-Apergi and Steven F. Dowdy

4.1 Introduction 94 4.1.1 RNA Interference 94 4.1.2 Modification of siRNAs 95 4.1.3 Off-Target Effects 96 4.2 Delivery of siRNA 96 4.2.1 Peptide Transduction Domains 96 4.2.2 Delivery of siRNA-PTD Nanoparticles 97 4.2.3 RNA Binding Proteins 98 4.2.4 Delivery of Single siRNA Molecules by

PTD-DRBD 99 4.3 Discussion 100 4.4 Conclusions 102

5. Cell-Specific Aptamer-Functionalized RNAi: A New Prospect for Targeted siRNA Delivery 107

Jiehua Zhou and John J. Rossi

5.1 Introduction 108 5.2 Generation of Cell-Specific Aptamers 111 5.2.1 Recombinant Protein-Based SELEX

Procedure 111 5.2.2 Whole Cell-Based SELEX Procedure 112 5.3 Cell-Specific Aptamer-Functionalized RNAi 114 5.3.1 Cell-Specific Aptamer-Functionalized

siRNAs 115 5.3.1.1 PSMA RNA aptamer-functionalized

siRNAs 115 5.3.1.2 HIV gp120 RNA aptamer-

functionalized siRNAs 116 5.3.1.3 CD4 RNA aptamer-functionalized

siRNAs 117 5.3.2 Cell-Specific Aptamer-Functionalized

Therapeutic Nanocarriers 117 5.3.2.1 CD4 RNA aptamer-functionalized

pRNA-nanoparticles 118

viii Contents

5.3.2.2 PSMA RNA aptamer-functionalized polymer nanocarriers 118

5.3.2.3 CD30 RNA aptamer-functionalized polymer nanocarriers 119

5.4 Conclusions and Perspectives 119

6. Bioresponsive Nanoparticles for the Intracellular Delivery of RNAi Therapeutics 129

Kenneth Alan Howard

6.1 Introduction 129 6.2 Repertoire of Potential RNAi Therapeutics 130 6.3 Nanoparticle-Based Delivery of RNAi Therapeutics 132 6.3.1 Polycation-Based Nanoparticles 132 6.3.2 Bioresponsive Systems 133 6.4 Copolypeptide System 134 6.5 Hyperbranched System 140 6.6 Conclusion 144

7. Lipid-Like Delivery Materials for Efficient siRNA Delivery 153

James Dahlman, Robert Langer, and Michael Goldberg

7.1 Introduction 154 7.2 Motivation: Need for Novel siRNA Carriers in vivo 155 7.3 Approach: Efficient Chemistry Allows for High-

Throughput Combinatorial Library Synthesis and Screening 155

7.4 Translation: Moving from in vitro to in vivo Screening 158

7.5 Optimization: Formulation Parameters Greatly Influence Carrier Efficacy 160

7.6 Synergy: Combining Existing Compounds to Achieve Improved Delivery 161

7.7 Next-Generation: Identifying Improved Carriers Using Innovative Chemistry 162

7.8 Applications: Using Lipidoids to Treat Disease Models 166

7.9 Future Directions and Conclusions 170

ixContents

8. Manipulation of Leukocytes Using Therapeutic RNAi Delivered by Targeted and Stabilized Nanoparticles 179

Dan Peer

8.1 Introduction 180 8.2 Strategies for RNAi Delivery into Leukocytes 182 8.3 CpG-Conjugated siRNA 184 8.4 Atelocollagen-Complexed siRNA 184 8.5 Cationic Nona-d-Arginine Peptide-Complexed

siRNA 185 8.6 I-tsNP as RNAi Delivery Vehicle for Leukocyte-

Associated Diseases 185 8.7 Leukocyte Integrins as Targets for siRNA Delivery 186 8.8 The Construction and Characterization of I-tsNP 186 8.9 In vivo Gene Silencing Using I-tsNP-Entrapping

siRNAs 187 8.10 Conclusion 188

9. Lowering the siRNA Delivery Barrier: Alginate Scaffolds and Immune Stimulation 193

Jana McCaskill, Sherry Wu, Norliana Khairuddin, and Nigel A. J. McMillan

9.1 Introduction 193 9.2 siRNA Delivery Systems: A Brief Overview 194 9.2.1 siRNA Conjugate Delivery 194 9.2.2 Peptide-Based Delivery Particles 195 9.2.3 Polymer-Based Delivery Vectors 196 9.2.4 Lipid-Based Delivery Particles 197 9.3 HDFM: A Novel Method for Formulating Stable

siRNA-Loaded Lipid Particles for in vivo Use 198 9.4 The Challenge of the Vaginal Tract 200 9.5 Vaginal Delivery of siRNA Using a Novel PEGylated

Lipoplex-Entrapped Alginate Scaffold System 202 9.6 Thinking Outside the Box: Bi-Functional siRNAs 205 9.7 siRNA-Induced Immunostimulation Promotes

Anti-tumoural Activity in vivo 208 9.8 Conclusion 210Index 217

Preface

More than three decades ago, Paul Zamecnik and his colleagues suggested that nucleic acids (NA) could be used to block gene function by virtue of Watson–Crick base pairing. Since the discovery of RNAi in 1998 by Andrew Fire and Craig Melo and soon after the discovery that RNAi is found in mammals in 2001 by Thomas Tuschl’s group, synthetic small RNAs were shown to treat disease in mice. Small RNAs were quickly proclaimed as the “next new class of drugs.” Eagerness sprinted high because of the potential of these molecules to knock down any gene of interest to treat almost any disease by targeting otherwise “undruggable” targets such as molecules without ligand-binding domains or enzymatic function. Despite the promise, developing any NA as therapeutics has proven challenging. Like most drug development, there is no quick fix. Although many of the hurdles to developing NA-based drugs have been easily addressed, the main obstacle is figuring out how to deliver these molecules into cells in a therapeutically acceptable way. Small RNAs being considered therapeutic drugs include not only siRNAs designed to knock down one gene at a time but also mimics of endogenous microRNAs to suppress the expression of many genes, but with less efficient suppression of each one. The delivery hurdle that needs to be solved to administer siRNAs and imperfectly paired microRNA mimics is essentially the same (although antagonizing endogenous micro RNAs using single-stranded antisense oligonucleotides may be somewhat easier). When injected intravenously, NA are rapidly cleared by renal filtration and are susceptible to degradation by extracellular RNases or DNases. The NA half-life can be increased—even to days—by chemical modifications to eliminate susceptibility to endogenous exonucleases and endonucleases and by incorpora-ting the NA into a larger moiety, above the molecular weight cutoff for kidney filtration. However, entering the cell is the biggest obstacle. Because of their large molecular weight and net negative charge, naked NA do not cross the plasma membrane. Although cells can endocytose many types of modified NA or NA-containing particles, another important bottleneck is getting these molecules

xii Preface

efficiently out of the endosome into the cytosol where the RNAi machinery resides or into the nucleus for DNA to work. NA therapeutics has been extensively studied both in the academia and in the pharmaceutical industry and is still considered the promise for new therapeutic modalities, especially in personalized medicine. The only hurdle that limits the translation of NA therapeutics from an academic idea to new therapeutic modality is the lack of efficient and safe delivery strategies. In this book, written by world experts in the field of nanotechnology for NA delivery, we bring together the state of the art in delivery strategies using lipids, polymers, chemical conjugates, NA aptamers, and proteins with strong emphasis on issues and aspects that are of essence to the pharmaceutical industry working in this area such as stability, general toxicity, immune-toxicity, pharmacokinetics and naturally efficacy and validation of new drug targets in vivo using unique approaches based on exquisite nanotechnology strategies. The work by Prof. Gerardo Byk and colleagues (Chapter 1) provides a tutorial overview of lipoplex and polyplex from a chemical standpoint. Discussions about lipopolyamines, lipopoly-aminoguanidines, and reduction-sensitive lipopolyamine and dendrimers provide new insights into chemical modifications toward non-electrostatic DNA complexing agents. The work by Prof. Avi Domb and colleagues (Chapter 2) provides an excellent overview on the major cationic polymers used for the delivery of nucleotides, among them polyethylenimine, poly (L-lysine), cationic polysaccharides (such as chitosan, cyclodextrins, and dextran-spermine), dendrimers, cationic polyesters, poly(amino ester)s, and poly(amido amine)s. Factors influencing cationic polymer-mediated nucleotide delivery are also discussed. In addition, several biomedical applications are discussed, such as siRNA delivery, DNA vaccination, lung and liver delivery, brain delivery, and tumor delivery. Chapter 3, authored by Prof. Leaf Huang and colleagues, provides an introduction to the challenges in nanocarriers systems for NA delivery. It details two strategies of membrane/core NPs based on lipids, the LPD, and the LCD and discusses several applications in siRNA delivery using these strategies. Another interesting strategy is the delivery of single siRNA molecules by peptide transduction domains as described by Steve Dowdy and colleagues in Chapter 4. Additional RNA binding proteins are also detailed.

xiii

Chapter 5, written by Prof. John Rossi and colleagues, reviews the current advances of cell-specific aptamers in cell recognition and targeted delivery, with a particular focus on the development of the aptamer-functionalized siRNA or nanocarrier for targeted gene silencing. Prof. Ken Howard details in Chapter 6 bioresponsive nano-particles based on copolypeptides and hyperbranched polymers for controlling the intracellular spatial and temporal effects of synthetic microRNA and siRNA. In Chapter 7, Prof. Robert Langer and Prof. Michael Goldberg describe the synthesis, screening, formulation, evolution, and application of “lipidoids,” a novel class of lipid-like molecules that highlights the utility of combinatorial approaches for the production of effective siRNA delivery vehicles. My personal contribution to this book is Chapter 8, in which I detail the use of integrin targeted and stabilized lipid-based nanoparticles for the manipulation of leukocytes’ function using RNAi. Finally, Prof. Nigel McMillan and his colleagues outline efforts to improve not only delivery but also RNAi efficacy in the vaginal mucosa as a means to treat genital infections, particularly virally driven cervical cancer, using various strategies. Clear, easy to understand, and focused on key issues for future research and development, this book provide new insights into the dynamic field of NA delivery using nanotechnology. I am grateful to all the authors who contributed to this book, among them Prof. Byk, from Bar-Ilan University, Prof. Domb from the Hebrew University in Jerusalem, Prof. Huang from the University of North Carolina at Chapel Hill, Prof. Dowdy from the University of California San Diego, Prof. Rossi from the City of Hope in California, Prof. Howard from the University of Aarhus, Prof. Langer from MIT, Prof. Goldberg from Harvard Medical School, and Prof. McMillan from the University of Queensland. Special thanks to my wife, Shlomit, and my children, Dor, Barak, and Naama, for their unrestricted support. This book is dedicated to the memory of my parents, Itta and Alexander Peer, who educated me to strive for knowledge and excellence.

Dan PeerTel Aviv, Winter 2012

Preface