Part 1 book “General and molecular pharmacology” has contents: Essential lexicon of pharmacology, a short history of pharmacology, cellular basis of pharmacokinetics , drug absorption and administration routes, drug distribution and elimination, drug metabolism.
General and Molecular Pharmacology General and Molecular Pharmacology Principles of Drug Action Edited By Francesco Clementi and Guido Fumagalli Co‐editors CHRISTIANO Chiamulera, Emilio Clementi, RICCARDO Fesce, Diego Fornasari, and Cecilia Gotti Translated and modified by Francesco Clementi and Guido Fumagalli Originally published in Italian under the title “Farmacologia Generale e Molecolare Il meccanismo d’azione dei farmaci”, 4th edition, by Francesco Clementi and Guido Fumagalli, © UTET SpA – Unione Tipografico-Editrice Torinese, Torino, Italy (2012) Copyright © 2015 by John Wiley & Sons, Inc All rights reserved Published by John Wiley & Sons, Inc., Hoboken, New Jersey Published simultaneously in Canada No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except as permitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, (978) 750-8400, fax (978) 750-4470, or on the web at www.copyright.com Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, (201) 748-6011, fax (201) 748-6008, or online at http://www.wiley.com/go/permissions Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose No warranty may be created or extended by sales representatives or written sales materials The advice and strategies contained herein may not be suitable for your situation You should consult with a professional where appropriate Neither the publisher nor author shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages For general information on our other products and services or for technical support, please contact our Customer Care Department within the United States at (800) 762-2974, outside the United States at (317) 572-3993 or fax (317) 572-4002 Wiley also publishes its books in a variety of electronic formats and by print-on-demand Not all content that is available in standard print versions of this book may appear or be packaged in all book formats If you have purchased a version of this book that did not include media that is referenced by or accompanies a standard print version, you may request this media by visiting http://booksupport.wiley.com For more information about Wiley products, visit us at www.wiley.com Library of Congress Cataloging-in-Publication Data General and molecular pharmacology : principles of drug action / Francesco Clementi, Guido Fumagalli, editors ; Christiano Chiamulera, Emilio Clementi, Riccardo Fesce, Diego Fornasari, Cecilia Gotti, co-editors online resource Includes bibliographical references and index Description based on print version record and CIP data provided by publisher; resource not viewed ISBN 978-1-118-76859-4 (pdf) – ISBN 978-1-118-76868-6 (epub) – ISBN 978-1-118-76857-0 (cloth : alk paper) I. Clementi, Francesco, editor. II. Fumagalli, Guido, editor [DNLM: 1. Chemistry, Pharmaceutical. 2. Molecular Biology. 3. Pharmacological Phenomena. QV 744] RS403 615.1′9–dc23 2015008591 Printed in the United States of America 10 9 8 7 6 5 4 3 2 1 1 2015 Contents LIST OF CONTRIBUTORS xlvi Preface xlix SECTION 1 INTRODUCTION TO PHARMACOLOGY 1 Essential Lexicon of Pharmacology Francesco Clementi and Guido Fumagalli The Social Impact of Pharmacology, Essential Lexicon, Active Substances, Pharmacological Disciplines, Drug–Receptor Interactions, Measure of the Clinical Response, Take‐Home Message, 2 A Short History of Pharmacology Vittorio A Sironi Birth and Historical Developments of Pharmacology, From Magical and Natural Remedies of Ancient Medicine to Arabic Alchemy, From Monastic Medicine to Botanical Gardens, From Anatomical Renaissance to the “Experienz”: Paracelsus’ Spagyric, 10 From Iatrochemistry to the Age of Enlightenment, 11 From the Search of the Active Principle to the Discovery of Alkaloids and Glucosides, 12 The Drug Synthesis Revolution: From Handmade to Industrial Production, 12 Modern Pharmacology, 13 Ehrlich and Chemotherapy: The Concept of Receptor, 13 The Birth of Modern Pharmacology, 14 The Biotechnology Era and the Pharmacology in the Third Millennium, 15 The Impact of New Biotechnologies: Molecular Biology, Bioinformatics, and Combinatorial Chemistry, 15 Biological Drugs and Pharmacology Perspectives, 16 vi Contents Personalized Therapies and New Sceneries in Pharmaceutical Industry, 17 Take‐Home Message, 18 Further Reading, 18 SECTION 2 GETTING THE DRUG TO ITS SITE OF ACTION 19 Cellular Basis of Pharmacokinetics 21 Riccardo Fesce and Guido Fumagalli A Quick Journey with the Drug in the Body, 21 Absorption, 21 Distribution, 21 Drug Elimination, 22 Crossing Cell Membranes, 23 Passive Diffusion across Cell Membranes, 24 Drug Transport across Cell Membranes, 25 Endocytosis, 25 Drug Diffusion to Organs and Tissues, 27 Properties of the Most Important Cell Barriers, 27 Take‐Home Message, 30 Further Reading, 30 Drug Absorption and Administration Routes 31 Riccardo Fesce and Guido Fumagalli General Rules About Drug Absorption Rate, 32 Partition Coefficient, 32 Drug Dispersion, 32 Extension of the Absorbing Surface, 32 Permeability of the Absorbing Surface, 32 Vascularization, 33 Enteral Routes of Administration, 33 Oral Route, 33 Sublingual and Rectal Routes, 35 Systemic Parenteral Routes of Administration, 35 The Intravascular Route, 35 i.m Injection, 36 Subcutaneous and Intradermal Injections, 36 Other Routes of Drug Administration, 36 Inhalation Route, 36 Topical/Regional Routes, 37 Intracavity Routes, 37 Dermal or Transcutaneous Route, 37 Mucosal Routes, 38 Absorption Kinetics, 38 General Rules, 38 Interrelation between Gene Therapy and Drug Delivery Techniques, 43 Take‐Home Message, 44 Further Reading, 44 Drug Distribution and Elimination Riccardo Fesce and Guido Fumagalli Distribution, 46 Tissues and Avidity for Drugs, 46 The Apparent Distribution Volume, 48 45 Contents Drug Binding to Plasma Proteins, 50 Factors That Determine the Distribution Rate of Drugs to the Various Compartments, 51 Elimination, 53 The Concept of Half‐Life, 53 The Concept of Clearance, 54 Renal Excretion of Drugs, 55 Glomerular Filtration, 56 Tubular Functions and Pharmacokinetics, 56 Active Transport of Organic Anions and Cations, 56 Factors Determining Renal Clearance of Drugs, 57 Hepatic Excretion and Enterohepatic Cycle, 58 Perfusion, Binding to Plasma Proteins, Enzymatic Activity, and Hepatic Clearance, 59 Take‐Home Message, 59 Further Reading, 60 Drug Metabolism 61 Enzo Chiesara, Laura Marabini, and Sonia Radice Metabolic Modification of Drug Activity, 61 Two Phases of Drug Metabolism, 62 Phase I Reactions, 62 Phase II Enzymatic Reactions, 66 Extrahepatic Biotransformations, 68 Biotransformation by the Intestinal Flora, 69 Pharmacometabolic Induction and Inhibition, 69 Induction of Drug Metabolism, 69 Inhibition of Drug Metabolism, 71 Take‐Home Message, 72 Further Reading, 72 Control of Drug Plasma Concentration Riccardo Fesce and Guido Fumagalli Time Course of Drug Plasma Concentration Following a Single Administration, 73 Drugs Distribute to Organs and Tissues and then are Eliminated, 74 Description of Drug Plasma Concentration Time Course Following a Single Administration, 74 Area under the Plasma Concentration Curve (AUC), 74 The Plasma Concentration Peak, 75 Drug Plasma Concentration Time Course During Repetitive Administrations, 75 During Repetitive Administrations, the Drug Plasma Concentration Time Course Is Given by the Sum of the Time Courses of the Single Doses, 75 In a Chronic Therapy at Steady State, Each New Dose Replaces the Drug Amount that has been Eliminated Since the Last Administration, 77 The Time to Reach the Steady State Depends on the Drug Half‐Life, 77 Plasma Concentration at Steady State, 78 The Single Dose to Administer is Computed as a Function of the Interval between Successive Administrations, 78 Fluctuations of Drug Plasma Concentration at Steady State, 79 Absorption Kinetics Influence the Amplitude of Oscillations in Plasma Concentration at Steady State, 80 Loading (Attack) Doses to Rapidly Attain Steady‐State Concentration, 80 Multicompartmental Kinetics, 81 Drug Binding to Plasma Proteins and Tissue Equilibration with Plasma, 81 73 vii viii Contents The Particular Case of the Nephron, 82 Drugs Redistribution among Compartments, 83 Corrections of the Therapeutic Regimen, 83 Normally Available Pharmacokinetic Data Are Average Values, 84 Varying Dosage as a Function of Body Weight and Physical Constitution, 84 Varying Dosage as a Function of Age, 84 Dosage Correction in the Presence of Hepatic Pathologies, 86 Dosage Correction in the Presence of Renal Pathologies, 86 Take‐Home Message, 87 Further Reading, 89 SECTION 3 RECEPTORS AND SIGNAL TRANSDUCTION 91 Drug–Receptor Interactions: Quantitative and Qualitative Aspects 93 Gian Enrico Rovati and Valérie Capra General Properties of Drug Receptors, 93 Drug Receptors Are Molecules Relevant for Cellular Functions, 93 Not All Drugs Interact with a Receptor, 94 Drug Activity Follows to Drug–Receptor Complex Formation, 94 Drug–Receptor Interaction Is Mostly Mediated by Weak Chemical Bonds, 94 Reversible or Irreversible Drug–Receptor Interactions, 95 Characteristics of Drug–Receptor Interaction, 95 The Relationship between Drug Concentration and Drug–Receptor Complex Is Similar to the Michaelis–Menten Equation, 97 The Binding Isotherm and Its Linear Transformations Allow to Obtain the Parameters of the Drug–Receptor Interaction, 97 Receptors Can Be Heterogeneous, 99 Drug Competition for a Same Receptor Binding Site, 99 Quantitative Aspects of Drug Effects: Dose–Response Curves, 99 Potency and Efficacy, 100 From Drug–Receptor Interaction to Drug Response, 101 Occupancy Theory, 101 Modifications to the Occupancy Assumption, 102 Efficacy Theory, 104 Nonlinear Function between Receptor Occupancy and Tissue Response: EC50 Different from Kd, 106 Constitutively Active Receptors and Inverse Agonists, 107 Two‐State Receptor Model and Beyond: Multiple Receptor States and “Biased” Signaling, 108 Take‐Home Message, 108 Further Reading, 108 Receptors and Modulation of Their Response Francesco Clementi and Guido Fumagalli Classes of Receptors and Strategies of Signal Transduction, 109 Intracellular/Intranuclear Receptors, 110 Membrane Receptors, 110 Control of Receptor Localization in the Cell Membrane, 116 Intracellular Traffic of Cell Receptors, 117 How Receptors Reach the Cell Membrane and how Their Number is Regulated, 117 Modulation of Receptor Responses, 117 Receptor Modulation By Drugs, 118 109 26 REGENERATIVE MEDICINE AND GENE THERAPY Luciano Conti and Elena Cattaneo By reading this chapter, you will: • Learn source, origin, and functional differences among different types of stem cells and their biological properties • Know the wide range of therapeutic opportunities arising from the discovery and use of stem cells in hematological, epithelial, cardiovascular, muscular, and neurodegenerative diseases • Acquire the main legal issues related to cell‐based drugs and their production • Know the theoretical and practical basic concepts on gene therapy approaches to monogenic diseases and cancer Recent advances in cell biology and stem cell research have led to the idea of using cells as therapeutics, in contrast to the traditional pharmacological approach employing molecules as drugs Cell‐based drugs are currently being developed as novel members of the large family of biological drugs that includes also monoclonal antibodies, ribozymes, and other biotechnological products with pharmacological properties This has prompted a revolution in biomedical research that is increasingly focusing on potential applications of stem cell‐ based therapies Significant advances have been made in this direction, but it is important to remember that to date clinical use of cellular therapies is restricted only to a small number of pathologies The concept of cell‐based therapy was introduced in the late 1950s when the bone marrow transplant procedure was being established The first transplant in humans was carried out by a French oncologist, Georges Mathé, who injected healthy bone marrow into six physicists whose bone marrow had been compromised by heavy radiation exposure during a nuclear reactor accident in Yugoslavia In the following years, the transplant technique was improved, and a significant contribution came from Edward Donnall Thomas’ studies demonstrating that bone marrow cells, delivered by intravenous infusion, can repopulate the bone marrow of receivers and produce new blood cells For these studies, in 1990, Edward Donnall Thomas was awarded of the Nobel Prize for Physiology or Medicine (shared with Joseph Murray, who performed the first kidney transplant in 1954) Since then, thousands of patients affected by leukemia or immunodeficiency have been cured by bone marrow transplant It has been estimated that currently about 70,000 bone-marrow transplants are performed worldwide every year During the last decade, cell‐based therapies have been developed also for nonhematological disorders, with those applied to epithelial and corneal diseases being particularly successful What diseases cell‐based therapies will be able to cure in the future are hard to predict, as well as which types of stem cells will prove more effective drugs Nevertheless, medical scientists are confident that a more in‐depth understanding of biological features and handling requirements of the different stem cell types will lead to new therapeutic strategies for cardiac diseases, diabetes, muscular dystrophy, and neurological diseases However, they are also aware that for such an ambitious goal to be reached, translation to clinic will always have to be supported by strong preclinical experimental evidence and decided in full respect of patient safety General and Molecular Pharmacology: Principles of Drug Action, First Edition Edited by Francesco Clementi and Guido Fumagalli © 2015 John Wiley & Sons, Inc Published 2015 by John Wiley & Sons, Inc 296 REGENERATIVE MEDICINE AND GENE THERAPY PRINCIPLES OF REGENERATIVE MEDICINE In general, stem cells are undifferentiated cells that can be distinguished from other cells for two main characteristics: 1. Self‐renewal, that is, the ability to reproduce themselves 2. Differentiation potential, that is, the ability to differentiate in a range of different specialized cell types The extent of such differentiation potential varies depending on the type of stem cells According to their differentiation potential, stem cells can be ordered in a hierarchical scheme, as shown in Figure 26.1 From a strictly functional point of view, a stem cell can be defined as a cell capable of generating all cells of the tissue in which it is found, throughout the life of an organism Skin or blood stem cells fully exhibit this functional feature that conversely cannot be demonstrated for most stem cells from other tissues, or it is not even compatible with the stem cell role (as in the case of brain stem cells) Stem cells can also be classified according to the timing of their appearance (Table 26.1) Stem cells not have all the same differentiation potentials, and depending on their plasticity, they can be classified as totipotent, pluripotent, or multipotent (Fig. 26.1) The only totipotent cell is zygote, the only cell capable of giving rise to a complete organism It is transient and cannot be expanded as such Differentiation potential Zygote (totipotent) ES/iPS cells (pluripotent) ng mi ram Precursors (mono- bi- potent) g pro Adult stem cells (multipotent) Re In the last decades, transplant medicine has provided tools to clinically test functional rescue after tissue and organ transplantation The aim of traditional transplant medicine is to replace defective tissues or organs, often at the final stages of a disease, with healthy ones to restore functional homeostasis Such approach has proved successful in curing otherwise incurable leukemias, kidney failure, cirrhosis, and cardiac conditions, but its application has always been limited by shortage of donated organs and adverse effects associated with immunosuppressive therapies This has prompted the search for alternative solutions to restore tissue and organ functionality, opening the way to the so‐called regenerative medicine Today, the goal of regenerative medicine is to exploit regenerative potential of stem cells to permanently heal and recover damaged tissues and organs Therefore, advancements in regenerative medicine today are tightly linked to progress in stem cell research Stem cells and specialized cells deriving from them, either normal or engineered, represent the “active principle” of regenerative therapies Stem cells are physiological constituents of the organism, providing tissues with a sort of internal repair system They are unspecialized cells with the ability to self‐renew (to a different extent depending on the stem cell type), and they are able to differentiate in specific cell types After p urification from the original tissue (in those cases in which this is possible), they can be expanded in vitro and “instructed” to differentiate in a specific cell type Therefore, stem cells potentially represent an unlimited source of cells to be transplanted to heal injured tissues Such cells could also promote recovery by releasing active molecules, such as anti‐inflammatory substances or growth factors, although more knowledge is needed in support of this hypothesis Moreover, to be better suited for the purpose, prior to transplantation, stem cells could be genetically modified to add useful genes or correct endogenous mutated genes To date, many of these therapeutic opportunities are still being investigated and are not clinically available yet The aim of regenerative medicine is to permanently restore damaged tissue functionality both in congenital and acquired pathologies, as well as in aging‐associated diseases The therapeutic outcome will depend on the ability of the employed stem cells to create a niche within the pathological tissue and to promote optimal levels of tissue regeneration Regenerative medicine follows two main strategies The former, the in vivo strategy, aims at the pharmacological stimulation of endogenous stem cells within the tissues of interest in order to trigger their regenerative potential However, this approach is still in its infancy The latter, the ex vivo strategy, involves the in vitro expansion and/or modification of stem cells and their subsequent transplantation in the region of interest to promote regeneration/healing In this chapter, we will focus mainly on ex vivo approaches, d iscussing issues and examples of cell therapy in regenerative medicine and gene therapy DEFINITION, CLASSIFICATION, AND FEATURES OF STEM CELLS Mature cells Figure 26.1 Hierarchical ordering of cells based on their differential potential The zygote is placed at the top of the pyramid as it is able to generate an entire organism, including the extraembryonic tissues (totipotent) Under the zygote, there are embryonic stem (ES) cells and iPS cells (obtained by cellular reprogramming), which are able to produce all cells deriving from the three germ layers (pluripotent) Next, adult stem cells are found, which can produce only the mature cell types of the tissue they belong and their progenitors (proliferating cells that can only differentiate into one or two specific cell types) At the bottom of the pyramid, we find mature specialized cell type; even when capable of diving, these cells can only produce mature cells of their own type 297 iPS Fetal cells, adult immature and differentiated cells Pluripotent Yes Phase I Not tested Yes Yes Yes Stem cell type Source Differentiation potential In vitro expansion Clinical use Clinical efficacy Genetic manipulation Cryopreservation Tumorigenic potential Pluripotent Yes Phase I Not tested Yes Yes Yes Blastocysts ESC Table 26.1 Characteristics of the main types of stem cells Multipotent Depending on the tissue of origin Yes Yes Limited Yes Some cases Fetal tissues Fetal SC Bone marrow, pheripheral blood Multipotent Limited Yes Yes Limited Yes Not seen Blood SC Multipotent Yes Yes Yes Limited Yes Not seen Skin Skin SC Multipotent Yes Not yet Not tested Limited Yes Not seen Brain Brain SC Multipotent No Yes Yes Limited Yes Not seen Umbilical cord Umbilical cord SC 298 REGENERATIVE MEDICINE AND GENE THERAPY Two cell types belong to the group of pluripotent stem cells: the embryonic stem cells (ESCs), which are transiently present in the blastocyst, and the induced pluripotent stem cells (iPSCs), which have been recently obtained by in vitro reprogramming of mature adult cells In contrast to zygotes, pluripotent cells cannot differentiate into a complete organism Nevertheless, they retain a high differentiation potential and can produce cells belonging to all three germ layers (ectoderm, mesoderm, and endoderm) This means that they can generate even more than 250 cell types making up fetal and adult organisms, which include both functionally mature specialized cell types and tissue‐specific stem cells However, in contrast to zygotes, pluripotent cells are unable to generate extraembryonic tissues (trophectoderm and placenta) The class of multipotent stem cells includes adult tissue‐ specific stem cells (also called somatic stem cells) that are found in fetal and adult tissues and can differentiate only in the specific cell types of the tissue they belong For example, a hematopoietic stem cell (HSC) can produce the eight main types of mature, differentiated blood cells It is important to note that even if adult stem cells from the blood, skin, muscle, kidney, nervous system, etc are grouped in a single class, each type has specific features and a different therapeutic potential and therefore is the focus of a specific research field Pluripotent Stem Cells Research on pluripotent stem cells started when Leroy Stevens, a developmental biologist working at Jackson Laboratory, discovered spontaneous testicular tumors (teratocarcinoma) in the inbred 129 mouse strain He found that cells from malignant teratocarcinoma (named embryonal carcinoma or EC cells), cultured in vitro and then injected into mouse blastocysts, were able to undertake multiple differentiation programs, thus contributing to the formation of the resulting chimeric mouse Later, in 1981, Sir Martins Evans and Matthew Kaufman succeeded in in vitro culturing ESCs isolated from the inner cell mass of mouse blastocysts (the preimplantation embryo), generating stable ESC lines In the absence of stimuli, ESCs can grow and multiply in vitro but retain the ability to differentiate in mesodermic, endodermic, and ectodermic cells when exposed to appropriate stimuli When ESCs are introduced into blastocysts that are then reimplanted in a pseudopregnant mouse female, they actively participate in embryo formation and give rise to chimeras, thus proving capable to differentiate in cells deriving from all three germ layers also in vivo The ability to produce chimeras is used to test the “stemness” of a given cell population It is important to note that mouse ESCs not contribute to extraembryonic tissues, such as the trophoblastic part of the placenta, indicating that their differentiation potential is not as wide as that of zygotes A specific feature of mouse ESCs consists in their ability to form teratomas when injected into immunodeficient mice Teratomas are benign tumors containing a variety of cells, either highly or partially differentiated, deriving from different germ layers; this observation confirms the huge differentiation plasticity of ESCs In 1998, 17 years after the isolation of mouse ESCs, a paper was published in Science in which James Thomson and coworkers reported the derivation of ESC lines from human blastocysts produced by in vitro fertilization and donated after informed consent and review board approval The result represented a significant achievement, but the procedure described by the authors involved human blastocyst dissociation This ignited a worldwide debate on whether destroying human blastocyst could be considered ethically acceptable The ethical controversy implies a choice between two moral duties: on one hand the duty to alleviate suffering (a goal that is pursued by the research on human embryonic stem (hES) cells) and on the other the duty to respect human life To this respect there is discussion on whether one should consider the blastocyst a “person” just like the patients who might benefit from therapies resulting from research on hES cells The debate is not resolved but different countries have decided to r egulate research on hES cell in different ways In the United Kingdom, Belgium, and Switzerland, it is legal to derive hES cells from blastocysts produced in excess during in vitro fertilization procedures In other countries, such as Germany, only cell lines obtained before a given date (2002) were initially permitted but in 2008, as a result of pressure from scientists, the German Act was amended to move the cut-off point to May 2007 In other countries, like Italy, derivation of new lines is not permitted but usage of lines obtained by others is accepted; therefore, research on hES cells is permitted but only on imported cell lines In the United States, during the Bush administration, no public funding was allocated to research on human embryonic cell lines produced after 2001 Nevertheless, such research was permitted and went on with the support of private funding (and led to significant discoveries such as the iPSCs) What is the reason of such an interest in hES cells? Like the murine counterpart, hES cells have the ability to differentiate in all cell types making up fetal and adult human body (except the extraembryonic tissues) hES cells can be induced to differentiate in vitro to generate (with different efficiency) epidermal and adrenocortical cells and keratinocytes, as well as endothelial, kidney, bone, muscle, heart, pancreas, and liver cells Moreover, differentiation of hES cells in electrophysiologically mature cardiomyocytes and neurons has also been reported Results obtained so far indicate that such differentiation potential of hES cells cannot be obtained with human adult stem cells Studies in animal models have shown that transplants of cells derived from hES cells can successfully treat congenital conditions such as Parkinson’s disease (PD) or diabetes This highlights the potential of hES cells in regenerative medicine However, given the ability of ESCs to form teratomas, the risk of tumor formation after stem cell therapies must be taken into account and is one of the highly studied topics of current CELL THERAPY AND REGENERATIVE MEDICINE ☞ research Today, stem cell research aims at the transplant of specialized cells derived from ESCs So far, two clinical trials of hES cell‐based therapies have received FDA approval It is important to remind that hES cells have also other applications and can be used, for instance, in toxicological and pharmacological studies or to investigate disease mechanisms or the physiology of human development One of the most revolutionary achievements in stem cell research is the discovery of the iPSCs (see Supplement E 26.1, “History and Development of iPS Cell Research”) iPSCs the result of mature adult cells, such as fibroblasts, that have been forced, by genetic reprogramming, to dedifferentiate and to reacquire the features of pluripotent cells Therefore, iPSCs are very much the same as ESCs, but they have a different origin that does not involve blastocyst dissociation iPSCs were first obtained by Shinya Yamanaka and his group, in Japan, and represent an outstanding breakthrough in stem cell research Using the iPS technology, in the future, it could be possible to obtain pluripotent cells from any individual This means that in the future iPSCs could be produced from patient’s fibroblasts and subsequently instructed to generate the specific cell types, including germ cells, required to cure diseases in the same patient Multipotent (or Adult) Stem Cells Many tissues in our body contain stem cells devoted to replacing damaged cells Their abundance varies depending on the tissue, and in general, they tend to be more abundant in tissues with a higher ability (and need) to regenerate For example, blood stem cells are professional stem cells; every day, they have to yield 2.5 billion erythrocytes, 2.5 billion platelets, and billion leukocytes per each kg of body weight in order to replace dying cells Skin is another tissue enriched in stem cells; every minute, 30,000 cells come off the skin superficial layer and need to be replaced for the organism to survive In contrast, brain stem cells are very few and poorly active and have been identified only in two brain areas, hippocampus and subventricular zone Out of the 100 billion neurons making up the brain, about 85,000 are believed to be lost every day in the subcortical area without being replaced Little is known about factors and mechanisms controlling the differentiation potential of adult stem cells, but there is an increasing interest in their isolation and characterization as possible therapeutic tools Compared to ESCs, some adult stem cells are more easily accessible, and some of them retain a remarkable ability to differentiate The HSC represent the best and most studied example of adult stem cells HSC can be isolated in a prospective way (using specific antibodies), enriched, and employed in either autologous or allogeneic transplants to treat inherited immunodeficiencies, autoimmune diseases, and other conditions of the immune system by replenishing specific blood cell types From a chronological point of view, fetal stem cells are found between ESCs and adult stem cells They are 299 multipotent and adult, as residing in differentiated tissues, and they are believed to be similar to stem cells isolated from the corresponding adult tissues but more plastic and therefore potentially more interesting for therapeutic p urposes For this reason, much attention is being given to fetal cord blood, and there is an increasing interest in placenta and in mesenchymal stem cells (MSC) However, it is important to remember that in many cases protocols for obtaining and expanding adult stem cells have not been established yet Moreover, with the only exceptions of HSC (often transplanted without previous in vitro expansion), skin stem cells (one of the very few example in which p rospective isolation and in vitro unlimited expansion can be obtained without loss of multipotency), or bone marrow‐derived MSC, isolation and in vitro culturing modify adult stem cell properties STEM CELL‐BASED DRUGS Stem cell‐based drugs belong to the class of biotechnological drugs, meaning drugs resulting from a biological process In contrast with most drugs, whose active principles consist of small molecules, in stem cell‐based drugs, the active principle is represented by living cells In the last few years, laws regulating their clinical usage have become very restrictive in order to ensure reproducibility, safety, and s tandardization of the protocols Stem cell drugs can be administered in combination with biomolecules and noncellular components, such as medical devices and matrices, and can be genetically modified The first generation of stem cell‐based drugs consists of unmodified human stem cells, in their original state, such as unmodified HSC taken from the bone marrow and employed in clinics to treat blood diseases The second generation typically consists of cells treated with growth factors, purified using tissue‐specific biomarkers, or genetically modified with the aim of enhancing their therapeutic potential and reducing risks of tumor formation or immunological incompatibility The third generation consists of stem cell with the additional property of producing and releasing soluble molecules in the transplanted tissue The released molecules may correct a genetic defect or promote tissue regeneration or enhance stress tolerance of both endogenous and transplanted cells, thus improving the therapeutic potential of the original stem cells CELL THERAPY AND REGENERATIVE MEDICINE As with many other biological products, immunotolerance is critical for the safe and effective application of stem cell‐based therapies Cellular therapies are classified as autologous, when they involve transplantation of tissues, cells, or proteins belonging to the same patient, and allogeneic (or heterologous) when a patient receives tissues, cells, or proteins from another individual of the same species 300 REGENERATIVE MEDICINE AND GENE THERAPY (a) (b) Hematopoietic stem cell Cerebral cortex Latearal ventricle Bone marrow Corpus callosum Mesenchymal cell Bone Cerebellum Hippocampus Adult human brain Bone marrow (c) (d) Iris Muscle fiber (e) Satellite cell (stem cell) Epidermids Isthmus stem cells Interfollicular stem cells Muscle fiber nuclei Pupil Muscle Hair Corneal limbus Sclera Eye Primary stem cells Dermin Bulge stem cells Skin Figure 26.2 Examples of adult tissues in which stem cells reside In the figure are the following: (a) hematopoietic stem cells from the bone marrow, (b) neural stem cells from the subventricular zone (SVZ) of the lateral ventricle and hippocampus, (c) muscle satellite cells, and (d) corneal stem cells from the limbus In the skin (e), three types of “specialized” stem cells were first identified: in the dermis basal layer (interfollicular stem cells regenerating epidermis), in the follicle bulge (for hair growth), and in the isthmus (for sebaceous gland formation) Recently, a new population of skin stem cells has been identified, from which the three specialized stem cell types originate ☞ Cell therapies based on the allogeneic transplantation of the bone marrow or hematopoietic cells to treat different blood diseases have been used with success for about 20 years (see Supplement E 26.2, “Use of Blood Stem Cells in Hematology”) In this section, we will discuss some examples of new therapeutic approaches employing adult stem cells and in particular one procedure that has been successfully applied to treat corneal lesions (Fig. 26.2) Regenerative Medicine Approaches to Epithelial Lesions After a lesion, skin can regenerate a perfectly functional corneal layer in a few days, starting from different types of specialized stem cells located in the dermis basal layer (stem cells responsible for epidermal regeneration), in the follicle bulge (stem cells responsible for hair growth), and in the isthmus (stem cells involved in sebaceous glands g eneration) More recently, in 2010, Hans Clevers’ group discovered a new population of skin stem cells that can produce all three most specialized stem cell types (Fig. 26.2) However, when endogenous skin repair is not sufficient for adequate regeneration of the lost tissue, such as in case of severe and large burns, autologous skin transplantation can be performed New skin layers can be grown in vitro on collagen and matrigel matrices starting from progenitors and skin stem cells obtained from small biopsies taken from the patient The first skin transplant using in vitro cultured cells was performed in 1983 by Howard Green, a pioneer in this research field, to treat three children severely burned Since then, the same procedure has been successfully applied to treat severe skin lesions In 1987, Yann Barrandon described a novel methodology to culture skin stem cells in vitro and to produce keratinocytes However, such procedure is very expensive (it costs more than 150,000 euro to treat an adult patient with 80% of his/her body surface burned) and requires many months to generate large skin layers Novel and cheaper methodologies to culture general skin stem cells may lead in the future to transplant of regenerated skin layers structurally and functionally identical to normal skin The eye corneal epithelium is another example of epithelial tissues that can be entirely regenerated in vitro and for which successful clinical protocols have been developed Corneal lesions, due, for example, to chemical burns, induce CELL THERAPY AND REGENERATIVE MEDICINE the conjunctival epithelium to generate a layer, called “pannus,” covering the entire eye bulb, eventually leading to blindness This can be prevented by replacing the damaged tissue with an in vitro regenerated layer Cornea can be reconstructed starting from stem cells taken from the limbus (Fig. 26.2), an area surrounding the cornea Corneal limbus stem cells are only a few thousands and represent about 10% of the limbus and are responsible for the cornea turnover occurring every 9 months When cultured in vitro, limbus stem cells can generate a layer of corneal epithelium in 3–4 weeks, which can be transplanted after removal of the damaged one The first pioneering study on corneal regeneration from stem cells was published in 1997 by Graziella Pellegrini and Michele De Luca In 2010, these researchers along with Paolo Rama refined the technique to allow treatment of large corneal lesions (3–4% of total lesions) for which normal stem cell transplantation is ineffective In these cases, corneal grafting is preceded by regeneration of the limbus area using stem cells taken either from the same patient or from a compatible donor This approach has proved very effective, leading to complete recovery in more than 75% of the treated patients The resulting product, Holoclar, has received the green light by the European Medicines Agency on February 2015, as the very first medicinal product based on stem cells to be approved and formally registered in the Western world Holoclar is manufactured by Holostem Advanced Therapies – a spin-off of the University of Modena and Reggio Emilia in Italy – at the Centre for Regenerative Medicine “Stefano Ferrari” (CMR) of the same University 301 iPSCs) Fetal cardiomyocytes not represent a possible source for future clinical applications, as they are few and heterologous; on the other hand, cardiomyocytes derived from ESCs and iPSCs have only been employed in preclinical animal studies, as safety concerns still need to be solved In spite of the lack of convincing preclinical data, a large number of clinical trials have been performed on a variety of cardiac pathologies using different types of stem cells However, so far, no clear evidence of significant and long‐ term functional rescue has been demonstrated, and no production of newly formed cardiomyocytes from transplanted cells has been observed The first clinical studies involving patients with myocardial infarction transplanted with m yoblasts taken from skeletal muscles reported ventricular arrhythmia and no significant morphofunctional recovery The largest clinical study carried out so far included about 20 studies on more than 2000 patients with acute myocardial infarction transplanted with autologous stem cells from either bone marrow or peripheral blood No significant clinical recovery was observed at the 3–12 months’ follow‐up In some cases, a limited (3%) increase of the left v entricular ejection fraction was reported but in the absence of any improvement in the damaged area Although unsuccessful, some would argue that this type of transplant is not dangerous for the patient and may facilitate remodeling of the infarcted tissue, probably because of the acute release of protective molecules leading to modest (and transient) enhancement of revascularization The increased revascularization resulting from stem cell transplantation may explain the more encouraging result of clinical trials in refractory angina, which are now in phase II Regenerative Medicine to Treat Cardiac Dysfunctions The heart has always been thought to be unable to self‐ regenerate, but recent studies have shown that progenitor stem cells, identified by stem cell markers such as c‐kit or sca‐1, are present in human myocardium According to these studies, under normal condition, such progenitors may be able to regenerate the entire cardiomyocyte population in about 4.5 years However, these data are controversial: other studies suggest that cardiomyocytes turnover is not as high as reported and only 1% of cardiomyocytes is actually replaced every year Nevertheless, there is an enormous interest in developing stem cell therapies to heal cardiac tissues, and this represents one of the main research focuses in regenerative medicine A critical question researchers need to answer is how to obtain cardiomyocytes the most similar to those damaged, as cardiomyocytes vary depending on the area in which they are found Atrial cardiomyocytes are different from ventricular cardiomyocytes; similarly, those residing on the surface of the ventricular wall are different from those in the deep layers Moreover, cardiac myocytes generating the electrical impulse responsible for heart beating are different from those only involved in muscle contraction So far, functionally, cardiomyocytes have been obtained only from fetal cardiomyocytes and pluripotent cells (ESCs and Stem Cell‐Based Therapies for Skeletal Muscle Diseases Skeletal muscle represents the largest organ of the body comprising about 40% of total body mass in humans This tissue contains a particular cell type named satellite cells constituting a stable, self‐renewing pool of stem cells (Fig. 26.2) devoted to the physiological repair of damaged skeletal muscles In some conditions, such as Becker and Duchenne muscular dystrophies (BMD and DMD), skeletal muscles progressively degenerate and lose their intrinsic regenerative capacity To treat these disorders, several preclinical studies have investigated the potential of cell therapies based on the transplantation of myogenic‐competent cells, including— besides satellite cells—CD133+ cells (isolated from skeletal muscle or bone marrow), endothelial progenitors, and mesoangioblasts (vessel‐associated stem cells) Notably, among this variety of cell populations, only satellite cells and mesoangioblasts exhibit clear myogenic competence both in vitro and in vivo However, massive early cell death and poor proliferation and migration following transplantation represent main hurdles to be overcome for satellite cells to become a valuable therapy option Mesoangioblasts, firstly isolated in 2003 in Giulio Cossu’s laboratory from the dorsal aorta of 302 REGENERATIVE MEDICINE AND GENE THERAPY mouse embryos, are able to differentiate into a variety of mesoderm tissues including skeletal, cardiac, and smooth muscle Their ability to contribute to anatomical and functional muscle regeneration has been tested by arterial injections in both mouse and dog models of DMD A phase I clinical trial based on intra‐arterial delivery of donor‐derived mesoangioblasts in six DMD patients has been just concluded, reporting no safety concerns related to the treatment Stem Cell‐Based Therapies for Brain Diseases Neurodegenerative disorders are among the most challenging and devastating illnesses in medicine They represent a heterogeneous group of chronic and progressive diseases characterized by disparate etiologies, anatomical impairments, and symptoms Some of these disorders, such as Huntington’s disease (HD), are acquired in an entirely genetic manner Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), and PD mainly occur sporadically, although familiar forms caused by inheritance of gene mutations are known On the other side, other neurodegenerative disorders, such as traumatic spinal cord injury and stroke, have no genetic (heritable) components By virtue of this extreme heterogeneity, different specific requirements should be envisaged when considering cell replacement as possible therapeutic strategy We can distinguish between (i) neuronal CNS degenerative disorders caused by a prominent loss of specific neuronal populations leading to destruction of precise cerebral circuitries and (ii) nonneuronal CNS degenerative conditions characterized by loss of nonneuronal elements In the case of neuronal degeneration, the success of cell replacement strictly depends on the complexity and p recision of the connectivity pattern that needs to be restored In PD, the affected dopaminergic neurons in substantia nigra (SN) exert a modulator action on the target circuits (striatum) mostly through the release of diffusible molecules (dopamine) In this type of system, defined as “paracrine,” even a partial pattern repair may lead to a significant functional recovery Indeed, in PD, donor cells can be transplanted directly into the target region to circumvent the problem of long‐distance neuritic growth in adult CNS Despite the ectopic location, if grafted cells acquire a dopaminergic identity reestablishing a regulated and e fficient release of dopamine, they can lead to a clinically relevant functional recovery Differently, other diseases with selective degeneration of specific neuronal populations, such as HD, ALS, and conditions exhibiting global neuronal degeneration (trauma, stroke, and AD), require a complex pattern repair and are therefore very difficult to treat with cell‐based strategies Differently, nonneuronal CNS degenerative syndromes, such as multiple sclerosis (MS), characterized by severe inflammation and oligodendroglial degeneration leading to axonal demyelination, represent a good target for cell replacement because of their limited requirements of pattern repair; for example, in MS, functional rescue requires grafted cells to produce oligodendrocytes capable of restoring axonal myelination It is important to emphasize that although pattern repair is critical to obtain permanent rescue, cells transplanted into the brain may also be beneficial via the release of molecules that may either stimulate the regenerative potential of local cells (where present) or increase the survival (and decrease disease progression) of the remaining host elements In addition, immunomodulation activity of grafted cells could be beneficial in diseases such as MS where a prominent disease‐associated inflammation contributes to establish ment and progression of the disease In the last two decades, a number of clinical studies employing grafting of human fetal tissues have provided a proof of concept for cell‐based therapies for PD and HD Nonetheless, fetal brain tissues not represent a practical source for large‐scale therapeutic applications due to scant availability, quality concerns, and ethical considerations Hence, the establishment of readily expandable neural stem cell (NSC) populations, maintaining their capability to generate cells belonging to the three major neural lineages and competence to regenerate the injured or diseased CNS, has represented a crucial milestone In the last years, several NSC populations, including fetal‐ and adult‐derived NSCs (Fig. 26.2), neural progenitors derived from human pluripotent cells (ESCs and iPSCs), have been generated, thus increasing the possibilities to ultimately uncover NSCs suitable (in terms of expandability, safety, and effectiveness) for clinical applications A large number of studies have explored grafting behavior of several NSC typologies (and their progeny) in a variety of preclinical studies and even in some clinical trials Nonetheless, besides the great expectations, it should be remarked that up to now an ideal NSC system is not yet available to the clinic In the last two years, protocols to obtain authentic dopaminergic neurons from hES cells have been developed by the groups of Lorenz Studer in New York and Malin Parmar in Lund, Sweden Upon transplantation into rodent models of PD, these donor cells exhibited important biological and functional capacities leading to behavioral recovery A first trial in PD patients with these cells is expected to start in the next few years Over the last 10 years, much attention has been given to the potential use of stem cells as therapeutic agents for neurodegenerative disorders, and significant progresses have been achieved To safely translate stem cell research into therapeutics, we need precise knowledge about molecules and signaling pathways that regulate proliferation, differentiation, and migration of NSCs in vivo Several attempts have already been made to move NSC discoveries from bench to bedside In a phase I/II clinical trial by StemCell Inc., chronic spinal cord injury patients have been transplanted with purified human adult NSCs The same company has completed a phase I clinical trial on six c hildren GENE THERAPY transplanted with human adult NSCs to treat Batten disease Other examples of clinical trials in the NSC field are Neuralstem phase I clinical trials for ALS patients and ReNeuron trial of a NSC therapy for disabled stroke patients (see https://clinicaltrials.gov) The first FDA‐approved clinical trial with human ESC‐derived cells, oligodendrocyte progenitors for spinal cord injury, by Geron Corporation started in 2010 but was recently stopped after phase I due to shortage of financial resources for stem cell programs These early stem cell‐based clinical trials are providing scientists and clinicians with initial results on the safety of these cells for treating the diseased brain and useful data for improving the design of future clinical trials It is important to add that a better communication of these careful and thorough clinical trials to the public is crucial to inform patients about controlled and reliable clinical research currently ongoing and alert them against the rising number of private clinics offering unproven stem cell treatments as safe and effective therapeutic options for a wide range of different brain diseases GENE THERAPY The goal of somatic gene therapy is to correct/compensate defective/abnormal gene functions responsible for disease Its purpose is to cure a variety of both inherited and acquired diseases by introducing nucleic acids (usually genes or gene fragments) into patient’s cell nuclei Initially, gene therapy was conceived to cure monogenic hereditary diseases with a recessive phenotype (such as cystic fibrosis, hemophilia, or muscular dystrophies), by transferring a normal copy of the mutated gene into the appropriate tissue In this context, the “drug” is represented by the encoded therapeutic protein or by the nucleic acid itself It is important to underline that the range of nucleic acids used in gene therapy approaches includes not only those with a replacement function but also genes, DNA, or RNA fragments regulating cell activity or modulating immune system functions Therefore, gene therapy is now extended to a vast number of acquired pathologies, such as tumors and cardiovascular diseases In the 1990s, the first protocol for gene therapy for the treatment of a hereditary immune system deficiency was approved in the United States Currently, almost a thousand of protocols are in progress all over the world In particular, most of them concern the treatment of cancers of different kind and grade Besides these, several clinical trials on cardiovascular diseases, infective diseases (almost all about HIV), and recessive autosomal single‐gene diseases, such as cystic fibrosis, are also in progress Overall, despite gene therapy is becoming safer for patients, its applicability remains limited, primarily because of technical issues that will be outlined in the next paragraphs 303 Protocols for Gene Therapy Two possible approaches are usually considered in gene therapy: nucleic acids transfer ex vivo or in vivo (see Fig. 26.3) In the ex vivo gene therapy, patient’s cells are isolated, cultivated in vitro, genetically modified to introduce the therapeutic gene, and then reinjected in the same patient This approach is feasible when target cells are easily a ccessible and isolable from the patient and can be handled and reinjected (i.e., throughout aerosol or injection into the bloodstream) A major advantage of this approach is its reduced probability to stimulate an immune response against the vector used to transfer the gene However, the method is laborious and expensive, as protocols have to be patient specific in order to avoid immune rejections The number of cell types that can be maintained and expanded ex vivo has greatly increased in the last years Besides lymphocytes and HSC, the list includes cells with a stem cell phenotype capable of generating different cellular subtypes, such as skin cells, vascular endothelial precursors, and muscular progenitors The possibility of transferring genes into stem cells is expanding the therapeutic opportunities as gene therapy and cell and regenerative therapy can cooperate to yield a more efficient treatment for both genetic (i.e., muscular dystrophies) and noninherited degenerative diseases (i.e., PD and myocardial infarction) With the in vivo gene therapy, the therapeutic gene is inserted directly into patient’ cells or tissues In theory, this approach is simpler than the ex vivo, and once optimized, it can be applied to an unlimited number of patients with the same disease In practice, this approach raises some concerns: 1. Many tissues are difficult to reach or to transduce in vivo quantities that may be clinically significant For example, it is difficult to conceive an extensive gene therapy for muscle tissue or cartilage; in these cases, the gene therapy is necessarily restricted to confined districts 2. Off‐target/side effects can be a problem due to gene transfer into cell types different from the target ones 3. When administered in vivo, the vector used for gene transfer is more exposed to inactivation (retroviral vectors or specific neutralizing antibodies can be inactivated by the complement), and it can stimulate patient’s immune responses 4. Most cell types in the organism (including muscular, neural, vascular endothelial, and hepatic cells) are generally quiescent This fact may limit the use of some type of vectors, such as retroviral vectors, that only infect cells actively dividing In Table 26.2, methods for gene transfer are reported with their specific characteristics, both positive and negative We will focus on applications based on safe viral vectors (see Supplement E 26.3, “Viral Vectors for Gene Therapy in Somatic ☞ 304 REGENERATIVE MEDICINE AND GENE THERAPY Therapeutic gene Production of viral particles carrying the therapeutic gene Ex vivo strategy Therapeutic gene delivery In vivo strategy Selection and expansion Direct injection into the patient Cells of interest withdrawal* Re-infusion of corrected cell Figure 26.3 The two main strategies of gene therapy There are two different strategies in gene therapy In the in vivo strategy, the corrected gene is injected directly into the patient, mainly using a virus as delivery system In the ex vivo strategy, cells are taken from the patient, genetically modified to introduce the curative gene, and then reinjected into the patient *Both autologous and heterologous cells can be used; theoretically, cells of interest can be derived also from pluripotent cells (ES cells and iPS cells, the latter can be an autologous source) Cells”) that currently represent the most practiced choice (about 80% of current clinical trials employ viral vectors) Gene Therapy for Monogenic Inherited Diseases In 1999 took place one of the saddest events in the history of gene therapy Jesse Gelsinger, an 18‐year‐old boy affected by a nonsevere form of ornithine transcarbamylase (OTC) deficiency, was the first patient ever treated with gene therapy He died few days after the treatment because of a hyperimmune reaction caused by the excessive amount of vector injected Indeed, he had been injected a very high dose of vector (40 billion viral particles, the highest dose ever administered in a clinical trial) carrying the normal OTC gene into his portal vein Hundreds of gene therapy protocols have been developed since then However, it is important to consider that about three‐fourth of current clinical trials are phase I Therefore, thorough and reliable data about their efficacy and therapeutic reproducibility are presently not available Some of the current optimized clinical protocols for gene therapy and their relative concerns are listed in the following Hereditary Immunodeficiency Syndromes This group of diseases represents the gold standard for gene therapy experimentation The first clinical trial for a hereditary immunodeficiency syndrome was developed in 1990 by three American researchers: French Anderson, Michael Blaese, and Kenneth Culver The ex vivo gene therapy protocol was conceived to treat a 4‐year‐old girl affected by severe combined immunodeficiency disease (SCID) This pathology is caused by lack of adenosine deaminase (ADA), an essential enzyme for purine metabolism ADA deficiency triggers selective T cell damage resulting in a severe form of immunodeficiency The ADA‐SCID patients generally suffer for recurrent severe infections, mostly concerning the a irways and gastrointestinal tract, chronic dysentery, and reduced growth rate The first symptoms onset in the n eonatal period, but sometimes, they can be delayed by several months, due to a partial immune protection provided by maternal antibodies Without early diagnosis and treatment, these patients not survive over 24 months Conventional treatments for ADA‐ SCID include hematopoietic cell transplantation and weekly ADA injection for the entire life Gene therapy was explored as an alternative because transplantation often failed and enzymatic replacement appears useless in many patients Another clinical trial was developed in 1991 Since no side effects emerged in the first two trials, eleven children were enrolled in a third gene therapy experimentation in 1996 In these initial ex vivo protocols, the cDNA encoding the human ADA enzyme was inserted into patients’ T cells by retrovirus‐mediated gene transfer The transduced T cells were then injected back into patients resulting in symptoms GENE THERAPY 305 Table 26.2 Gene delivery methods for gene therapy Method Vector Advantages Disadvantages Direct injection Naked DNA Low efficiency Transient transgene expression Chemical Liposomes Physical Electroporation Viral transduction Retrovirus Not pathogenic Easy to handle Unlimited transgene size Not pathogenic Easy to handle Unlimited transgene size Not pathogenic Easy to handle Unlimited transgene size High transduction efficiency Stable integration in host genome Poor immunogenicity Transduction efficiency Transduction of both dividing and quiescent cells Stable integration in host genome Poor immunogenicity Transduction efficiency Transduction of both dividing and quiescent cells Episomal maintenance Accommodate large DNA fragments High levels of transgene expression Not pathogenic for humans Lentivirus Adenovirus Adeno‐associated virus Herpesvirus Poor immunogenicity Transduction of both dividing and quiescent cells Accommodate large DNA fragments Transduction of both dividing and quiescent cells High tropism for nerve cells Episomal maintenance improvement, as long as the genetically modified cells persisted In order to achieve a long‐term solution, recent protocols have been developed by Italian researchers employing ex vivo manipulation of HSC It is important to note that in this gene therapy protocols, patients are weekly administered their pharmacological ADA dose to avoid the ethical dilemma of blocking a potentially effective therapy (enzymatic replacement) in order to try a new treatment Gene therapy has also been used to treat an X‐linked form of SCID (X‐SCID) This lethal condition is caused by mutations in the gene encoding the common chain gamma, a protein involved in the production of different receptors in the immune system Because of a differentiation block, this disease is characterized by deficiency of T lymphocytes and natural killer cells; lymphocyte precursors are produced, but they rapidly die by apoptosis because of the lack of gamma chain In 2000, in France, two X‐SCID pediatric patients were treated by gene therapy Both were severely affected, manifesting pneumonia, dysentery, and skin rashes HSC were extracted from their bone morrow, manipulated ex vivo using a retroviral vector carrying the healthy gene cDNA, and injected back into the patients Notably, treated patients did not receive any other treatment Given the encouraging initial results, additional patients were enrolled in the clinical Low efficiency Transient transgene expression Low efficiency Transient transgene expression Low viral titer production Possible insertional mutagenesis Targets dividing cells only Low viral titer production Possible insertional mutagenesis Targets dividing cells only Transient transgene expression High immunogenicity Accommodate small DNA fragments (