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“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) Barenholz
Hebrew 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 Torchilin
Northeastern 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, immunetoxicity, pharmacokinetics, efficacy, and validation of new drug targets using unique
approaches based on exquisite nanotechnology strategies.
V335
ISBN 978-981-4411-04-2
9-78981-4411042
Nanotechnology
for the Delivery of
Therapeutic Nucleic Acids
Peer
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.
Nanotechnology for the Delivery 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 Ferrari
The Methodist Hospital Research Institute, USA
Pan Stanford
Series on
Biomedical
Nanotechnology
Volume 4
Dan Peer
Editor
Nanotechnology
for the Delivery of
Therapeutic Nucleic Acids
Pan Stanford Series on Biomedical Nanotechnology
Series Editors
Vladimir Torchilin and Monsoor Amiji
Titles in the Series
Vol. 1
Handbook of Materials
for Nanomedicine
Vladimir Torchilin and Monsoor Amiji,
eds. 2010
Vol. 5
Inorganic Nanomedicine
Bhupinder Singh Sekhon, ed.
2014
978-981-4267-55-7 (Hardcover)
978-981-4267-58-8 (eBook)
Vol. 6
Nanotechnology for Cancer
Vol. 2
Nanoimaging
Julia Ljubimova, ed.
2014
Beth A. Goins and William T. Phillips,
eds. 2011
978-981-4267-09-0 (Hardcover)
978-981-4267-91-5 (eBook)
Vol. 3
Biomedical Nanosensors
Joseph Irudayraj, ed.
2013
978-981-4303-03-3 (Hardcover)
978-981-4303-04-0 (eBook)
Vol. 4
Nanotechnology for the Delivery
of Therapeutic Nucleic Acids
Dan Peer, ed.
2013
978-981-4411-04-2 (Hardcover)
978-981-4411-05-9 (eBook)
Vol. 7
Nanotechnology for Delivery of
DNA and Related Materials
Bengt Fadeel, ed.
2015
Vol. 8
Translation Industrial
Nanotechnology
Thomas Redelmeier, ed.
2015
Nanotechnology
for the Delivery of
Therapeutic Nucleic Acids
Dan Peer
Editor
Published by
Pan Stanford Publishing Pte. Ltd.
Penthouse Level, Suntec Tower 3
8 Temasek Boulevard
Singapore 038988
Email: [email protected]
Web: www.panstanford.com
British Library Cataloguing-in-Publication Data
A 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
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For photocopying of material in this volume, please pay a copying fee
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required from the publisher.
ISBN 978-981-4411-04-2 (Hardcover)
ISBN 978-981-4411-05-9 (eBook)
Printed in the USA
Contents
Preface
1. Lipoplexes and Polyplexes: From Gene Delivery to
Gene Expression
Gerardo Byk, Mirit Cohen-Ohana, and Fiana Mirkin
1.1 Introduction
1.2 Lipopolyamines
1.3 Lipopolyamine Co-formulation with DNA
Complexing Peptides
1.4 Lipopolyaminoguanidines
1.4.1 Biodegradable Lipoplexes: Reduction-Sensitive
Lipopolyamines
1.4.2 Biodegradable Polyplexes: Reduction-Sensitive
Dendrimers
1.5 Towards Non-Electrostatic DNA Complexing
Agents
1.6 Site-Specific Chemical Ligation of Targeting
Peptides to Plasmid DNA
1.7 Concluding Remarks and Future Directions
Wahid Khan, Saravanan Muthupandian, and Abraham J. Domb
2.1 Introduction
2.2 Cationic Polymer Targeted Delivery of Nucleotides
2.3 Major Cationic Polymers Used for Delivery of
Nucleotides
2.3.1 Polyethylenimine
2.3.2 Poly(L-lysine)
2.3.3 Cationic Polysaccharides
2. Cationic Polymers for the Delivery of Therapeutic
Nucleotides
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1
1
4
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15
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27
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31
31
35
37
vi
Contents
2.3.3.1 Chitosan
2.3.3.2 Cyclodextrins 2.3.3.3 Dextran, dextran-spermine 2.3.4 Dendrimers
2.3.5 Other Cationic Polymers
2.3.5.1 Cationic polyesters
2.3.5.2 Poly(amino ester)s
2.3.5.3 Poly(amido amine)s 2.4 Factors Influencing Cationic Polymer Mediated
Nucleotides Delivery
2.5 Biomedical Applications
2.5.1 Tumor Therapy
2.5.2 siRNA Delivery
2.5.3 DNA Vaccination
2.5.4 Lung and Liver Delivery
2.5.5 Brain Delivery
2.6 Conclusion
Younjee Chung and Leaf Huang
3.1
3.2
3.3
3.4
3. Membrane/Core Nanoparticles for Delivery of Therapeutic
Nucleic Acid
Introduction
Challenges in Nanocarrier Systems
Current Non-Viral Carrier Systems
Membrane/Core NPs
3.4.1 LPD 3.4.1.1 Formulation of LPD
3.4.1.2 The effect of surface modification
of LPD 3.4.1.3 Therapeutic applications of LPD
3.4.1.4 Modified LPD formulations
3.4.2 LCP 3.4.2.1 Physicochemical characteristic
of LCP
3.4.2.2 Potential therapeutic effect of LCP
3.5 Conclusion
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Contents
4. Delivery of Single siRNA Molecules
Caroline Palm-Apergi and Steven F. Dowdy
4.1 Introduction
4.1.1 RNA Interference
4.1.2 Modification of siRNAs
4.1.3 Off-Target Effects
4.2 Delivery of siRNA
4.2.1 Peptide Transduction Domains
4.2.2 Delivery of siRNA-PTD Nanoparticles
4.2.3 RNA Binding Proteins
4.2.4 Delivery of Single siRNA Molecules by
PTD-DRBD
4.3 Discussion
4.4 Conclusions
Jiehua Zhou and John J. Rossi
5.1 Introduction
5.2 Generation of Cell-Specific Aptamers
5.2.1 Recombinant Protein-Based SELEX
Procedure
5.2.2 Whole Cell-Based SELEX Procedure
5.3 Cell-Specific Aptamer-Functionalized RNAi
5.3.1 Cell-Specific Aptamer-Functionalized
siRNAs 5.3.1.1 PSMA RNA aptamer-functionalized
siRNAs
5.3.1.2 HIV gp120 RNA aptamerfunctionalized siRNAs
5.3.1.3 CD4 RNA aptamer-functionalized
siRNAs
5.3.2 Cell-Specific Aptamer-Functionalized
Therapeutic Nanocarriers 5.3.2.1 CD4 RNA aptamer-functionalized
pRNA-nanoparticles
5. Cell-Specific Aptamer-Functionalized RNAi: A New
Prospect for Targeted siRNA Delivery
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viii
Contents
5.3.2.2 PSMA RNA aptamer-functionalized
polymer nanocarriers
5.3.2.3 CD30 RNA aptamer-functionalized
polymer nanocarriers
5.4 Conclusions and Perspectives
6. Bioresponsive Nanoparticles for the Intracellular
Delivery of RNAi Therapeutics
Kenneth Alan Howard
6.1 Introduction
6.2 Repertoire of Potential RNAi Therapeutics 6.3 Nanoparticle-Based Delivery of RNAi Therapeutics
6.3.1 Polycation-Based Nanoparticles
6.3.2 Bioresponsive Systems
6.4 Copolypeptide System
6.5 Hyperbranched System
6.6 Conclusion
James Dahlman, Robert Langer, and Michael Goldberg
7.1 Introduction
7.2 Motivation: Need for Novel siRNA Carriers in vivo
7.3 Approach: Efficient Chemistry Allows for HighThroughput Combinatorial Library Synthesis
and Screening
7.4 Translation: Moving from in vitro to in vivo
Screening
7.5 Optimization: Formulation Parameters Greatly
Influence Carrier Efficacy
7.6 Synergy: Combining Existing Compounds to
Achieve Improved Delivery
7.7 Next-Generation: Identifying Improved Carriers
Using Innovative Chemistry
7.8 Applications: Using Lipidoids to Treat Disease
Models
7.9 Future Directions and Conclusions
7. Lipid-Like Delivery Materials for Efficient siRNA
Delivery 118
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Contents
8. Manipulation of Leukocytes Using Therapeutic RNAi
Delivered by Targeted and Stabilized Nanoparticles
Dan Peer
8.1
8.2
8.3
8.4
8.5
8.6
8.7
8.8
8.9
8.10
Introduction Strategies for RNAi Delivery into Leukocytes
CpG-Conjugated siRNA
Atelocollagen-Complexed siRNA
Cationic Nona-d-Arginine Peptide-Complexed
siRNA I-tsNP as RNAi Delivery Vehicle for LeukocyteAssociated Diseases
Leukocyte Integrins as Targets for siRNA Delivery The Construction and Characterization of I-tsNP In vivo Gene Silencing Using I-tsNP-Entrapping
siRNAs
Conclusion
9. Lowering the siRNA Delivery Barrier: Alginate Scaffolds
and Immune Stimulation
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193
Jana McCaskill, Sherry Wu, Norliana Khairuddin, and
Nigel A. J. McMillan
9.1 Introduction
9.2 siRNA Delivery Systems: A Brief Overview
9.2.1 siRNA Conjugate Delivery
9.2.2 Peptide-Based Delivery Particles
9.2.3 Polymer-Based Delivery Vectors
9.2.4 Lipid-Based Delivery Particles
9.3 HDFM: A Novel Method for Formulating Stable
siRNA-Loaded Lipid Particles for in vivo Use 9.4 The Challenge of the Vaginal Tract 9.5 Vaginal Delivery of siRNA Using a Novel PEGylated
Lipoplex-Entrapped Alginate Scaffold System
9.6 Thinking Outside the Box: Bi-Functional siRNAs
9.7 siRNA-Induced Immunostimulation Promotes
Anti-tumoural Activity in vivo
9.8 Conclusion
Index 193
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217
ix
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 incorporating 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, lipopolyaminoguanidines, 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.
Preface
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 nanoparticles 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 Peer
Tel Aviv, Winter 2012
xiii