Table Of ContentGene Therapy Protocols
M E T H O D S I N M O L E C U L A R M E D I C I N ETM
John M. Walker, SERIES EDITOR
69. Gene Therapy Protocols, 2nd ed., edited 51. Angiotensin Protocols, edited by Donna
byJeffrey R. Morgan, 2002. H. Wang, 2001
68. Molecular Analysis of Cancer, edited by 50. Colorectal Cancer: Methods and
Jacqueline Boultwood and Carrie Fidler, 2002 Protocols, edited by Steven M. Powell,
67. Meningococcal Disease: Methods and 2001
Protocols,edited by Andrew J. Pollard 49. Molecular Pathology Protocols, edited by
and Martin C. J. Maiden, 2001 Anthony A. Killeen, 2001
66. Meningococcal Vaccines: Methods and 48. Antibiotic Resistance Methods and
Protocols,edited by Andrew J. Pollard and Protocols,edited by Stephen H. Gillespie,
Martin C. J. Maiden, 2001 2001
65. Nonviral Vectors for Gene Therapy: 47. Vision Research Protocols,edited by
Methods and Protocols, edited by Mark A, P. Elizabeth Rakoczy, 2001
Findeis,2001
46. AngiogenesisProtocols,edited by
64. Dendritic Cell Protocols, edited by Stephen J. Clifford Murray, 2001
P. Robinson and Andrew J. Stagg, 2001 45. Hepatocellular Carcinoma: Methods and
63. Hematopoietic Stem Cell Protocols, Protocols,edited byNagy A. Habib, 2000
edited by Christopher A. Klug and Craig
44. Asthma:Mechanisms and Protocols,edited by
T. Jordan, 2001 K. Fan Chung and Ian Adcock, 2001
62. Parkinson’s Disease: Methods and Protocols, 43. Muscular Dystrophy: Methods and
edited by M. Maral Mouradian, 2001
Protocols,edited byKatherine B. Bushby
61. Melanoma Techniques and Protocols: and Louise Anderson, 2001
Molecular Diagnosis, treatment, and 42. Vaccine Adjuvants: Preparation Methods
Monitoring,edited by Brian J. Nickoloff, 2001
and Research Protocols, edited by Derek
60. Interleukin Protocols, edited by Luke A. T. O’Hagan, 2000
J. O’Neill and Andrew Bowie, 2001 41. Celiac Disease: Methods and Protocols,
59. Molecular Pathology of the Prions, edited by Michael N. Marsh, 2000
edited by Harry F. Baker, 2001
40. Diagnostic and Therapeutic Antibodies,
58. Metastasis Research Protocols: Volume edited by Andrew J. T. George and
2, Cell Behavior In Vitro and In Vivo, Catherine E. Urch, 2000
edited by Susan A. Brooks and Udo
39. Ovarian Cancer: Methods and Protocols,
Schumacher,2001 edited by John M. S. Bartlett, 2000
57. Metastasis Research Protocols: Volume 1, 38. Aging Methods and Protocols, edited by
Analysis of Cells and Tissues,edited by
Yvonne A. Barnett and Christopher
Susan A. Brooks and Udo Schumacher, 2001 R. Barnett, 2000
56. Human Airway Inflammation: Sampling 37. Electrochemotherapy, Electrogenetherapy,
Techniques and Analytical Protocols,
and Transdermal Drug Delivery:
edited by Duncan F. Rogers and Louise Electrically Mediated Delivery of Molecules
E. Donnelly, 2001
to Cells, edited by Mark J. Jaroszeski,
55. Hematologic Malignancies: Methods and Richard Heller, and Richard Gilbert, 2000
Protocols,edited by Guy B. Faguet, 2001 36. Septic Shock Methods and Protocols,
54. Mycobacterium tuberculosis Protocols, edited edited by Thomas J. Evans, 2000
byTanya Parish and Neil G. Stoker, 2001
35. Gene Therapy of Cancer: Methods and
53. Renal Cancer: Methods and Protocols, Protocols, edited by Wolfgang Walther
edited by Jack H. Mydlo, 2001 and Ulrike Stein, 2000
52. Atherosclerosis: ExperimentalMethods and
Protocols,edited by Angela F. Drew, 2001
M E T H O D S I N M O L E C U L A R M E D I C I N ETM
Gene Therapy
Protocols
2nd Edition
Edited by
Jeffrey R. Morgan
Brown University, Providence, RI
Humana Press Totowa, New Jersey
© 2002 Humana Press Inc.
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Due diligence has been taken by the publishers, editors, and authors of this book to assure the accuracy of
the information published and to describe generally accepted practices. The contributors herein have care-
fully checked to ensure that the drug selections and dosages set forth in this text are accurate and in accord
with the standards accepted at the time of publication. Notwithstanding, since new research, changes in
government regulations, and knowledge from clinical experience relating to drug therapy and drug reactions
constantly occur, the reader is advised to check the product information provided by the manufacturer of each
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Library of Congress Cataloging-in-Publication Data
Gene therapy protocols. -- 2nd ed. / edited by Jeffrey R. Morgan.
p. ; cm. -- (Methods in molecular medicine ; 69)
Includes bibliographical references and index.
ISBN 0-89603-723-1 (hardcover; alk. paper) ISBN 0-89603-869-6 (comb binding, alk. paper)
1. Gene therapy--Methodology. I. Morgan, Jeffrey Robert. II. Series.
[DNLM: 1. Gene Therapy--methods. 2. Gene Transfer Techniques. 3. Genetic Vectors. QZ 50
G3213 2002]
RB155.8 .G465 2002
616'.042--dc21
2001026441
Preface
Efforts in gene therapy have grown dramatically in recent years. Basic
research as well as clinical activity have made exciting progress and are begin-
ning to offer renewed hope that gene therapy may be able to deliver novel
approaches for the treatment of inherited as well as such acquired diseases as
cardiovascular disease and cancer. With the sequencing of the human genome
complete, we now have a comprehensive catalog of genes that further expands
the potential role of gene therapy into such new fields as tissue engineering.
Central to gene therapy is the process of gene transfer; thus, advances in the
technology of gene transfer are at the heart of this field’s progress. Numerous
technologies, based on a variety of methods (e.g., viral-mediated, physical/
chemical), have been developed to achieve gene transfer. Some of the earliest
methods, such as recombinant retroviruses, are still widely used, have undergone
significant improvements, and have given rise to new vectors based on lentiviruses.
Others, such as molecular conjugates, are exciting new methods that blur the
distinctions between the fields of gene therapy and drug delivery. Regardless of
the method chosen, gene therapy is performed in either one of two settings, namely
ex vivo or in vivo. Individual gene transfer methods have inherent advantages
and limitations in these settings and this is an important consideration in planning
an overall strategy. Another, perhaps more important, distinction between gene
transfer technologies is whether or not the genetic modification is permanent or
temporary and this has a large impact on any new medical therapy envisioned.
Each of these criteria plays a significant role as the field tries to weigh the advan-
tages and disadvantages of each gene transfer technology and find a match with
an intended medical application.
The field of gene therapy is advancing simultaneously on several fronts.
The core technologies of gene transfer have undergone significant improvements
in critical areas such as gene transfer efficiency, gene regulation, and vector
production. Industry has joined academia, and a new emphasis is being placed on
a quantitative understanding of gene transfer. And the applications of gene
transfer continue to expand into new medical areas. In this new and entirely
revised second edition, Gene Therapy Protocolspresents detailed methods and
protocols covering a comprehensive range of technologies and techniques used
v
vi Preface
by leaders of the gene therapy field. The first part of the book covers the new
molecular conjugates that show great promise for targeting gene transfer and regu-
lating transgene expression. The second part of the book presents completely
revised methods based on retroviruses and adenoviruses as well as new and prom-
ising methods based on lentiviruses and adeno-associated viruses. The third part
of the book details protocols used in exciting applications of gene transfer in such
areas as delivery of therapeutic proteins, vaccination, and tissue engineering.
This collection of protocols should make a valuable and indispensable
resource for graduate students, postdoctoral fellows, as well as basic and clinical
researchers in industry and academia.
Jeffrey R. Morgan
Contents
Preface .............................................................................................................v
Contributors.....................................................................................................xi
1 Poly-L-Lysine-Based Gene Delivery Systems:
Synthesis, Purification, and Application
Charles P. Lollo, Mariusz G. Banaszczyk, Patricia M. Mullen,
Christopher C. Coffin, Dongpei Wu, Alison T. Carlo,
Donna L. Bassett, Erin K. Gouveia, and Dennis J. Carlo.............1
2 Targeted Gene Transfer to Liver Using Protein-DNA Complexes
Catherine H. Wu, Cherie M. Walton, and George Y. Wu.................15
3 Receptor-Directed Molecular Conjugates for Gene Transfer
Assem G. Ziady and Pamela B. Davis...............................................25
4 Gene Transfer into Muscle by Electroporation in Vivo
Jun-ichi Miyazaki and Hiroyuki Aihara.............................................49
5 Viral Liposomes: Preparation and Use
Yasufumi Kaneda.................................................................................63
6 LPD Nanoparticles—Novel Nonviral Vector
for Efficient Gene Delivery
Yadi Tan, Mark Whitmore, Song Li, Peter Frederik,
and Leaf Huang................................................................................73
7 Solvoplex Synthetic Vector for Intrapulmonary Gene Delivery:
Preparation and Use
Klaus Schughart and Ulla B. Rasmussen........................................83
8 Gene Correction Frequency by Chimeric RNA-DNA Oligonucleotide
Using Nuclear Extracts
Olga A. Igoucheva and Kyonggeun Yoon........................................95
9 Regulated Expression of Plasmid-Based Gene Therapies
Ronald V. Abruzzese, Fiona C. MacLaughlin, Louis C. Smith,
and Jeffrey L. Nordstrom.............................................................109
10 Photochemical Transfection:
Light-Induced, Site-Directed Gene Delivery
Lina Prasmickaite, Anders Høgset, and Kristian Berg.................123
vii
viii Contents
11 Direct Gene Transfer and Vaccination
Via Skin Transfection Using a Gene Gun
Chia-Feng Kuo, Jeng Hwan Wang, and Ning-Sun Yang...............137
12 Preparation of Pseudotyped Retroviral Vector
Hong Yu..............................................................................................149
13 Quantitative Measurement of the Concentration
of Active Recombinant Retrovirus
Stelios T. Andreadis and Jeffrey R. Morgan..................................161
14 Minigene-Containing Retroviral Vectors
Using an Alphavirus/Retrovirus Hybrid Vector System:
Production and Use
Jarmo Wahlfors and Richard A. Morgan........................................173
15 Retrovirus-Mediated Gene Transfer
to Human Hematopoietic Stem Cells
William P. Swaney, Enrico M. Novelli, Alfred B. Bahnson,
and John A. Barranger.................................................................187
16 Genetically Modified Skin Substitutes: Preparation and Use
Karen E. Hamoen, Gulsun Erdag, Jennifer L. Cusick,
Hinne A. Rakhorst, and Jeffrey R. Morgan................................203
17 Bioartificial Muscles in Gene Therapy
Courtney Powell, Janet Shansky, Michael Del Tatto,
and Herman H. Vandenburgh......................................................219
18 Cytokine Gene-Modified Cell-Based Cancer Vaccines
R. Todd Reilly, Jean-Pascal H. Machiels, Leisha A. Emens,
and Elizabeth M. Jaffee.................................................................233
19 HIV-Based Vectors: Preparation and Use
Antonia Follenzi and Luigi Naldini..................................................259
20 Packaging Cell System for Lentivirus Vectors: Preparation and Use
Narasimhachar Srinivasakumar......................................................275
21 Lentiviral Vectors: Preparation and Use
Lung-Ji Chang and Anne-Kathrin Zaiss.........................................303
22 Simian Foamy Virus Vectors: Preparation and Use
Jeonghae Park and Ayalew Mergia.................................................319
23 Recombinant Feline Immunodeficiency Virus Vectors:
Preparation and Use
Michael A. Curran and Garry P. Nolan............................................335
24 Lentivirus Vector Based on Simian Immunodeficiency Virus:
Development and Use
Klaus Überla.......................................................................................351
Contents ix
25 Cytoplasmic RNA Vector Derived
from Nontransmissible Sendai Virus: Production and Use
Akihiro Iida and Mamoru Hasegawa...............................................361
26 Preparation of Helper-Dependent Adenoviral Vectors
Philip Ng, Robin J. Parks, and Frank L. Graham...........................371
27 Construction of First-Generation Adenoviral Vectors
Philip Ng and Frank L. Graham........................................................389
28 Preparation of Ovine Adenovirus Vectors
Peter Löser, Daniel Kümin, Moritz Hillgenberg, Gerald W. Both,
and Christian Hofmann.................................................................415
29 Highly Purified Recombinant Adeno-Associated Virus Vectors:
Preparation and Quantitation
Bruce Schnepp and K. Reed Clark..................................................427
30 High-Titer Stocks of Adeno-Associated Virus
from Replicating Amplicons and Herpes Vectors
Ajay R. Mistry, Mahesh De Alwis, Elisabeth Feudner,
Robin R. Ali, and Adrian J. Thrasher..........................................445
31 Herpes Simplex Virus/Adeno-Associated Virus Hybrid Vectors
for Gene Transfer to Neurons: Preparation and Use
Lauren C. Costantini, Cornel Fraefel, Xandra O. Breakefield,
and Ole Isacson.............................................................................461
32 Development of Replication-Defective Herpes Simplex Virus Vectors
William F. Goins, David M. Krisky, Darren P. Wolfe,
David J. Fink, and Joseph C. Glorioso.......................................481
Index............................................................................................................509
Contributors
RONALD V. ABRUZZESE • Valentis, The Woodlands, TX
HIROYUKI AIHARA • Department of Nutrition and Physiological Chemistry,
Osaka University Medical School, Suita, Osaka, Japan
ROBIN R. ALI • Molecular Immunology Unit, Institute of Child Health,
London; Institute of Ophthalmology, London, United Kingdom
STELIOS T. ANDREADIS • Bioengineering Laboratory, Department of
Chemical Engineering, University of New York at Buffalo, Amherst, NY
ALFRED B. BAHNSON • Department of Human Genetics, University
of Pittsburgh, Pittsburgh, PA
MARIUSZ G. BANASZCZYK • The Immune Response Corporation, Carlsbad, CA
JOHN A. BARRANGER • Department of Human Genetics and Department
of Molecular Genetics and Biochemistry, University of Pittsburgh,
Pittsburgh, PA
DONNA L. BASSETT • The Immune Response Corporation, Carlsbad, CA
KRISTIAN BERG • Department of Biophysics, Institute for Cancer Research,
The Norwegian Radium Hospital, Oslo, Norway
GERALD W. BOTH • CSIRO, North Ryde, Australia
XANDRA O. BREAKEFIELD • Molecular Neurogenetics Unit, Massachusetts
General Hospital and Harvard Medical School, Boston, MA
ALISON T. CARLO • The Immune Response Corporation, Carlsbad, CA
DENNIS J. CARLO • The Immune Response Corporation, Carlsbad, CA
LUNG-JI CHANG • Powell Gene Therapy Center, Department of Molecular
Genetics and Microbiology, University of Florida, Gainesville, FL
K. REED CLARK • Division of Molecular Medicine, Department of Pediatrics;
Department of Molecular Virology, Immunology, and Molecular
Genetics, Ohio State University, Columbus, OH
CHRISTOPHER C. COFFIN • The Immune Response Corporation, Carlsbad, CA
LAUREN C. COSTANTINI • Neuroregeneration Laboratory, Harvard Medical
School; McLean Hospital, Belmont, MA
MICHAEL A. CURRAN • Program in Immunology, Stanford University,
Stanford, CA
xi
