Gene Therapy

318 Pages·2010·4.134 MB·English

by  Mauro Giacca (auth.)

Gene Therapy
Mauro Giacca
Gene Therapy
123
Mauro Giacca
International Centre for Genetic
Engineering and Biotechnology (ICGEB)
Trieste
Italy
ISBN 978-88-470-1642-2 e-ISBN 978-88-470-1643-9
DOI 10.1007/978-88-470-1643-9
Springer Dordrecht Heidelberg London Milan New York
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This book is dedicated to
Serena, Massimo and Giovanna,
for making everything possible.
Preface
I entered the gene therapy field in the mid-1990s, being fascinated by the immense
potential of genes as drugs for the treatment of human disease. Since then, I have
experienced the ups and downs of this discipline, and tried to contribute with my
work and that of my laboratory to the development of innovative approaches to the
treatment of cardiovascular disorders. During these years, I have had several oppor-
tunities to speak on gene therapy at lectures and academic lessons, and have often
noticed that the field is very attractive to scientists of all disciplines. However, as
yet no comprehensive book on the subject has been published. Indeed, most books
in the field are either a collection of gene transfer laboratory protocols or deal with
the subject in a rather superficial manner. Hence the idea to write a gene therapy
textbook that is broad and comprehensive, but at the same time provides sufficient
molecular and clinical detail to be of interest to students, professors, and specialists
in the various disciplines that contribute to gene therapy. I have tried to keep the
language plain and, whenever possible, non-technical. Since the book is intended
to be a textbook in the field of gene therapy in both the basic science and clinical
areas, whenever technical descriptions are required, they are provided. For exam-
ple, clinical readers might find it difficult to understand the principles of viral vec-
tor design without knowing some molecular details on viral genome organization
and virus life cycle, and basic scientists might be unaware of the clinical and ther-
apeutic advancements for the various disorders discussed in the book. In both cases,
basic explanations are provided.
The book starts by providing a broad overview of the field of gene therapy
(Chapter 1), and then moves on with a description of the gene therapy “tools”,
namely the nucleic acids with curative potential (Chapter 2) and the methods for
their delivery into patients’ cells (Chapter 3). Chapter 4 contains an extensive
description of the clinical conditions so far addressed by gene therapy clinical tri-
als, describing the successes and frustrations over the last 20 years of study. In this
respect, I wish to point out that the purpose of the book is to be truly a gene “ther-
apy” book, namely to only consider gene transfer approaches that have already pro-
ceeded to the clinic or are close to doing so. Basic research on gene transfer or
vii
viii Preface
nucleic acid modification is only discussed when close to clinical application.
Finally, the social and ethical problems related to the use of genes to combat dis-
ease and to the possibility of engineering human genetic material are the subject of
Chapter 5. Along with the problem of enhancing safety and minimizing health risk
in gene therapy clinical applications, this chapter also deals with issues such as
gene transfer into germ cells, in utero gene therapy, and the possibility of exploit-
ing gene therapy for non-disease conditions, such as in the case of gene doping.
A bibliography list is provided for each section at the ends of the chapters. This
list is divided into Further Reading, which cites papers specifically addressing indi-
vidual topics presented in that session, and a Selected Bibliography, listing the most
relevant sources of original information in the scientific literature.
The content of this book has already constituted the basis for a series of aca-
demic lessons that I have delivered in various Molecular Medicine courses held at
the Scuola Normale Superiore in Pisa and the University of Trieste, Italy, over the
past 5 years. The audience of these courses, and thus the potential readership of this
book, included graduate students in biology, biotechnology, biochemistry, and med-
icine in their final years of study, PhD students in the above disciplines, and med-
ical doctors specializing in different fields of internal and specialist medicine, as
well as research scientists in various fields of current biomedical research.
Curing human disease using nucleic acids constitutes one of the most demand-
ing challenges of science and medicine, and many hurdles remain to be overcome.
However, I am deeply convinced that, once clinical success is fully attained, gene
therapy will offer unprecedented possibilities for curing the vast range of human
disorders that are currently beyond the scope of traditional medicine.
Trieste, March 2010 Mauro Giacca
Acknowledgments
I am indebted to Oscar Burrone, Serena Zacchigna, Lorena Zentilin, and Miguel
Mano for their critical reading of various parts of the manuscript, and to Suzanne
Kerbavcic for her valuable help. Many thanks to them and to all the other members
of my laboratory at the International Centre for Genetic Engineering and
Biotechnology in Trieste for their constant contributions to the maintenance of a
scientifically and intellectually stimulating environment.
ix
Contents
Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv
1 Introduction to Gene Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1 Genes as Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2 Gene Therapy: An Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2 Therapeutic Nucleic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.1 Protein-Coding Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.1.1 Proteins Substituting Missing or Mutated Cellular Proteins . . . . . . . . . . 10
2.1.2 Proteins Modulating Cellular Functions . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2.1.3 Secreted Growth Factors and Cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2.1.4 Proteins Regulating Cell Survival and Apoptosis . . . . . . . . . . . . . . . . . . . 12
2.1.5 Antigens for Vaccination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2.1.6 Antibodies and Intracellular Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2.1.7 T-Cell Receptor (TCR) Subunits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2.1.8 Control of Therapeutic Gene Expression . . . . . . . . . . . . . . . . . . . . . . . . . 16
2.2 Non-Coding Nucleic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
2.2.1 Oligonucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
2.2.2 Modified Oligonucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
2.2.3 Clinical Applications of Oligonucleotides . . . . . . . . . . . . . . . . . . . . . . . . 27
2.2.4 Catalytic Nucleic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
2.2.5 Small Regulatory RNAs (siRNAs, microRNAs) . . . . . . . . . . . . . . . . . . . 31
2.2.6 Decoys . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
2.2.7 Aptamers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
2.2.8 Modes of Delivery or Intracellular Synthesis of Small Regulatory RNAs . 37
Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Selected Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
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xii Contents
3 Methods for Gene Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
3.1 Cellular Barriers to Gene Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
3.1.1 Endocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
3.1.2 Escape from the Intracellular Vesicle Compartment . . . . . . . . . . . . . . . . 49
3.1.3 Nuclear Targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
3.1.4 Methods for Gene Delivery: An Overview . . . . . . . . . . . . . . . . . . . . . . . . 51
3.2 Direct Inoculation of DNAs and RNAs . . . . . . . . . . . . . . . . . . . . . . . . . . 53
3.3 Physical Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
3.3.1 Electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
3.3.2 Hydrodynamic Intravascular Injection . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
3.3.3 Sonoporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
3.3.4 Bombardment with DNA-Coated Microparticles (“Gene Gun”) . . . . . . . 55
3.3.5 Injection of DNA using High-Pressure Jets (“Jet Injection”) . . . . . . . . . . 56
3.4 Chemical Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
3.4.1 Liposomes and Cationic Lipids (Lipofection) . . . . . . . . . . . . . . . . . . . . . 56
3.4.2 Cationic Polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
3.4.3 Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
3.4.4 Chemical Methods for Gene Transfer: Pros and Cons . . . . . . . . . . . . . . . 63
3.5 Viral Vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
3.5.1 Vectors Based on Gammaretroviruses . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
3.5.2 Vectors Based on Lentiviruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
3.5.3 Vectors Based on Adenoviruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
3.5.4 Vectors Based on the Adeno-Associated Virus (AAV) . . . . . . . . . . . . . . . 112
3.5.5 Vectors Based on the Herpes Simplex Virus (HSV) . . . . . . . . . . . . . . . . . 123
3.5.6 Viral Vectors for Gene Therapy: Fields of Application
and Comparative Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
Selected Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
4 Clinical Applications of Gene Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
4.1 Clinical Applications of Gene Therapy: General Considerations . . . . . . . 139
4.1.1 Number of Clinical Trials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
4.1.2 Phases of Clinical Trials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
4.1.3 Diseases Addressed by Gene Therapy Clinical Trials . . . . . . . . . . . . . . . 142
4.1.4 Therapeutic Genes Used in the Clinical Trials . . . . . . . . . . . . . . . . . . . . . 145
4.1.5 Modality of Therapeutic Gene Delivery in Gene Therapy Clinical Trials . . 145
4.2 Gene Therapy of Hematopoietic Stem Cells . . . . . . . . . . . . . . . . . . . . . . 146
4.2.1 Bone Marrow and Hematopoiesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
4.2.2 Hematopoietic Stem Cell Transplantation . . . . . . . . . . . . . . . . . . . . . . . . 147
4.2.3 Gene Therapy of Hematopoietic Stem Cells: Major Applications . . . . . . 149
4.2.4 Procedures for Gene Transfer into Hematopoietic Stem Cells . . . . . . . . . 154
4.2.5 Gene Therapy Clinical Trials Entailing Gene Transfer
into Hematopoietic Stem Cells: General Considerations . . . . . . . . . . . . . 154