Molecular Genetic Medicine. Volume 3

188 Pages·1993·4.78 MB·English

by  Theodore Friedmann (Eds.)

Volume 3
Molecular
Genetic
Medicine
Edited by
Theodore Friedmann
Department of Pediatrics
Center for Molecular Genetics
School of Medicine
University of California, San Diego
La Jolla, California
Academic Press, Inc.
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Contributors
Francis S. Collins, Department of Internal Medicine, and the Howard Hughes
Medical Institute, University of Michigan, Ann Arbor, Michigan
48109
Mitchell L. Drumm, Department of Human Genetics, and the Howard Hughes
Medical Institute, University of Michigan, Ann Arbor, Michigan
48109
Theodore Friedmann, Department of Pediatrics, Center for Molecular Genetics,
School of Medicine, University of California, San Diego, La JoUa,
California 92093
James F. Gusella, Department of Genetics, Harvard Medical School, Cam­
bridge, Massachusetts 02138; and Molecular Neurogenetics Laboratory,
Massachusetts General Hospital, Charlestown, Massachusetts 02129
Gary Landreth, Alzheimer Center and Department of Neurology, University
Hospitals of Cleveland, Case Western Reserve University, Cleveland,
Ohio 44106
Marcy E. MacDonald, Department of Neurology, Harvard Medical School, Cam­
bridge, Massachusetts 02138; and Molecular Neurogenetics Laboratory,
Massachusetts General Hospital, Charlestown, Massachusetts 02129
Tom Mikkelsen, MidWest Neuro-Oncology Center, Henry Ford Hospital, De­
troit, Michigan 48202
Mark A. Rothstein, Health Law and Policy Institute, University of Houston,
Houston, Texas 77204
Peter J. Whitehouse, Alzheimer Center and Department of Neurology, Univer­
sity Hospitals of Cleveland, Case Western Reserve University, Cleve­
land, Ohio 44106
Steven Younkin, Department of Pathology, University Hospitals of Cleveland,
Case Western Reserve University, Cleveland, Ohio 44106
IX
Preface
Our ability to apply the tools of molecular genetics to an understanding
of human disease is making rapid and, at times, breathtaking progress. To the
suφrise of even its most enthusiastic proponents, the human genome project
(reviewed in Volume 1 of this series) is roaring ahead of schedule. The entire
contents of two human chromosomes, the Y chromosome and chromosome 22,
have been connected by overlapping yeast artificial chromosomes (YACs) to
produce contiguous physical maps. It is likely that the contents of most or all of
the human chromosomes will be connected into contiguous overlapping YAC
clones long before the original projected date of 2005.
Treatments of most human diseases are only partially effective. In the
case of human genetic disease, this may be because all therapies, until very
recently, were limited to attempts to manipulate the metabolic or biochemical
consequences of the basic underlying genetic defect rather than seeking a defini-
tive correction of the responsible defect. Certainly, until several decades ago,
the application of molecular genetics to such a genetic correction of human
disease has also been considered improbable and out of our grasp, both concep­
tually and technically. It has only been approximately 20 years since the con­
cepts of human gene therapy were first delineated in "modern" molecular terms.
The first truly efficient gene transfer vectors for mammalian cells have only been
available for 10 years. And yet, today, there are more than 35 human gene
therapy studies that have been approved by federal regulatory agencies, includ­
ing the Recombinant DNA Advisory Committee of the National Institutes of
Health and the Food and Drug Administration.
In the first chapter of this volume of Molecular Genetic Medicine, I
review the birth and early development of the field of human gene therapy and
the earliest conceptual and technical descriptions of the issues and opportunities
in this new area of medicine. This chapter does not include a discussion of many
of the technical developments in vectorology and disease models, because the
goal is more historical and because a number of recent reviews deal very effec­
tively with up-to-date technical developments. Furthermore, because the pres­
ent human clinical studies are at very preliminary stages and because clinical
results have not yet been reported, I have not included a description of these
initial human studies. They will be summarized in a forthcoming volume, by
which time some definitive results should be available.
The genetic components of a growing number of human disorders are
being discovered. One of the most remarkable successes has been the character-
XI
xii Preface
ization of the gene responsible for one of our society's most important genetic
diseases—cystic fibrosis (CF). The illumination of the underlying defect in this
disease represents the first pure application of what used to be called "reverse
genetics" and is now known as positional cloning. This phrase describes the use
of molecular and cytogenetic techniques to isolate a disease-related marker based
entirely on knowledge of its chromosomal localization. Earlier work with several
other diseases, such as Duchenne muscular dystrophy and chronic granu­
lomatous disease, has certainly used "reverse genetic" concepts, but in those
previous cases molecular probes became available with the discovery of patients
with chromosomal aberrations. In this volume, M. L. Drumm and P. Collins
review the startling speed with which genetic mapping information of CF was
combined with improved physical mapping techniques to isolate the responsible
gene and learn about its role in the pathogenesis of this important disease.
Cancer is a disease, or more properly, a collection of diseases, charac­
terized by the accumulation of multiple genetic defects, all of which interact
with each other and cooperate to bring about the unregulated growth and
replication of cells. The activation of protooncogenes, the shut down of tumor-
suppressor genes, and epigenetic phenomena such as imprinting affect the ex­
pression of key cellular genes. The final outcome of these events is an alteration
in the ability of cells to heed their customary growth regulatory signals. Exactly
how a cell progresses from a state of normal growth to one of altered regulation
and even metastatic spread is certainly not well understood, but important
progress has been made recently on the genetic and epigenetic mechanisms
responsible for tumor progression. Astrocytes represent one important and useful
model system for studying this phenomenon; T. Mikkelsen describes some of the
complicated interactions and genetic phenomena that accompany the progres­
sion of astrocytic tumors.
Alzheimer's disease has only recently been recognized as an enormous
medical and social problem and has now become one of the most intensely
studied of human diseases. In many of its forms it appears sporadically and does
not seem to have overriding genetic components. In some families, there is an
important genetic component, and candidate loci have now been localized by
linkage analysis on several human chromosomes. P. J. Whitehouse and his
colleagues summarize recent progress in the study of this exceedingly important
disease and indicate that major new insights are imminent.
One of the disease models that raised the greatest excitement a decade
ago and seemed to promise an immediate payoff to a molecular attack on an
otherwise insoluble problem is Huntington's disease (HD). Ten years ago J. F.
Gusella and his colleagues reported linkage of an anonymous piece of DNA to
Huntington's disease. Many thought that the gene would soon be identified by
"reverse genetics" and that we would have, for the first time, a tool to understand
this puzzling and terrible disease, but the HD gene remained a will-o-the-wisp.
Preface xiii
eluding the best efforts at isolation and characterization. It was known that the
gene was at or near the tip of the short arm of chromosome 4, but, illustrating
the severest of the potential problems with positional cloning, it remained
unidentified. Many human geneticists became inured to the difficulties of the
region and came to expect, more or less facetiously, that there simply was no
gene there. All that has now changed, almost as if overnight. The gene responsi­
ble for this disorder is yet another representative of the new and growing class of
disease-causing mutations characterized by unstable trinucleotide repeats such as
those recently described in fragile X syndrome (reviewed by W. Ted Brown and
Edmund C. Jenkins in Volume 2 of this series), myotonic dystrophy, and Ken­
nedy's disease. In this volume. Dr. Gusella reviews the frustrations and the final
triumphal conclusion of the search for the HD gene and the role of genetic
instability in this disease.
Finally, the pace of advances in screening and detection of genetic
disease that we have witnessed over the past several decades is certainly going to
pale compared with the explosion that we are already beginning to see from the
outfall of molecular genetics in general and the human genome project in
particular. The burden of all this new genetic knowledge will fall not only on the
shoulders of the biomedical establishment, but also on other areas of our society
concerned with health care. In a nation where health care is lamentably driven
as much or more by financial considerations as by medical or scientific ones, the
medical insurance industry is having to confront the knowledge that many of the
previous uncertainties about diagnosis and risk prediction will come to be sub­
ject to precise calculation. The genetics industry, likewise, will be forced to
rethink its conflicts and its role in delivering predictive tests. M. Rothstein
discusses the ways in which both industries will have to respond to changes in
the power of genetic information and its ability to predict coronary vascular
disease, cancer, neurological disease, and all the other common afflictions that
constitute the bulk of their businesses.
Overall, as in previous volumes, these summaries of important recent
developments emphasize the obvious—that the impact that molecular genetics
is having on human disease is already enormous and growing at an astounding
rate. Areas such as the pathogenesis of neurodegenerative and other disorders of
the central nervous system, cancer, and the previously impenetrable diseases
cystic fibrosis and Huntington's disease are now becoming vulnerable to rational
therapeutic attack. Amazing!
Theodore Friedmann
Milestones and Events in the
Early Development of Human
Gene Therapy
Theodore Friedmann
Department of Pediatrics
Center for Molecular Genetics
School of Medicine
University of California, San Diego
La JoUa, California 92093
From the earliest days of the modem science of genetics to the present time,
most approaches to the treatment of human genetic disease have been based on
the manipulation of metabolic pathways made ineffective or aberrant through
damage to the genetic material. These concepts of theory were derived most
strongly from the rediscovery at the beginning of the 20th century of the work of
Gregor Mendel (1901) and his elucidation of the laws of genetic inheritance. In
a remarkably insightful body of work carried out long before the nature of the
genetic material or the protein products of the genes were becoming understood,
the British clinician Sir Archibald Garrod intuited that many aspects of human
individuality, even health and disease, were determined by differences in the
function of the enzymes that catalyze biochemical pathways (Childs, 1970;
Garrod, 1902a,b, 1923). In the case of some inherited human disorders, Garrod
understood that such differences were caused by errors in the genetic material
and that such errors were propagated according to the rules discovered by Men­
del. Through his studies of human families with a number of rare genetic dis­
eases, he came to recognize what he called "inborn errors of metabolism." Based
on this concept, approaches to disease therapy over the ensuing half century or
so came to be based on biochemical manipulation of these aberrant pathways,
including replacement of metabolic products, elimination of stored cellular tox­
ins, reduction of substrates for defective pathways, pharmacological enhancing
or interference with a metabolic step, enzyme replacement, and even tissue and
organ transplantation (Friedmann, 1991). These approaches have resulted in
Molecular Genetic Medicine, Vol. 3 1
Copyright © 1993 by Academic Press, Inc.
All rights of reproduction in any form reserved.
2 Theodore Friedmann
the development of some highly effective forms of treatment. However, as ele­
gant as most of these treatments are in principle, only rarely have they led in
practice to truly effective therapy. More definitive treatments have long been
needed for most human genetic disease.
One such conceptually new approach to the treatment of human dis­
ease has emerged during the past several decades (Anderson, 1984, 1992;
Nichols, 1988; Friedmann, 1989; Verma, 1990; Miller, 1992). It is a change in
the approach to therapy that represents a conceptual break with all forms of
therapy that have preceded it in the history of medicine, since it proposes for the
first time truly curative approaches to human disease through a direct attack on
the genetic aberrations responsible for so much of human disease. The concept is
that of gene therapy, the correction of the genetic aberration underlying a
human disease rather than the mere manipulation of the aberrations in metabol­
ic or biochemical functions of the causative genetic defects resulting from the
genetic alteration. In the short period of no more than 2 or 3 decades, the
concepts and techniques of gene therapy have progressed from being entirely
fanciful to the beginnings of human clinical applications. The idea that many
forms of human genetic disease and even some degenerative and infectious
disease will become amenable to correction at the genetic level has cleared its
initial conceptual and technical obstacles and has now become widely accepted
in most molecular genetic, medical, and public policy circles. The first phase of
human gene therapy, i.e., the emergence and acceptance of the general con­
cept, is over. We are now in an explosive second phase—one of technical
implementation. A number of recent reviews have summarized many of the
technical and ethical aspects of the development of human gene therapy, but
there have been few descriptions of the discrete periods and pivotal events that
have led to this remarkable new concept of medicine.
The current concepts and tools of gene therapy can be envisioned to
have occurred in three more or less distinct phases. The first phase included the
development of some of the cellular reagents for studying the feasibility of gene
transfer, the clarification of the mechanisms of malignant cellular formation by
several classes of tumor viruses, the first suggestion that modified viruses be used
as gene delivery vehicles, and the discovery of the tools of recombinant DNA
manipulation. During this time, the concepts of designed therapeutic genetic
change in humans were being expounded by only very few proponents and were
generally not widely accepted. The second phase was initiated by a failed human
gene therapy experiment and, equally importantly, by the development of the
first truly efficient and useful gene transfer tools—the retroviral vectors. These
vectors permitted the first demonstrations of the complementation of genetic
defects in human cells and the correction of human disease phenotypes in vitro.
It was during this second phase that the basic science and clinical aspects of gene
therapy first began to merge and during which the rationale of gene therapy
1. Early Development of Human Gene Therapy
began to be accepted by the scientific, medical, public policy, and ethics com­
munities. The third and present phase, a phase that has begun only within the
past 3 or 4 years, is that of rapid application of the in vitro experience to the
increasingly varied clinical problems of genetic, degenerative, and infectious
diseases and rapid delivery of the techniques to the bedside. This phase has been
accompanied by a very broad acceptance of the concepts of somatic cell human
gene therapy for both genetic and nongenetic disease.
I. PHASE 1. PRIOR TO 1970: GENETICALLY MARKED CELLS AND
DNA-MEDIATED TRANSFORMATION
Important advances in cell biology during the 1960s paved the way for early
experiments in the possibilities for stable introduction of DNA into mammalian
cells with the puφose of introducing new and permanent genetic functions—an
obvious prerequisite for gene therapy. There was good reason to suspect that this
kind of manipulation might in fact become possible. In 1944, Avery and col­
leagues at the Rockefeller Institute reported that DNA introduced into pneu-
mococci could alter the phenotype of the recipient bacteria permanently, herita­
bly, and stably (Avery et al, 1944). These studies proved that it was DNA rather
than protein or other cellular macromolecules that served as the repository of
genetic information. During the same period, cell biologists were developing
mutant or auxotrophic lines of mammalian cells that could be used to deter­
mine whether genes carried into mammalian cells by high-molecular-weight
DNA provide permanent new genetic functions for cells. The gene encoding
the purine salvage enzyme hypoxanthine guanine phosphoriboxyl transferase
(HPRT) became one of the most useful after Szybalska and Szybalski (1962)
developed the HAT medium (hypoxanthine, aminopterin, and thymidine) for
the chemical selection of cells expressing HPRT. In addition, Seegmiller and
colleagues demonstrated the clinical relevance of HPRT mutations by discover­
ing the HPRT deficiency in patients suffering from the Lesch-Nyhan syndrome
(Seegmiller et al, 1967). Additional mammalian cell lines became available for
studies of stable gene transfer, including mutagenized nonhuman HPRT-
deficient cell lines and other selectable auxotrophic cells, such as thymidine
kinase-deficient cells (Kao and Puck, 1968). Studies with these new reagents
began to suggest that mammalian cells could indeed incoφorated and express
foreign DNA (Borenfreund and Bendich, 1961; Kay, 1961; Rabotti, 1963;
Szybalska and Szybalski, 1962; Bradley et α/., 1962; Bendich et al, 1971; Hill
and Huppert, 1970). However, because these gene transfer mechanisms were
exceedingly inefficient, none of these systems could be used to demonstrate
really convincing permanent genetic transformation. For instance, after Lesch-
Nyhan cells were exposed in vitro to DNA of normal cells, rare cells, possibly 1
Theodore Friedmann
in 10^, could be found by very sensitive autoradiographic methods to express the
HPRT enzyme. However, the genetic modification was unstable, and it was not
possible to grow genetically corrected cells in the selective medium, due presum­
ably to the rarity of the genetic complementation event and to the likelihood
that the foreign genetic material had not been integrated stably in the host cell
genome and was therefore only transiently expressed.
By the middle-late 1960s, investigators began to be optimistic that
more efficient genetic transformation might be possible. During the mid- and
late 1960s, studies by Dulbecco and colleagues of the mechanisms of neoplastic
transformation of murine and hamster cells by the DNA tumor viruses such as
SV40 and polyoma revealed that the entire genomes, or specific transforming
portions of the genomes of these transforming viruses, became integrated into
the host cell DNA in the course of malignant cell transformation (Sambrook et
αι., 1968; Hill and Hillova, 1972). At least some of the newly introduced viral
genes continued to be expressed in the "transformed" cells (Topp et al, 1981).
These infectious agents had obviously evolved to perform exactly the function
that would have been required for therapeutically useful gene transfer and thera­
py. With this in mind, investigators began to consider the possibility that these
or other viruses could be modified to allow them to act as Trojan horses, vehicles
to carry therapeutic foreign genes rather than their own deleterious genes into
defective cells.
Since these early cell transformation studies were carried out without
the benefit of the tools or concepts of the recombinant DNA era, it was far from
clear that vector modifications to allow engineered viruses to incoφorate would
be feasible. It was still not known how these or other viral vectors could be
modified to incoφorate and express foreign genes in any mammalian cells, but a
number of possible avenues emerged, including the ligation of foreign sequences
into the genomes of viruses. As with so many phenomena in genetics, this
concept was inspired partly by work with phage transduction and with the
enzymology of nucleic acid biosynthesis. Rogers reported that polylysine was
synthesized in infected plant cells from poly (A) ligated to the 3' end of the
TMV genome with terminal transferase (Rogers and Pfuderer, 1968). He sug­
gested that similar manipulations might eventually be useful in introducing
therapeutic new genetic information into human cells. But, of course, there was
little or no insight at that time into how such modifications might be carried
out. There were then no methods available to isolate single genes, to determine
their nucleotide sequence, to prepare workable amounts of purified genes, or to
ligate them to the vectors.
Several other workers suggested that artificially constructed pseu-
dovirions (pseudotypes) might be used to carry therapeutic foreign DNA into
cells (Aposhian et αί., 1972; Aposhian, 1970; Friedmann, 1971). This approach
was inspired by the knowledge that the papovaviruses matured by template-free