Manufacturing of Gene Therapeutics Methods, Processing,
Regulation, and Validation, 1st Edition
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Preface
Advances in molecular biology and recombinant DNA technology have
accelerated progress in many fields of life science research including gene
therapy. A large number of genetic engineering approaches and methods are
readily available for gene cloning and therapeutic vector construction.
Significant progress is being made in genomic, DNA sequencing, gene
expression, gene delivery and cloning. Thus gene therapy has already shown
that it holds great promise for the treatment of many diseases and disorders.
In general it involves the delivery of recombinant genes or transgenes into
somatic cells to replace proteins with a genetic defect or to transfer with the
pathological process of an illness. The viral and non-viral delivery systems
may hold the potential for future non-invasive, cost effective oral therapy of
genetically based disorders.
Recent years have seen considerable progress in the discovery and early
clinical development of a variety of gene therapeutic products. The
availability, validation and implementation have enabled success but also for
testing and evaluation. New challenges will need to be overcome to ensure
that products will also be successful in later clinical development and
ultimately for marketing authorisation. These new challenges will include
improvements in delivery systems, better control of in-vivo targeting,
increased level transduction and duration of expression of the gene, and
manufacturing process efficiencies that enable reduction in production costs.
Perhaps profound understanding of regulated gene design may result in
innovative bioproducts exhibiting safety and efficacy profiles that are
significantly superior to those achieved by the use of naturally occurring
genes. This procedure may considerably contribute to fulfil standards set by
regulatory authorities.
This book aims to project an overview of the current advances in the field
of gene therapy and the methods that are being successfully applied in the
manufacture of gene therapeutic products.
I am indebted to the international group of contributors who have shared
their practical knowledge and experience. Each chapter represents an
overview of its chosen topic. Chapters one and two provide an overview of
gene therapy and gene therapy for cancer. Gene self-assembly and gene
expression are discussed in chapters three and four. Genotype and response
to cytotoxic gene therapy is reviewed in chapter five. Vector assembly and
gene transfer is discussed in chapters six and seven. Plasmid manufacturing
is reviewed in chapter eight. The importance of quality control and
assurances and the analytical methods are discussed in chapters nine and ten.
Chapter eleven provides an insight into validation aspects in gene therapy
and gene delivery is reviewed in chapter twelve. The importance of
regulatory issues and guidelines are reviewed for the American market in
chapter thirteen and the European market in chapter fourteen, and chapter
fifteen discusses the regulatory compliance in contract manufacturing
environment. Finally, chapter sixteen discusses the risk assessment in gene
therapy.
My thanks to the contributors for the extensive diligence and their
patience and goodwill during the production of the book; they deserve the
full credit for the source of the book.
It is hoped that this book will be of great value to all those who are
engaged in the field of gene therapy and that it will stimulate further
progress and advancement in this field to meet the ever increasing demands.
I should be most grateful for any suggestion, which could serve to improve
future editions of this book.
My deep appreciation to Jo Lawrence of Kluwer Academic/Plenum
Publishers for her continuous patience, encouragement and help in guiding
all of us through the preparation and the completion of this book.
G. Subramanian
Contributors
Akshay Anand
Department of Immunopathology
Post Graduate Institute of Medical Education and Research
Chandigarh, India
Sunil K. Arora
Department of Immunopathology
Post Graduate Institute of Medical Education and Research
Chandigarh, India
Joy A. Cavagnaro
President, Access Bio LC
Leesburg
VA 20177-1400, USA
Nancy Chew
President, Regulatory Affairs, North America LLC
P.O.BOX 72375
Durham
NC 2772, USA
Odile Cohen-Haguenauer
Laboratorie TGOM & Service d'Oncologie Medicale
Hopital Saint-Louis
1, avenue Claude Vellefaux
75475 Paris CEDEX 10, France
Peter Daniel
Department of Haematology, Oncology and Tumour Immunology
University Medical Centre Charite
Campus Berlin-Buch
Humboldt University of Berlin
Lindenberger Weg
13125 Berlin, Germany
Linh Do
Berlex Biosciences
15049 San Pablo Avenue
P.O.BOX 4099
Richmond
CA 94804-4089, USA
Vladimir I. Evtushenko
Laboratory of Genetic Engineering
Research Institute of Roentgenology and Radiology
St. Petersburg 189646, Russia
James G. Files
Berlex Biosciences
15049 San Pablo Avenue
P.O.Box 4099
Richmond
CA 94804-4089, USA
Erwin Flaschel
PlasmidFactory GmbH & Co. KG
Meisenstrasse 96
D-33607 Bielefeld, Germany
Karl Friehs
University of Bielefeld
Postfachl00131
D-33501 Bielefeld, Germany
Bernhard Gillissen
Department of Haematology, Oncology and Tumour Immunology
University Medical Centre Charite
Campus Berlin-Buch
Humboldt University of Berlin
Lindenberger Weg
D-13125 Berlin, Germany
Clague P. Hodgson
Nature Technology Corporation
4701 Innovation Drive
Lincoln
Nebraska 68521,USA
John Irving
Berlex Biosciences
15049 San Pablo Avenue
P.O.Box 4099
Richmond
CA 94804-4089, USA
Juan Irwin
Berlex Biosciences
15049 San Pablo Avenue
P.O.Box 4099
Richmond
CA 94804-4089, USA
John Jenco
Dow Biopharmaceutical Contract Manufacturing Services
50 East Loop Road
Stony Brook
NY 11790, USA
Jaspreet Kaur
Department of Biochemistry
Postgraduate Institute of Medical Education and Research
Chandigarh, India
Steven S. Kuwahara
Kuwahara Consulting
PMB #506
1669-2 Hollenbeck Avenue
Sunnyvale
CA 94087-5042, USA
Mark Lawler
Department of Haematology and Oncology
St Patrick Dun Research Labs
James's Street
Dublin 8, Ireland
Elisabeth Lehmberg
Berlex Biosciences
15049 San Pablo Avenue
P.O.Box 4099
Richmond
CA 94804-4089, USA
Bruce Mann
Berlex Biosciences
15049 San Pablo Avenue
P.O.Box 4099
Richmond
CA 94804-4089, USA
Michael T. McCaman
Berlex Biosciences
15049 San Pablo Avenue
P.O.Box 4099
Richmond
CA 94804-4089, USA
Stephen Morris
BioReliance
14920 Broschart Road
Rockville
MD 20850-3349, USA
Munishi Mukesh
National Bureau of Animal Genetics Resources (ICAR)
Karnal, India
Peter K. Murakami
Berlex Biosciences
15049 San Pablo Avenue
P.O.BOX 4099
Richmond
CA 94804-4089, USA
Chris Murphy
BioReliance
14920 Broschart Road
Rockville
MD 20850-3349, USA
Jeffrey W. Nelson
Berlex Biosciences
15049 San Pablo Avenue
P.O.Box4099
Richmond
CA 94804-4089, USA
Eirik Nestaas
Berlex Biosciences
15049 San Pablo Avenue
P.O.BOX 4099
Richmond
CA 94804-4089, USA
Erno Pungor, Jr.
Berlex Biosciences
15049 San Pablo Avenue
P.O.Box 4099
Richmond
CA 94804-4089, USA
Martin Schleef
PlasmidFactory GmbH & Co. KG
Meisenstrasse 96
D-33607 Bielefeld, Germany
Torsten Schmidt
PlasmidFactory GmbH & Co. KG
Meisenstrasse 96
D-33607 Bielefeld, Germany
Gail Sofer
BioReliance
14920 Broschart Road
Rockville
MD 20850-3349, USA
Isrid Sturm
Department of Haematology, Oncology and Tumour Immunology
University Medical Centre Charite
Campus Berlin-Buch
Humboldt University of Berlin
Lindenberger Weg
D-13125 Berlin, Germany
Mei P. Tan
Berlex Biosciences
15049 San Pablo Avenue
P.O.Box 4099
Richmond
CA 94804-4089, USA
Joseph A. Trai-Na
Berlex Biosciences
15049 San Pablo Avenue
P.O.Box 4099
Richmond
CA 94804-4089, USA
Spencer Tse
Berlex Biosciences
15049 San Pablo Avenue
P.O.BOX 4099
Richmond
CA 94804-4089, USA
Dominic K. Vacante
BioReliance
14920 Broschart Road
Rockville
MD 20850-3349, USA
Martin Weber
Group Manager R&D
Qiagen GmbH
Max-Volmer Strasse 4
D-40724 Hilden, Germany
TaoYu
Berlex Biosciences
15049 San Pablo Avenue
P.O. Box 4099
Richmond
CA 94804-4089, USA
Contents
Preface .............................................................................. v
Contributors ....................................................................... vii
Somatic Gene Therapy, Paradigm Shift or Pandora's
Box: A Perspective on Gene Therapy ..................... 1
Gene Therapy for Cancer: Deceiving the Malignant
Cell ............................................................................. 17
Gene Self-Assembly (GENSA): Facilitating the
Construction of Genes and Vectors ........................ 33
Gene Expression ............................................................. 45
Tumour Genotype and Response to Cytotoxic Gene
Therapy ...................................................................... 59
Protein Binding Matrices: Tools for Phenol-free
Cloning and Vector Assembling .............................. 99
Gene Transfer into Eukaryotic Cells .............................. 135
Plasmid DNA Manufacturing .......................................... 155
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xvi Contents
Quality Assurance and Quality Control for Viral
Therapeutics ............................................................. 169
Analytical Assays to Characterise Adenoviral
Vectors and Their Applications ............................... 201
Validation of Gene Therapy Manufacturing
Processes: A Case Study for Adenovirus
Vectors ....................................................................... 227
Gene Delivery ................................................................... 245
Regulatory Issues in Gene Therapy. Good Science –
Good Sense ............................................................... 273
Regulatory Aspects in Gene Therapy: Special
Highlights on European Regulation ........................ 289
Regulatory Issues for Process Development and
Manufacture of Plasmids under Contract ............... 311
Risk Assessment in Gene Therapy ................................ 331
Index ................................................................................. 339
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Somatic Gene Therapy, Paradigm Shift or
Pandora's Box
A perspective on gene therapy
MARK LAWLER
Department ofHaematology, St Patrick Dun Research Labs, St James's Hospital and Trinity
College Dublin, James's Street, Dublin 8, Ireland
l. DEFINITIONS
Gene therapy may be defined as the introduction of genetic material into
the cells of a patient in an effort to help cure the disease either by producing
a gene product which is missing or in reduced amounts in the patient due to a
genetic mutation in the individual (eg Factor VIII protein for haemophilia) or
by introduction of new genetic material which either directly or indirectly
will help to combat the disease (eg genetic vaccination). Therapeutic genes
are delivered using a carrier (called a vector) which may be a non functional
viral vector or by using non viral vector approaches such as liposomes or
other carrier molecules. All gene therapy protocols involve the introduction
of genetic material into cells that have a finite life span such as blood cells,
liver cells etc (termed somatic tissue), thus the introduced gene is not passed
on to the next generation. This type of gene therapy is known as somatic
gene therapy and is in contrast to the concept of germ line gene therapy
(which would involve a gene being introduced into sperm or ova so that the
gene could be inherited by the children of the patient). Germ line gene
therapy is subject to an international moratorium.
Somatic gene therapy
Introduction of a gene into a specific tissue or tissues to provide
therapeutic benefit to the patient.
Germ line gene therapy
Introduction of genetic material into the egg cells or sperm cells of an
individual such that the gene will also be passed on to the next generation.
Ex vivo gene therapy
Collection of the patient's cells, introduction of therapeutic genetic
material into these cells and reintroduction of these cells into the patient.
In vivo gene therapy
Direct injection of therapeutic gene to the relevant tissue via a vector.
The promise of gene therapy lies in its proposed ability to treat the causes
of disease rather than the symptoms. The first decade of gene therapy has
been somewhat of a "roller coaster" ride, with early excitement of the
potential of this approach being tempered somewhat by disappointing
clinical results. Recently, however, improvements in vector construction and
vector delivery to the appropriate tissue has led to better pre clinical and
clinical results.
2. INTRODUCTION
Somatic gene therapy involves the amelioration of a disease by
introduction of genetic material with therapeutic potential into a somatic
tissue. It is only in the last few years that gene transfer could be
contemplated in the clinic. Advances have been made which allow the
transfer and stable existence of genetic material in a foreign host. Gene
therapy has benefited from (A) the gene transfer and expression techniques
of molecular genetics; (B) the natural ability of retro viruses to infect foreign
replicating cells and stably integrate their genetic material into the host
genome and (C) the fact that Bone Marrow Transplantation (BMT) provides
a straightforward delivery of in vitro manipulated material into the blood
stream. Thus a retrovirus can be engineered to contain an appropriate
segment of DNA and ex vivo transduction of haemopoietic cells allows
subsequent introduction of foreign material into the host by the BMT route.
There are a variety of gene delivery systems currently available and some
systems may be more useful in targeting particular tissues. Retro viruses were
the initial vehicles of choice but many studies have made use of
adenoviruses or herpes viruses and new developments such as the use of
adeno associated virus and non viral delivery systems have great potential.
These systems together with the early work in animal studies have been
crucial in bringing gene therapy protocols to the clinic. While much of the
earlier preclinical gene transfer work has concentrated on murine models, it
has become clear that use of large animal models including sheep, dogs, pigs
and monkeys may be more relevant as the final preclinical step before
proceeding to human trials.
While a large number of single gene defects are candidates for a gene
therapy protocol and several, including Adenosine Deaminanse (ADA)1 and
Cystic Fibrosis (CF)2'3 have been treated with such protocols it is the
realisation that gene therapy has potential in acquired disease which is
perhaps the most interesting. The ability to tackle malignant disease either
directly through the introduction of genes such as Tumour Necrosis Factor
(TNF) or indirectly by introducing immune stimulatory genes may have
enormous clinical applications. Gene therapy for malignant diseases will be
discussed in Chapter 5. The use of immunostimulatory molecules may also
yield dividends in the fight against AIDS. Protocols for cardiovascular
disease using either vector or antisense based approaches have shown
promise4'5 while gene therapy protocols for neurodegenerative disorders are
also being developed and applied6'7.
2.1 Vectors and target cells
Retroviruses have been the most extensively used vectors to date
particularly due to the fact that the therapeutic gene is integrated into the
host genome. Haemopoietic cells are probably the easiest to target due to the
well characterised hierarchical structure of the haemopoietic system. Thus
many of the clinical protocols have involved the introduction of a new gene
into haemopoetic cells. A second important advantage is that ex vivo gene
transfer can be performed - haemopoietic cells can be taken from the
individual, infected and then re-introduced to the individual by BMT. Ex
vivo gene transfer is very efficient as cells can be externally stimulated to
allow higher infection rates with retroviral constructs. The other important
advantage of the haemopoietic system is the ability to target stem cells as
well as lineage specific cells.
Other targets for retroviral vectors include skin fibroblasts where
retroviral infection rates of 50% have been reported and several clinically
important genes including ADA, purine nucleoside phosphorylase (PNP) and
Factor IX have been successfully transferred. The skin may be a highly
important target for delivery of therapeutic genes - collagen beads with
genetically modified fibroblasts secreting Factor IX have been produced and
using skin keratinocytes, therapeutic genes could be delivered directly to the
circulation.
The major disadvantage of retroviral vectors is their inability to infect
non dividing cells. The other major disadvantages relate to the safety aspects
of retroviruses. It is necessary to do stringent testing of cell lines for
potential replication competent retroviruses. Clearly insertional mutagenesis
and the worry that retroviral activation of cellular oncogenes has occurred in
murine systems must also be considered. Finally the fact that the maximum
size of DNA which can be efficiently packaged and transduced is
approximately 7 kb limits their potential for certain diseases. Specialised
packaging cell lines capable of producing high titres of replication deficient
recombinant virus free of any wild type viral contaminants have been
produced. However, outbreaks have been reported presumably due to
contaminating replication competent viruses. The prevention of such
outbreaks must of course be avoided. This has been aided by the
development of better modifications to packaging lines and by more
stringent monitoring of the products of such lines.
2.2 Adenoviruses
Adenoviruses are potentially more useful in in vivo gene therapy. They
can infect non dividing cells, larger sizes of exogenous DNA (> 30 kbs) can
be incorporated and high titres of the virus can be produced which is of great
importance for in vivo applications. The virus particle itself is relatively
stable. One worry is the presence of similar sequences in the human genome
which could combine with the inserted sequences leading to development of
malignancy. Replication deficient adenovirus can be generated and
propagated by growth in cells engineered to express the El replication
region, thus allowing the development of adenovirus vectors expressing
large amounts of a foreign gene product in vitro. Pre clinical studies have
indicated that efficient transduction in vivo occurs and gene expression can
be seen for significant periods post transduction. Adenoviruses have been
used to deliver the Cystic Fibrosis gene to airway epithelium and the ability
of adenoviral vectors to target brain, liver and muscle cells have indicated
that adenoviruses may become major vectors in clinical protocols. However
adenovirus vectors tend to be recognised by the host's immune system and
so need to be modified greatly to avoid immunisation and clearance of the
therapeutic vector.
2.3 Other viral vectors
The most important of these include adeno-associated virus (where their
ability to integrate at a particular locus on chromosome 19 might allow
controlled precise expression of any inserted gene), and herpes simplex virus
which could be highly important in neurodegenerative disorders8. Adeno
associated viruses are very simple viruses to produce and their broad host
range in conjunction with adenovirus make them useful vector systems. The
scope of such systems is enormous and will mean that even vector systems
that we might consider enemies (such as the Human Immunodeficiency
virus) may prove to be friends at the gene therapy level due to their target
cell specificity9.
2.4 Non viral delivery systems
The majority of the work currently reported in the literature has focused
on the use of viral vectors to deliver the desired gene product to its target cell
or tissue; however there is a growing body of evidence that non viral
methods may be useful in the potential treatment of several single or
polygenic disorders. While a variety of methods including the direct
injection of naked DNA into muscle cells, arterial walls or the heart itself
have been shown to be feasible, an approach which would have general
implications for a variety of diseases has involved the delivery of DNA
protein complexes to a specific cell via a receptor molecule intermediate.
The main advantages associated with non viral methods are (A) their ability
to deliver large transgenes (up to 50 kb) to their target; (B) their ability to
target different receptors; (C) a safer approach since viral integration is
avoided. This receptor mediated delivery system has been used to deliver the
low density lipoprotein receptor (LDLr) to the circulation of Watanebe rats
and the lowering of subsequent total serum cholesterol by this treatment
provide a good animal model for a gene therapy protocol for
hypercholesteremia10. Recent studies are also indicating that it may be much
easier than we at first believed to get DNA into the cell through the use of
anitisense approaches or "naked DNA " injection11.
2.5 Tissue specific gene delivery
While the majority of pre-clinical work has focused on delivery of
therapeutic genes to the haemopoietic system, there have been many
attempts to target other tissues also. In the liver, the primary candidates
would be the hepatocyte but while transducing hepatocytes is relatively
straightforward, the transplantation of such hepatocytes has proven
problematic. This may be overcome by using methods for ectopically
grafting hepatocytes or by in vivo transduction by retrovirus. Delivery of
gene products to the circulation will also be important for a wide variety of
diseases and so a method for efficient delivery of transgenes is necessary.
Retro viral transduction of keratinocytes has been achieved but expression of
exogenous genes was short lived. Both factor IX and growth hormone can be
expressed in myoblast cell lines using vectors which contained non viral
