Design and recombinant production of combinatorial peptide libraries for gene delivery

113 Pages·2013·9.76 MB·English

Design and recombinant production
of combinatorial peptide libraries
for gene delivery
Markus de Raad
Design and recombinant production
of combinatorial peptide libraries
for gene delivery
Ontwerp en recombinante productie
van een verzameling combinatoriële peptiden
The printing of this thesis was financially supported by: voor genafgifte
(met een samenvatting in het Nederlands)
Proefschrift
Design and recombinant production
of combinatorial peptide libraries for gene delivery
Markus de Raad
Ph.D. Thesis
Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS),
ter verkrijging van de graad van doctor
Faculty of Science, Utrecht University, The Netherlands
aan de Universiteit Utrecht
November 2013
op gezag van de rector magnificus, prof.dr. G.J. van der Zwaan,
ingevolge het besluit van het college voor promoties
ISBN: 978-90-393-6059-0
in het openbaar te verdedigen op woensdag 20 november 2013
des middags te 12.45 uur
Cover
Promotie In Zicht en Markus de Raad
Layout
Promotie In Zicht, Arnhem door
Print
CPI Wöhrmann Print Service, Zutphen
Markus de Raad
Copyright © 2013 Markus de Raad geboren op 13 maart 1984
te Gouda
All rights reserved. No parts of this book may be reproduced in any form or by any means
without permission of the author.
Promotoren
Prof.dr. D.J.A. Crommelin
Prof.dr. P.J.M. Rottier
Copromotor
Dr. E. Mastrobattista
“Here comes a delivery, straight from the heart to you”
Delivery - Babyshambles
This research is supported by the Dutch Technology Foundation STW, which is part
of the Netherlands Organisation for Scientific Research (NWO), and which is partly
funded by the Ministry of Economic Affairs (project number 10243).
Table of contents
Chapter 1 General Introduction 9
Chapter 2 Peptide Vectors for Gene Delivery: From Single Peptides 19
to Multifunctional Peptide Nanocarriers
Chapter 3 A Solid-Phase Platform for Combinatorial and Scarless 43
Multipart Gene Assembly
Chapter 4 High-Content Screening of Peptide-Based Non-Viral Gene 81
Delivery Systems
Chapter 5 Multimodular Peptide Libraries for Gene Delivery 105
Chapter 6 EGFR Targeted Multimodular Peptide Libraries for 129
Gene Delivery
Chapter 7 EGFP fused Multimodular Peptide Libraries for Gene Delivery 157
Chapter 8 Summarizing Discussion and Perspectives 185
Nederlandse samenvatting 207
Curriculum Vitae 217
List of Publications 219
Dankwoord | Acknowledgements 221
1
Chapter
General Introduction
GENERAL INTRODUCTION
Introduction
Gene therapy can be defined as the introduction of exogenous nucleic acids into cells
with the intention of altering gene expression to prevent, halt or reverse a pathological
process. It forms an attractive approach for therapeutic intervention of a wide range
of diseases, including genetic diseases, metabolic disorders, infectious diseases,
chronic illnesses, cardiovascular diseases and cancer (1,2). Gene therapy can be
carried out by three routes: gene addition/replacement, gene modulation/knockdown
and gene correction/repair (1,3).
Gene addition/replacement therapy is used to provide therapeutic benefit or to
supply a protein that is missing and is mainly applied to correct monogenic loss-of-
function mutations (1,3). Other applications are suicide therapy, where the expression
of a suicide gene leads to induced cell death, or the expression of ligands for surface
display to activate cytotoxic T cells (1). For gene addition/replacement therapy,
plasmid DNA (pDNA) or messenger RNA (mRNA) can be used.
Gene modulation/knockdown refers to the downregulation or complete inhibition
of gene expression by intervening at either the transcriptional or translational level.
Gene modulation/knockdown can be applied to reverse the deleterious effects
caused by the abnormal expression of a mutated protein, an oncogene or a virulence
factor (3). Gene modulation/knockdown can be mediated through several mechanisms.
Triple-helix forming oligodeoxynucleotides (TFOs), DNA- and RNA decoys can be used
for physically blocking transcription or translation (4). RNA interference (RNAi) using
small interfering-RNA (siRNA) or short hairpin-RNA (shRNA)-expressing pDNA,
microRNA (miRNA), ribozymes, DNAzymes and antisense RNA or DNA can be used to
destabilize and destruct mRNA, thereby inhibiting translation (3-5).
Gene correction/repair is a method to restore wild type functions in dominant
negative mutations and can be implemented at the mRNA level or at the genome
level (3). Oligonucleotides or small DNA fragments can be used to promote mismatch
repair, site-specific recombination or splice site modulation (3,5).
In order for gene-based therapeutics to become effective, the therapeutic nucleic
acids must be delivered into target cells and have to reach their site of action within
the cell. However, due to the high charge density and large molecular weight, nucleic
acids are generally impermeable to cellular membranes and require assistance in
order to reach their target site (6). To facilitate the uptake by target cells and delivery
of nucleic acids at their target site, a sophisticated delivery system is required which
must be capable of targeting the diseased cell, facilitate uptake and intracellular
trafficking of the nucleic acid cargo to their site-of-action.
11
CHAPTER 1 GENERAL INTRODUCTION
Viral and non-viral gene delivery Aim
Gene delivery systems can be divided into two broad classes: viral vectors and
non-viral (synthetic) vectors. Viral delivery systems are derived from viruses, whereas The aim of this thesis is to set up a high-throughput screening method to select out of
non-viral systems are based on macromolecular complexes. About 70% of all a large library of multimodular peptides those candidates that are able to efficiently
approved gene therapy clinical trials worldwide used viral vectors (7). In 2003, China deliver therapeutic nucleic acids into target cells at their site of action. To achieve
became the first country to approve a viral based gene therapy product (Gendicine™) this, we propose a design strategy that follows a random, integrative approach
for clinical use and just recently, the EMA recommended for the first time a virus selecting multimodular peptides containing combinations of functional traits that are
based gene therapy product for approval in the European Union (8). Viruses are highly optimal for efficient gene transfer (Fig.1). By randomly combining peptides with
complex and are adapted to infect cells and deliver their RNA/DNA cargo (9). The properties needed for gene delivery (e.g. DNA condensing peptides and membrane
transfection efficiencies of viral vectors remain unprecedented and outperform their disrupting peptides), a combinatorial library encoding multimodular peptides will be
non-viral counterparts. However, viral vectors have several drawbacks, including generated. This library will be used for the recombinant production and screening of
their immunotoxicity and the chances for insertional mutagenesis (10). Subsequently, these multimodular peptides for their transfection efficiency. Several rounds of
non-viral delivery systems have emerged as potential alternatives to viral vectors. screening and selection will be performed to obtain multimodular peptides for
Most non-viral delivery systems are based on self-assembling complexes of effective gene delivery.
nucleic acids with positively charged molecules, such as polymers, lipids and peptides, We opt for recombinant production of such multimodular peptides for several
through electrostatic interactions (11). In general, non-viral vectors have a low reasons. First, synthesis of long synthetic peptides of over >50 amino acids is difficult
immunogenicity, are nonpathogenic, and thus lack the major safety issues associated and often results in low yields and high heterogeneity (15,16). Second, by using
with their viral counterparts (12). Also, non-viral vectors can be produced at relatively recombinant protein synthesis technology, the precision of the cellular machinery
low costs, and have no limitation on the DNA-size and carrying capacity (13). However, can be exploited to reproducibly create an exactly specified protein-based material (17).
compared to viral vectors, the gene delivery efficiency of non-viral vectors is poor.
Peptide-based gene delivery systems Outline
Peptide-based gene delivery systems offer advantages compared to polymer- or lipid
based non-viral gene delivery systems. Both cationic polymers and lipids are known Chapter 2 provides an overview of functional peptides used for gene delivery, either
to be cytotoxic (14). Also, functionalization of polymers and lipids with targeting individually or in multifunctional/multi-component vectors. The chapter is concluded
ligands, endosomal escape agents or polyethylene glycol (PEG) is often required in with a discussion on why an integrative approach is beneficial for the generation of
order to mediate efficient gene transfer. Reproducible incorporation and/or attachment new successful non-viral gene delivery systems.
of functional components is a significant challenge and often results in compositional
variations, which is unfavorable from a pharmaceutical point of view. The development of a method for “scarless” ligation of multipart gene segments in a
Peptide-based gene delivery systems, on the other hand, may offer a versatile truly sequence-independent fashion is described in Chapter 3. This method is based
platform for efficient gene delivery. Peptides are biodegradable, biocompatible and on the ligation of single-stranded or double-stranded oligodeoxynucleotides (ODN)
various peptides have been identified that can perform several basic functions for and PCR products immobilized on a solid support. Different settings were tested to
gene delivery, such as DNA condensation or membrane disruption. By assembling optimize the solid-support ligation.
different functional peptides required for effective gene delivery into a single-chain,
the ideal gene delivery system can be created, thereby eliminating compositional In Chapter 4, the development of a high-content screening (HCS) assay for rapid
variations, facilitate pharmaceutical formulation, and achieve reproducibility at the screening of non-viral gene delivery systems is described. For a proof-of-principle, a
molecular level. small library of peptide-based transfectants was simultaneously screened for
transfection efficiency, cytotoxicity, induction of cell permeability and the capacity to
transfect non-dividing cells.
12 13
CHAPTER 1 GENERAL INTRODUCTION
The generation of a genetic library encoding multimodular peptides is reported in
Chapter 5. Individual multimodular peptides were obtained after recombinant
expression and purification of the generated library.
In Chapters 6 and 7, the multimodular peptide libraries were fused to a fusion protein
in order to increase the recombinant protein yield of the individual constructs.
Chapter 6 describes the generation of targeted multimodular peptide libraries for
gene delivery by fusing of the anti epidermal growth factor receptor (EGFR)
biparatopic nanobody to the multimodular peptide library. Chapter 7 describes the
generation of enhanced green fluorescent protein (EGFP) fused multimodular peptide
libraries for gene delivery.
Chapter 8 summarizes this thesis and discusses the findings and conclusions.
Figure 1 The proposed random, integrative approach that selects for optimal combinations
of functional peptides for efficient gene transfer using combinatorial protein engineering.
Functional domains (DNA condensing peptides (DCP), cell penetrating peptides (CPP), NLS
peptides (NLS) and targeting peptides (NP)) can be randomly combined on a genetic level to
generate a combinatorial gene library encoding for multimodular peptide vectors. The gene
library can then be inserted into an expression vector and expressed in a suitable host
organism. After subsequent high-throughput expression and purification, the multimodular
peptide vectors can directly be screened and most efficient candidates selected.
14 15
CHAPTER 1 GENERAL INTRODUCTION
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2
Chapter
Peptide Vectors for Gene Delivery:
From Single Peptides to Multifunctional
Peptide Nanocarriers
Markus de Raada, Erik A. Teunissena and Enrico Mastrobattistaa
a D epartment of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Faculty of Science,
Utrecht University, Universiteitsweg 99, 3584 CG Utrecht, The Netherlands
Submitted for publication