Biomedical Engineering Reference
In-Depth Information
Therapeutic gene transfer effectively generates
transgenic human cell clones and, for this reason,
only somatic cells can be used as targets. The prospect
of germ-line transgenesis in humans raises serious
ethical concerns and, with the rapid advances in
technology allowing germ-line transformation and
nuclear transfer in numerous mammals, these
concerns will need to be addressed in the very near
future ( Johnson 1998). As an alternative to perman-
ent gene transfer, transient gene therapy can be
achieved using oligonucleotides, which can disrupt
gene expression at many levels but do not per-
manently change the genetic material of the cell
(Pollock & Gaken 1995).
The tools and techniques for gene therapy are
essentially similar to those used for gene transfer to
any animal cells. Transfection, direct delivery or
transduction (see Chapter 11) can be used to intro-
duce DNA into cells. Viral vectors are most popular
because of their efficiency of gene transfer
in vivo
.
However, extreme precautions need to be taken to
ensure the safety of such vectors, avoiding potential
problems, such as the production of infectious viruses
by recombination and the pathological effects of
viral replication. A number of viral vectors have
been developed for gene therapy, including those
based on oncoretroviruses, lentiviruses, adenovirus,
adeno-associated virus, herpes virus and a number
of hybrid vectors combining advantageous elements
of different parental viruses (Robbins
et al.
1998,
Reynolds
et al.
1999). The risks associated with viral
vectors have promoted research into other delivery
methods, the most popular of which include direct
injection of DNA into tissues (e.g. muscle), the injec-
tion of liposome-DNA complexes into the blood and
direct transfer by particle bombardment. Although
inherently much safer than viruses, such procedures
show a generally low efficiency (Scheule & Cheng
1996, Tseng & Huang 1998).
met. Tumour-infiltrating lymphocytes (cells that
naturally seek out cancer cells and then kill them by
secreting proteins such as tumour necrosis factor
(TNF)) were isolated from patients with advanced
cancer. The cells were then genetically marked with
a neomycin-resistance gene and injected back into
the same patient (Rosenberg
et al.
1990).
The first clinical trial using a therapeutic gene-
transfer procedure involved a 4-year-old female
patient, Ashanthi DeSilva, suffering from severe com-
bined immune deficiency, resulting from the absence
of the enzyme adenosine deaminase (ADA). This dis-
ease fitted many of the ideal criteria for gene-therapy
experimentation. The disease was life-threatening
(therefore making the possibility of unknown treat-
ment-related side-effects ethically acceptable), but
the corresponding gene had been cloned and the
biochemical basis of the disease was understood.
Importantly, since ADA functions in the salvage
pathway of nucleotide biosynthesis (p. 177), cells
in which the genetic lesion had been corrected had
a selective growth advantage over mutant cells,
allowing them to be identified and isolated
in vitro
.
Conventional treatment for ADA deficiency involves
bone-marrow transplantation from a matching donor.
Essentially the same established procedure could be
used for gene therapy, but the bone-marrow cells
would be derived from the patient herself and would
be genetically modified
ex vivo
(Fig. 14.10). Cells
from the patient were subjected to leucophaeresis
and mononuclear cells were isolated. These were
grown in culture under conditions that stimulated
T-lymphocyte activation and growth and then
transduced with a retroviral vector carrying a nor-
mal
ADA
gene as well as the neomycin-resistance
gene. Following infusion of these modified cells, both
this patient and a second, who began treatment in
early 1991, showed an improvement in their clin-
ical condition as well as in a battery of
in vitro
and
in
vivo
immune-function studies (Anderson 1992).
However, the production of recombinant ADA in
these patients is transient, so each must undergo
regular infusions of recombinant T lymphocytes.
Research is ongoing into procedures for the trans-
formation of bone-marrow stem cells, which would
provide a permanent supply of corrected cells.
Gene-augmentation therapies for a small number
of recessive single-gene diseases are now undergoing
Gene-augmentation therapy for recessive diseases
The first human genetic-engineering experiment was
one of
gene marking
, rather than gene therapy, and
was designed to demonstrate that an exogenous
gene could be safely transferred into a patient and
that this gene could subsequently be detected in
cells removed from the patient. Both objectives were
