Gene Therapy of Cancer. Translational Approaches from Preclinical Studies to Clinical Implementation

513 Pages·2002·22.066 MB·English

by  Edmund C. Lattime, PhD, and Stanton L. Gerson, MD (Eds.)

Contributors
Numbers in parentheses indicate the pages on which the authors' Joseph R. Bertino (365) Department of Medicine, Division
contribution begin. of Hematologic Oncology and Lymphoma, and Programs
of Molecular Pharmacology and Therapeutics, Memorial-
Rafat Abonour (355) Department of Medicine, Indiana
Sloan Kettering Cancer Center, New York, New York
University School of Medicine, Indianapolis, Indiana
12001
46202
Scott I. Abrams (145) Laboratory of Tumor Immunol- Patrick Blanco (167) Baylor Institute for Immunology
ogy and Biology, Center for Cancer Research, National Research, Dallas, Texas 75204
Cancer Institute, National Institutes of Health, Bethesda,
Maryland 20892 Tulin Budak-Alpdogan (365) Department of Medicine,
Programs of Molecular Pharmacology and Therapeutics,
Laura K. Aguilar (513) Harvard Gene Therapy Initiative, Memorial-Sloan Kettering Cancer Center, New York, New
Harvard Medical School, Boston, Massachusetts 02115 York 12001
Estuardo Aguilar-Cordova (513) Department of Radiology,
E. Brian Butler (513) Department of Radiology, Baylor
Baylor College of Medicine, Houston, Texas 77030 and
College of Medicine, Houston, Texas 77030
Harvard Gene Therapy Initiative, Harvard Medical School,
Boston, Massachusetts 02115
Lisa It. Butterfield (179) Division of Surgical Oncology,
Steven M. Albelda (493) Thoracic Oncology Research Lab- UCLA Medical Center, University of California, Los
oratory, Pulmonary, Allergy, and Critical Care Division, Angeles, California 90095
University of Pennsylvania Medical Center, Philadelphia,
Alfred E. Chang (241) Department of Surgery, Division
Pennsylvania 19104
of Surgical Oncology, Comprehensive Cancer Center,
Mark R. Albertini (225) The University of Wisconsin, Com- University of Michigan, Ann Arbor, Michigan 48109
prehensive Cancer Center, Madison, Wisconsin 53792
Saswati Chatterjee (53) Division of Virology, City of Hope
Gustavo Ayala (513) Department of Pathology, Baylor
National Medical Center, Duarte, California 91010
College of Medicine, Houston, Texas 77030
Jacques Banchereau (167) Baylor Institute for Immunology K. .V Chin (393) Departments of Medicine and Pharmacol-
Research, Dallas, Texas 75204 ogy, The Cancer Institute of New Jersey, UMDNJ-Robert
Wood Johnson Medical School, Piscataway, New Jersey
Christopher Baum )3( Medizinische Hochschule, Abt.
10980
Haematologie, 30625 Hannover, Germany
Virginia K. Clements (127) Department of Biological
Christian M. Becket (421) Department of Surgery,
Sciences, University of Maryland, Baltimore, Maryland
Children's Hospital, Harvard Medical School, Boston,
21250
Massachusetts 02115
Carmela Beger (95) Department of Medicine, University of Mark J. Cooper (31) Copernicus Therapeutics, Inc.,
San Diego, La Jolla, California 92093 Cleveland, Ohio 44106
VX
xvi srotubirtnoC
Kenneth Cornetta (355) Department of Medicine, Indiana William N. Hait (393) Departments of Medicine and Phar-
University School of Medicine, Indianapolis, Indiana macology, The Cancer Institute of New Jersey, UMDNJ-
46202 Robert Wood Johnson Medical School, Piscataway, New
Jersey 08901
James M. Croop (355) Department of Pediatrics, Indiana
University School of Medicine, Indianapolis, Indiana Mien-Chie Hung (465) Departments of Molecular and Cel-
46202 lular Oncology and Surgical Oncology, M. D. Anderson
Cancer Center, The University of Texas, Houston, Texas
Samudra Dissanayake (127) Department of Biological 77030
Sciences, University of Maryland, Baltimore, Maryland
21250 Kevin D. Judy (505) HUP-Department of Neurosurgery, The
University of Pennsylvania Medical Center, Philadelphia,
Stephen L. Eck (505) HUP-Department of Neurosurgery, Pennsylvania 19004
The University of Pennsylvania Medical Center, Philadel-
Dov Kadmon (513) Department of Urology, Baylor College
phia, Pennsylvania 19004
of Medicine, Houston, Texas 77030
James S. Economou (179) Division of Surgical Oncol-
Thomas Kearney (393) Department of Surgery, The Cancer
ogy, and Department of Immunology, Microbiology, and
Institute of New Jersey, UMDNJ-Robert Wood Johnson
Molecular Genetics, UCLA Medical Center, University of
Medical School, Piscataway, New Jersey 08901
California, Los Angeles, California 90095
Edsel .U Kim (257) Department of Otolaryngology, Univer-
Laurence C. Eisenlohr (207) Department of Microbi-
sity of Michigan, Ann Arbor, Michigan 48109
ology and Immunology, Thomas Jefferson University,
Philadelphia, Pennsylvania 19107 David M. King (225) The University of Wisconsin, Com-
prehensive Cancer Center, Madison, Wisconsin 53792
Wafik S. EI-Deiry (273, 279, 299) Laboratory of Molecu-
lar Oncology and Cell Cycle Regulation, Howard Hughes David Kirn (449) Imperial Cancer Research Fund, Program
Medical Institute, Departments of Medicine and Genetics, for Viral and Genetic Therapy of Cancer, Imperial College
Cancer Center and The Institute for Human Gene Therapy, School of Medicine, Hammersmith Hospital, London W 11
University of Pennsylvania School of Medicine, Philadel- OHS, United Kingdom
phia, Pennsylvania 19104
Omer N. Koq (341) Division of Hematology/Oncology, De-
partment of Medicine and Ireland Cancer Center at Case
Filip A. Farnebo (421) Departments of Surgery and Genet-
Western Reserve University and University Hospitals of
ics, Children's Hospital, Harvard Medical School, Boston,
Cleveland, Cleveland, Ohio 44106
Massachusetts 02115
Martin Kriiger (95) Department of Medicine, University of
Andrew L. Feldman (405) Surgery Branch, National Cancer
San Diego, La Jolla, California 92093
Institute, Bethesda, Maryland 20892
Calvin J. Kuo (421) Departments of Surgery and Genet-
Judah Folkman (421) Department of Surgery, Children's
ics, Children's Hospital, Harvard Medical School, Boston,
Hospital, Harvard Medical School, Boston, Massachusetts
Massachusetts 02115
02115
C. Lampert (81) Department of Hematology and Med-
Stanton L. Gerson (341 ) Division of Hematology/Oncology,
ical Oncology, .tS Peter's University Hospital, New
Department of Medicine and Ireland Cancer Center at Case
Brunswick, New Jersey 08901
Western Reserve University and University Hospitals of
Cleveland, Cleveland, Ohio 44106 Edmund C. Lattime (207,393) Departments of Surgery and
Molecular Genetics & Microbiology, The Cancer Institute
Mileka Gilbert (127) Department of Biological Sciences,
of New Jersey, UMDNJ-Robert Wood Johnson Medical
University of Maryland, Baltimore, Maryland 21250
School, Piscataway, New Jersey 08901
Leonard G. Gomella (207) Department of Urology, Thomas Irina ¥. Lebedeva (315) Department of Medicine and Phar-
Jefferson University, Philadelphia, Pennsylvania 19107 macology, Columbia University, College of Physicians
and Surgeons, New York, New York 10032
David H. Gorski (435) The Cancer Institute of New Jersey,
UMDNJ-Robert Wood Johnson Medical School, New Steven K. Libutti (405) Surgery Branch, National Cancer
Brunswick, New Jersey 08901 Institute, Bethesda, Maryland 20892
srotubirtnoC xvii
H. Kim Lyerly (199) Department of Surgery, Duke Univer- Wolfram Ostertag (3) Heinrich-Pette-Institut ftir Experi-
sity Medical Center, Durham, North Carolina 27710 mentelle Virologie und Immunologie an der ti~tisrevinU
Hamburg, 20251 Hamburg, Germany
Michael J. Mastrangelo (207) Division of Medical Oncol-
ogy, Department of Medicine, Thomas Jefferson Univer- Suzanne Ostrand-Rosenberg (127) Department of Bio-
sity, Philadelphia, Pennsylvania 19107 logical Sciences, University of Maryland, Baltimore,
Maryland 21250
Helena J. Mauceri (435) Department of Radiation and Cel-
lular Oncology, University of Chicago Hospitals, Chicago, A. Karolina Palucka (167) Baylor Institute for Immunology
Illinois 60637 Research, Dallas, Texas 75204
A. M. McCall (81) Fox Chase Cancer Center, Philadelphia, Beth A. Pulaski (127) Department of Biological Sciences,
Pennsylvania 91010 University of Maryland, Baltimore, Maryland 21250
Kevin .T McDonagh (241) Department of Internal Medicine, Ling Qi (127) Department of Biological Sciences, University
Division of Hematology/Oncology, Comprehensive Can- of Maryland, Baltimore, Maryland 21250
cer Center, University of Michigan, Ann Arbor, Michigan
48109 Alexander L. Rakhmilevich (225) The University of
Wisconsin, Comprehensive Cancer Center, Madison,
R. Scott Mclvor (383) Gene Therapy Program, Institute of Wisconsin 53792
Human Genetics, Department of Genetics, Cell Biology
and Development, University of Minnesota, Minneapolis, Jane S. Reese (341) Division of Hematology/Oncology, De-
Minnesota 55455 partment of Medicine and Ireland Cancer Center at Case
Western Reserve University and University Hospitals of
Raymond D. Meng (273,279, 299) Laboratory of Molecu- Cleveland, Cleveland, Ohio 44106
lar Oncology and Cell Cycle Regulation, Howard Hughes
Medical Institute, Departments of Medicine and Genetics, Michael Reiss (393) Department of Medicine, The Cancer
Cancer Center and The Institute for Human Gene Therapy, Institute of New Jersey, UMDNJ-Robert Wood Johnson
University of Pennsylvania School of Medicine, Philadel- Medical School, Piscataway, New Jersey 08901
phia, Pennsylvania 19104
Antoni Ribas (179) Division of Surgical Oncology, and Di-
Brian Miles (513) Department of Urology, Baylor College vision of Hematology/Oncology, UCLA Medical Center,
of Medicine, Houston, Texas 77030 University of California, Los Angeles, California 90095
Frederick L. Moolten (481) Edith Nourse Rogers Memo- Isabelle Rivi~re (109) Laboratory of Gene Transfer and
rial Veterans Hospital, Bedford, Massachusetts 01730 Gene Expression, Department of Medicine and Immunol-
and Boston University School of Medicine, Boston, ogy Program, Memorial Sloan-Kettering Cancer Center,
Massachusetts 02118 New York, New York 10021
Michael A. Morse (199) Department of Medicine, Division Justin C. Roth (341) Division of Hematology/Oncology,
of Medical Oncology and Transplantation, Duke Univer- Department of Medicine and Ireland Cancer Center at Case
sity Medical Center, Durham, North Carolina 27710 Western Reserve University and University Hospitals of
Cleveland, Cleveland, Ohio 44106
Paula J. Mroz (481) Edith Nourse Rogers Memorial
Veterans Hospital, Bedford, Massachusetts 01730 Michel Sadelain (109) Laboratory of Gene Transfer and
Gene Expression, Department of Medicine and Immunol-
James J. Muir (257) Department of Surgery, University of ogy Program, Memorial Sloan-Kettering Cancer Center,
Michigan, Ann Arbor, Michigan 48109 New York, New York 10021
Smita K. Nair (199) Department of Surgery, Duke University Ruping Shao (465) Department of Molecular and Cellular
Medical Center, Durham, North Carolina 27710 Oncology, M. D. Anderson Cancer Center, The University
of Texas, Houston, Texas 77030
Owen A. O'Connor (365) Department of Medicine, Di-
vision of Hematologic Oncology and Lymphoma, and C. A. Stein (315) Department of Medicine and Pharma-
Developmental Chemotherapy Services, Memorial-Sloan cology, Columbia University, College of Physicians and
Kettering Cancer Center, New York, New York 10021 Surgeons, New York, New York 10032
xviii Contributors
Daniel H. Sterman (493) Thoracic Oncology Research Lab- L. M. Weiner (81) Fox Chase Cancer Center, Philadelphia,
oratory, Pulmonary, Allergy, and Critical Care Division, Pennsylvania 91010
University of Pennsylvania Medical Center, Philadelphia,
Pennsylvania 19104 Thomas Wheeler (513) Department of Pathology, Baylor
College of Medicine, Houston, Texas 77030
Carol Stocking )3( Heinrich-Pette-Institut fur Experi-
mentelle Virologie und Immunologie an der ti~tisrevinU Lee G. Wilke (257) Department of Surgery, University of
Hamburg, 20251 Hamburg, Germany Michigan, Ann Arbor, Michigan 48109
Bin S. Teh (513) Department of Radiology, Baylor College
K. K. Wong, Jr. (53) Division of Hematology and Bone
of Medicine, Houston, Texas 77030
Marrow Transplantation, and Division of Virology, City
Timothy C. Thompson (513) Department of Urology, of Hope National Medical Center, Duarte, California
Baylor College of Medicine, Houston, Texas 77030 91010
Deborah Toppmeyer (393) Department of Medicine, The
Flossie Wong-Staal (95) Department of Medicine, Univer-
Cancer Institute of New Jersey, UMDNJ-Robert Wood
sity of San Diego, La Jolla, California 92093
Johnson Medical School, Piscataway, New Jersey 08901
Catherine M. Verfaillie (331) Stem Cell Institute, Can- Duen-Hwa Yan (465) Departments of Molecular and Cel-
lular Oncology and Surgical Oncology, .M D. Anderson
cer Center, and Division of Hematology, Oncology and
Cancer Center, The University of Texas, Houston, Texas
Transplantation, Department of Medicine, University of
Minnesota, Minneapolis, Minnesota 55455 77030
Maria T. Vlachaki (513) Department of Radiology and Steven P. Zielske (341) Division of Hematology/Oncology,
Veterans Affairs Medical Center, Baylor College of Medi- Department of Medicine and Ireland Cancer Center at Case
cine, Houston, Texas 77030 Western Reserve University and University Hospitals of
Cleveland, Cleveland, Ohio 44106
Dorothee yon Laer )3( Chemotherapeutisches Forschung-
sinstitut, Georg-Speyer-Haus, 60596 Frankfurt, Germany
Robert CH Zhao (331) Stem Cell Institute, Cancer Center,
Ralph R. Weichselbaum (435) Department of Radiation and Division of Hematology, Oncology and Transplanta-
and Cellular Oncology, University of Chicago Hospitals, tion, Department of Medicine, University of Minnesota,
Chicago, Illinois 60637 Minneapolis, Minnesota 55455
Preface
The second edition of Gene Therapy of Cancer comes at of clinical trials and appears to have accomplished its goal of
a pivotal transition point in the development of this exciting achieving stem cell protection in both preclinical and clinical
technology. Much has occurred in the past 4 years to cata- settings.
pult preclinical and basic scientific concepts into therapeutic Gene delivery remains an important aspect of gene therapy
trials. In addition, while the outcome of the initial phase of of cancer and a number of chapters focus on gene delivery
clinical trials using gene therapy to target cancers has not systems using both viral and nonviral approaches.
yielded the amazing results initially hoped for, as with ev- The reader will find this to be a comprehensive assess-
ery new therapeutic venture in medicine, initial results pro- ment of the current state of gene therapy of cancer offering
vide the fodder for critical experiments, new targets, and new state of the art research, a review of basic mechanisms and
questions that propel the field forward. approaches, and a compilation of current clinical trial efforts.
We have reorganized the presentations in the second edi- We have not encumbered this text with a number of the cur-
tion to reflect the continued new emerging strategies that will rent controversies in gene therapy but would note the impor-
ultimately lead to the success of this therapeutic approach and tance of these issues in the field. Conflict of interest remains
have added introductory chapters to a number of the sections an active area of discussion at every major cancer center in the
with the goal of setting the contributions in their proper basic country and has been the recent focus of the American Society
scientific context. of Gene Therapy. The careful monitoring of patients under-
Immune therapeutics takes on added emphasis given some going clinical trials in gene therapy will remain a priority for
of the recent breakthroughs in vaccine development and all clinical trialists in this field. Linking preclinical models to
targeted delivery. Oncolytic virus therapeutics have also clinical endpoints is an important aspect of this focus and will
emerged in a very promising light with initial positive re- enable intermediate assessments to define whether the gene
suits observed in head and neck cancer leading to a number therapy effect has been achieved prior to relying on clinical
of preclinical advances. Therapies directed towards onco- cancer response and will help drive both Phase I and Phase
genes, be it by expression of normal oncogenes, use of ri- II clinical trial design.
bozymes and antisense therapeutics, and the use of E1A The next 5 years will be explosive in the next generation
continue to be promising in preclinical and early clinical mod- of preclinical and clinical developments of gene therapy of
els. Hematopoietic stem cells are being used in gene therapy cancer. We hope this second edition will provide an important
both in the antisense setting, for instance use of BCR/ABL reference for investigators and observers alike in this exciting
antisense to block CML stem cell proliferation, and in genet- field.
ically modified stem cells as immunotherapies. In addition,
bone marrow protection by introduction of a drug-resistant Edmund .C Lattime, PhD
gene into hematopoietic stem cells is entering its next phase Stanton .L Gerson, MD
xix
C H A P T E R
1
Retroviral Vector Design for Cancer
Gene Therapy
CHRISTOPHER BAUM WOLFRAM OSTERTAG CAROL STOCKING
Medizinische Hochschule Heinrich-Pette-lnstitut fiir Heinrich-Pette-Institut fiir
Abt. Haematologie Experimentelle Virologic und lmmunologie Experimentelle Virologie und Immunologie
30625 Hannover, Germany an der Universitiit Hamburg, an der Universitiit Hamburg,
20251 Hamburg, Germany 20251 Hamburg, Germany
DOROTHEE VON LAER
Chemotherapeutisches Forschungsinstitut
Georg-Speyer-Haus
60596 Frankfurt, Germany
I. Introduction 3 ogy, including poor vector design [2]. Nevertheless, many
II. Applications for Retroviral Vectors former skeptics were turned to true believers, not only due
in Oncology 4 to the enormous public and economical interest [3]. Thus,
III. Biology of Retroviruses 6 strong international competition in the field was generated,
.A Classification 6 with an increasing number of researchers following valuable
.B Retroviral Genes and Their Products 6 long-term concepts, including improvement of basic vector
.C Retroviral cis Elements 6 technology.
.D Retroviral Life Cycle 7
An ideal vector should (1) allow efficient and selective
IV. Principles of Retroviral Vector Systems
transduction of the target cell of interest, (2) be maintained,
.A Packaging Cells 9
(3) be expressed at levels necessary for achieving therapeutic
.B Basic Vector Architecture 01
effects, and, last but not least, (4) be safe in terms of avoid-
V. Advances in Retroviral Vector Tailoring 11
ing unexpected side effects in the host. Viruses are a perfect
.A Components Active in trans 11
tool for gene transfer as they have evolved to deliver their
B. cis-Acfive Elements 61
genome efficiently to target cells with subsequent high-level
VI. Outlook 22
References 32 gene expression. Vector systems for therapeutic gene transfer
have been developed from different virus groups, each sys-
tem having specific advantages and drawbacks. Retroviruses
have several unique features that render them highly suitable
.1 INTRODUCTION for vector development. Retroviral vectors are, therefore, the
prevalent system for gene transfer in human cells. Retro-
In the past years, oncology was the center of gene therapy viruses integrate and express their genome in a stable manner,
research [ 1 ]. However, despite generous support by, for ex- thus allowing long-term manipulation with transferred genes.
ample, the National Institutes of Health and related institu- This is a prerequisite for many gene therapy applications,
tions in Europe, there is still a wide gap between the hopes including some approaches in cancer gene therapy. Integra-
raised and the results achieved. Most of the failures of gene tion usually does not alter host cell functions and is well toler-
therapy trials can be attributed to a discordant combination ated. In the retroviral genome, cis-acting elements, responsi-
of overinterpreted clinical concepts and immature technol- ble for reverse transcription, integration, and packaging, can
Copyright © 2002 by Academic Press
eneG yparehT of ,recnaC dnoceS noitidE All rights of reproduction in any form reserved.
4 Christopher Baum et al.
be well separated from coding sequences. Such a genome distinguish between diagnostic and therapeutic approaches;
structure facilitates the design of safe vectors and packaging in either case, both healthy tissues or tumor cells may be
cell lines. targeted. Each strategy has special implications for vector
However, with the transition to applications in human design.
gene therapy, severe limitations of conventional retroviral Gene marking uses stable retroviral transduction of het-
vector systems have become apparent. These include low erologous genetic sequences to analyze the biological (stem
and variable particle titers, lack of appropriate vector tar- cell function, antiviral effects) or pathogenic (tumor cell con-
geting to specific cell types and genomic loci, failure to tamination, graft-versus-host reaction) capacity of blood cell
transduce quiescent cells, and relatively inefficient, position- transplants 4-6. Here, efficient transduction of long-lived
dependent transcription. Fortunately, substantial progress in hematopoietic cells is required. Moreover, long-term trans-
vector development has been made, based on deeper un- gene expression is necessary for follow-up analyses involv-
derstanding of the biology of retroviruses and target cells. ing phenotyping and preparative sorting of transduced cells,
Here, we review some of the work relevant to cancer gene based on cell surface or cytoplasmic markers encoded by the
therapy. vector 7-9.
We start with a short overview of potential applications of Besides this entirely diagnostic approach, several thera-
retroviral vectors in oncology. Then, we describe aspects of peutic strategies target healthy tissues. These strategies are
retrovirus biology relevant to gene therapy, to create a basis also relevant to gene therapy of some inborn genetic disorders
for discussing principles of and specific recent advances in or acquired viral infections, due to the use of marker genes
retroviral vector design. that allow selection of transduced cells in vivo. Positive se-
lection is established in the context of drug resistance gene
transfer, negative selection in adoptive immunotherapy.
.II APPLICATIONS FOR RETROVIRAL Drug resistance gene transfer in nontumor tissues such as
VECTORS IN ONCOLOGY bone marrow is aimed at augmenting the therapeutic index
of anticancer chemotherapy 10,11 . Protection at the level
In oncology, several different strategies involving somatic of hematopoietic progenitor cells reduces short-term toxic-
gene transfer are currently considered (Table .)1 We can ity, and protection at the stem cell level might even prevent
TABLE 1 Somatic Gene Transfer in Oncology and Implications for Vector Design
Approach Aim tegraT cells rotceV requirements Vector system
Gene marking Diagnostic Healthy hematopoietic or Transduction of long-lived Retroviral vectors
lymphocytic cells, tumor stem cells, stable gene
cells (both ex vivo) expression
Drug resistance gene Therapeutic (paradigm for Healthy hematopoietic cells Transduction of repopulating Retroviral vectors
transfer positive selection of xe( )oviv cells, stable and high gene
transduced cell ni vivo) expression
Adoptive immunotherapy Therapeutic (paradigm for Donor lymphocytes (ex vivo) Transduction of lymphocytes, Retroviral vectors
negative selection of stable and high gene
transduced cell in vivo) expression
Mini-organs Therapeutic Healthy autologous or Stable or inducible gene Retroviral vectors
xenogenic cells (ex vivo) expression
Suicide gene transfer Therapeutic (but not Tumor cells (usually in vivo) Applicability in vivo, Retroviral vectors;
systemic) targeting to tumor cells; alternatively herpes virus
strong, but not necessarily vectors or adenoviral
stable gene expression vectors
Oncogene antagonism Therapeutic (but not Tumor cells (usually in vivo) Applicability in vivo, Retroviral vectors;
systemic) targeting to tumor cells; alternatively herpes virus
strong, but not necessarily vectors or adenoviral
stable gene expression vectors
Tumor vaccination Therapeutic Tumor cells, antigen- Applicability in vivo; Retroviral vectors;
presenting cells (ex vivo moderate, but not alternatively herpes virus
or in vivo) necessarily stable gene vectors, adenoviral
expression vectors, or
physicochemically
larivorteR Vector Design rof Cancer Gene Therapy 5
long-term toxicity and the mutagenicity of chemotherapy. tients, these strategies might offer interesting perspectives.
The benefit for the patient will depend on the numbers of pro- Usually, vectors have to be applied in vivo to become
tected cells obtainable. These are expected to increase with effective, and sometimes even replication-competent vectors
each cycle of chemotherapy, because cells acquire a selective will be needed. Here, nonretroviral systems may offer im-
advantage upon expression of the drug resistance gene. Thus, portant alternatives, given that the problem of instability of
this approach sets a paradigm for forced expansion of trans- persistence or expression is of minor importance.
duced cells in vivo. Similar to gene marking, this approach Transfer of prodrug-converting enzyme genes (suicide
requires a number of technological improvements: First, gene transfer) is performed to render tumor cells susceptible
helper functions of the vector systems and transduction con- to cytotoxic compounds requiring activation by a heterolo-
ditions have to mediate efficient uptake (see Section V.A) gous enzyme 23,24. Alternatively, toxin genes may be used
and nuclear translocation (see Section V.B) in primitive 25,26. Another approach to control tumor cells by gene
hematopoietic cells (reviewed in Baum et al. 12). Second, transfer is oncogene antagonism 27,28. Here, one attempts
the vector needs to be equipped with cis-regulatory elements to counteract tumor-promoting mutations of cellular genes.
mediating dominant gene expression levels and thus strong This is achieved by transducing tumor cells with wild-type
penetrance of the phenotype (see Section V.B) 13,14. Third, copies of tumor suppressor genes or dominant negative pro-
coexpression of a second gene (see Section V.B.7) is impor- teins, antisense nucleotides, or ribozymes directed against
tant in this approach, because coordinated transfer of two oncogenes and their products. In compact tumor masses,
complementary drug resistance genes greatly widens its flexi- some of these strategies may profit from the so-called by-
bility 15-18. Finally, malignant cells must be excluded from stander effect. This refers to cytostatic effects observed in
productive transduction by cell purging or vector targeting nontransduced cells that result from delivery of proteins or ac-
(see Sections V.A.I.b and V.B.1). tivated cytotoxic drugs through direct intercellular exchange.
A paradigm for negative selection of transduced cells in However, this exchange might also dilute the effects in trans-
vivo is established in adoptive immunotherapy. Here, ex vivo duced cells 29. With either approach, immune responses
selected populations of allogenic donor lymphocytes are used may be triggered that are expected to promote antitumor
to elicit an antiviral or antileukemic effect 19. Gene transfer efficiency. Transfer of suicide or toxin genes,in contrast to
in lymphocytes serves for both positive and negative selec- oncogene antagonism, should exclude healthy tissues. Be-
tion. After transduction, positive selection of gene-modified sides direct targeting of retroviral vectors to tumor cells, cel-
lymphocytes is performed ex vivo using cell surface mark- lular vehicles may be used to deliver tumor-antagonistic gene
ers. After reinfusion, concomitant expression of a negative products into tumor masses; these may be either cytotoxic
selection marker (a suicide gene) is instrumental for treating T cells 26 or normal progenitor cells with homing capacity
eventually occurring graft-versus-host disease. Here, the key 30, tumor cells themselves 13 , and possibly also endothe-
issue is to design vectors with reliable and persisting coex- lial cells or their precursors 32.
pression of two genes (see Sections V.B.2, V.B.4, and V.B.7). Application of retroviral vectors in vivo requires pro,
An extension of this approach is the transfer and expression duction of complement-resistant particles at high titers.
of "designer" T-cell receptors in autologous or allogeneic Selectivity can be achieved already at the level of transduc-
T cells, in order to generate effector cells with a new, prede- tion, taking advantage of preferential infection of dividing
fined target cell specificity 20. cells by vectors based on murine retroviruses 33. Specific
Positive or negative selection and monitoring of trans- targeting using engineered envelope proteins may, however,
duced cells in vivo is crucial for the development of artificial be superior (see Section V.A.I.b). At the level of transcrip-
mini-organs (derived from genetically manipulated cells tional regulation, selectivity can be accomplished by insertion
21). In oncology, these are of interest for systemic delivery of promoters preferentially activated in tumor cells or tumor
of tumor-antagonistic factors such as immunotoxins or vasculature (see Section V.B.3). Depending on the tumor
inhibitors of angiogenesis. Further applications extend to type, herpesviruses or adenoviruses (some of the latter specif-
genetic or acquired disorders that can be treated by delivery ically replicating in p53-negative cells) may represent alter-
with enzymes, hormones, or ligands. Equipping mini-organs native vectors 34. Key aspects of targeting using specific
with regulatable promoters might allow the adjustment of receptors or promoters also apply to these nonretroviral
supply according to individual clinical requirements (see systems.
Section V.B.5) 22. Finally, tumor vaccination is performed to evoke a sys-
Other therapeutic concepts rely on direct genetic ma- temic immune response to tumor-specific antigens 35,36.
nipulation of tumor cells. Some of these suffer from poor This is accomplished by transfer and expression of genes that
predictability, mostly due to the tremendous variability of tu- increase antigen presentation or improve effector cell func-
mor evolution among and within individual patients. More- tions. Target cells for transduction are tumor cells, antigen-
over, not all of these strategies acknowledge the systemic presenting cells, or tumor-infiltrating T cells. Thus, tumor
character of tumor diseases. Nevertheless, in selected pa- vaccination strategies are highly variable with respect to the
6 Christopher Baum et al.
target cell population and to the type and numbers of activat- receptor 38. Except for ecotropic viruses, gene transfer into
ing genes to be transferred. "Vectors may be applied either ex human cells is possible with all groups of viruses mentioned.
vivo or in vivo (after injection in tumor masses), and sustained For historical reasons, most retroviral vectors applied thus
gene expression in target cells is not necessarily required. For far in human gene therapy have utilized the amphotropic
this approach, alternative vector systems (e.g., adenoviral or receptor, although this is not the most efficient envelope for
herpes vectors or physicochemical methods such as biolis- many targets.
tics) may also be valuable.
In all these different strategies, important variables to ac-
B. Retrovirai Genes and Their Products
count for in vector design are the route of gene transfer, the
target cell population, the efficiency and specificity of trans- Retroviruses within a group share a very similar provi-
duction, and the level, duration, and specificity of transgene ral structure. In the first three groups (see Table 2), includ-
expression (Table 1). Therefore, appropriate components of ing mammalian C-type retroviruses, the genome codes only
the vector system have to be identified for each applica- for the virion structural proteins Gag, Pol, and Env (Fig. )1
tion ("tailored vectors"). Fortunately, substantial progress 37. The gag gene products constitute the viral matrix and
has been made toward all aspects of vector design rele- package the two retroviral RNA genomes into a viral nucle-
vant to cancer gene therapy. These vector improvements are ocapsid. Encoded by the pol gene, the virion also includes
based on detailed insights into the biology of retrovirus-host several enzymes necessary for virus replication. These are
interactions. the reverse transcriptase, the integrase, and the viral protease,
which cleaves the Gag and Pol precursors into the individ-
ual proteins. Receptor utilization is determined mainly by
!!!. BIOLOGY OF RETROVIRUSES the glycosylated env gene product SU, which is anchored
in the viral envelope by the transmembrane protein TM.
A. Classification The viruses of the HTLV-BLV group, the lentiviruses, and
the spumaviruses are more complex and also encode spe-
Sequence data and genome structure are the basis for the
cific nonvirion proteins with different regulatory functions
classification of retroviruses (Table 2) 37. Each group con-
39,40. Examples are the viral transcriptional activators of
tains several virus strains that differ in biological properties,
gene expression, such as tax in HTLV, tat in HIV, and bel-1
such as receptor utilization and pathogenicity. Until recently,
in foamy viruses.
most retroviral vector systems discussed for human gene ther-
apy were based on murine leukemia viruses (MLVs). MLVs
belong to the mammalian C-type retroviruses and are fur- C. Retroviral cis Elements
ther classified according to the species distribution of their
The cis-acting elements that regulate viral gene expres-
receptors. Ecotropic MLVs replicate only in rodent cells
sion, reverse transcription, and integration of the provirus
and xenotropic MLVs only in nonmurine cells, while poly-
into the cellular DNA are organized very similarly in all
tropic and amphotropic MLVs can infect murine and non-
retroviruses 37. The provirus is flanked by the long termi-
murine cells. The 10A1 strain has an overlapping but dis-
nal repeats (LTRs), carrying the terminal att sites, which are
tinct host range, due to the use of the receptor for the gibbon
recognized by the integration machinery. The LTR is further
ape leukemia virus (GALV) in addition to the amphotropic
divided into the three sections U3, R, and U5 (Fig. .)1 The
U3 region carries the viral enhancer and promoter elements.
In the 3' LTR, initiation of transcription is suppressed, possi-
TABLE 2 Retrovirus Genera
bly due to interference with the 5' LTR. The polyadenylation
Genus Example viruses signal resides in the R or U3 region. A recent report suggests
that the retroviral splice donor may play an important role in
naivA sisokuel amocras suoR amocras suriv )VSR(
suppressing the utilization of the polyadenylation signal of
nailammaM C epyt eniruM aimekuel suriv ,)VLM( lareves the 5' LTR 41. Transcription of viral genomic RNA thus
:sniarts hcus sa ,-yenoloM ,-yevraH
begins at the R region in the 5' LTR and ends with R in the
,-noslebA .VLM-A704
3 t LTR. The RNA genome is thus flanked by identical redun-
enileF aimekuel suriv )VLeF(
dant regions (R), which play an important role during reverse
nobbiG epa aimekuel suriv )VLAG(
transcription (see Section II.D). The U5 region contains se-
epyt-D sesuriv rezifP-nosaM yeknom suriv )VMPM(
quences necessary for reverse transcription and terminates
epyt-B sesuriv esuoM yrammam romut suriv )VTMM( with the att site.
VLB-VLTH puorg namuH T cell aimekuel suriv 1-)VLTH( dna 2 The untranslated leader comprises R and U5 regions of
surivitneL namuH ycneicifedonummi suriv -)VIH( 1 dna 2- the 5 p LTR and sequences upstream of gag, including 81 nu-
surivamupS namuH ymaof suriv )VFH( cleotides that form the primer binding site (PBS). The PBS
is perfectly complementary to the 3 ~ terminus of the tRNA
7
Retroviral Vector Design for Cancer Gene Therapy
Replication-competent simple retrovirus • combination of cis and trans
trans: gag pol env
5'LTR MA p12 CA NC PR RT IN SU TM 3'LTR
-i-u -u;l-
m II I • ~ " II BI! /
cis: att E P I dD SA ' PP -- pA ' att '
PBS
Components active in trans
gag ~ MA (matrix, p15), p12, CA(capsid, p30), NC (nucleocapsid, pl0)
pol ~ protease (PR), reverse transcriptase/RNaseH (RT), integrase (IN)
env ~ SU (surface protein, gp70), TM(transmembrane protein, p15SE )
Components active in cis
att ~ integration signal
---~ dimerization and packaging signal
E P ~ enhancer / promoter
PP ~ polypurine tract
PBS ~ tRNA-primer binding site
pA ---~ polyadenylation signal
SD, SA ~ splice donor, splice acceptor
ERUGIF 1 Scheme of eht larivorp form of a tnetepmoc-noitacilper elpmis -C
epyt .surivorter secneuqeS gnidoc rof trans-acting snietorp era detacidni evoba eht
,gniward cis-acting secneuqes .woleb
primer that initiates reverse transcription of the RNA genome transcriptase (RT) [37]. The nucleocapsid protein NC is also
into the minus strand of proviral DNA. Leader sequences required for this process. Reverse transcription is initiated at
downstream of the PBS contain the splice donor site for the PBS. RT synthesizes the negative strand complementary
generating the subgenomic RNAs, as well as the packag- to the U5 and R regions of the 5' LTR, while the RNAse
ing and dimerization signal, which directs incorporation of H activity of RT degrades the genomic RNA. The nascent
two viral RNA genomes into virions. Optimal packaging DNA strand is transferred to the 3' end of the RNA genome,
was originally reported to require additional sequences, such where, starting with the U5 region, the negative strand is
as the first 400 nucleotides of gag in MLV-based vectors completed with concomitant degrading of the RNA genome.
(so-called gag + vectors) [42], although more recent data sug- The PP tract escapes digestion and serves as a primer for
gest that viral coding sequences are dispensable for high-
titer packaging of MLV vectors [43,44]. In human immuno-
deficiency virus (HIV), the minimal sequences sufficient for
I
packaging have not yet been defined [45,46]; in addition to
part of the leader, a region in the 5' end of the gag gene
and sequences within env encompassing the Rev responsive
element (RRE) appear to improve packaging [47].
The untranslated region between env and the 3' LTR con-
tains the polypurine (PP) tract, a run of at least nine A and
G residues. Synthesis of the plus strand of proviral DNA is A
initiated here.
D. Retroviral Life Cycle
The retroviral life cycle is illustrated in Fig. 2. Initially,
retroviruses bind through the Env protein SU to a specific vi-
ral receptor on the cell surface. All known retroviral receptors
are membrane proteins, and several have been cloned [48].
The receptors for amphotropic MLV (Pit-2) and for GALV
(Pit- )1 are phosphate transporters found on most human cells.
Interaction of viral SU with the receptor exposes a fusion FIGURE 2 Life elcyc of a tnetepmoc-noitacilper :surivorter )1( noiriv
peptide in the TM and triggers fusion of the viral and cellu- ,gnidnib )2( noiriv noitartenep dna ,gnitaocnu )3( esrever noitpircsnart fo
ANR emoneg into larivorp ,AND )4( raelcun tropsnart of noitargetnierp
lar membranes with subsequent release of the nucleocapsid
xelpmoc dna noitargetni of ,surivorp )5( noitpircsnart fo cimoneg dna -egbus
into the cytoplasm [49]. Here, the viral RNA genome is re-
cimon ANRm dna noitalsnart of lariv eneg ,stcudorp dna )6( dispacoelcun
verse transcribed into the proviral DNA by the viral reverse ,ylbmessa ,gniddub dna noitarutam fo .noiriv