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Blood, Vol. 94 No. 12 (December 15), 1999:
pp. 3965-3967
INTRODUCTION: FOCUS ON HEMATOLOGY
Gene Transfer by Adenovectors
By
Malcolm Brenner
From the Baylor College of Medicine, Houston, TX.
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ARTICLE |
CLINICAL GENE THERAPY is going through an
uncomfortable adolescence. Many observers have already been
disappointed by the lack of clear-cut successes with this strategy. To
this sense of frustration has now been added a feeling of dismay
because of the death of an 18-year-old with ornithine transcarbamylase deficiency, who received an intrahepatic arterial injection of an
adenoviral vector that encoded a wild-type version of the defective enzyme.1 The primate study by Lozier et al2 in
this issue of BLOOD, further emphasizes the limitations of many
adenoviral vector systems for the treatment of human diseases. This
group used an adenovector in which the E1 genes had been
deleted to inhibit replication of the vector in human cells. After
incorporating the human factor IX gene into this, a first-generation
vector, they injected the construct intravenously into macaque monkeys. The virus had no effect at its lowest dose, but at the highest, it
produced significant levels of human factor IX for approximately 3 weeks. Unfortunately, this benefit was secured at the cost of severe
and likely permanent liver damage, manifest by high enzyme levels and
persistent hypofibrinogenemia. The experience of Lozier et
al2 underscores the difficulty of finding a
"Goldilocks" dose for this gene-vector combination. Indeed, as
the investigators point out, the window between an effective and a
toxic dose is exceedingly narrow. It is important to emphasize that
this type of adverse event is not unique to the model used by Lozier et al.2 A baboon receiving 1.2 × 1013 viral
particles/kg intravenously also developed severe endothelial injury
with coagulopathy and hepatocellular damage.3
Even if toxicity could be avoided, another adenovector-associated
problem may become apparent. In the study of Lozier et al,2 the transgenic human factor IX produced was highly immunogenic, which
is in striking contrast to its lack of effects in the same monkeys when
administered as a purified protein. The investigators suggest that the
adenovector proteins trigger an acute-phase danger response during
infusion that is manifest by secretion of inflammatory cytokines that
recruit an immune response both to the adenovector and to any
associated transgenic proteins. Regardless of the mechanism, the end
result appears to be a cellular and humoral immune response that,
within a few days or weeks, destroys the transgenic cells and may
neutralize any human factor IX they have produced. Moreover, these
effects are seen even in animals that have never been exposed to the
adenoviruses on which the vectors are based and thus constitute a
primary immune response.4-7 Because most humans
already have immunity to adenoviruses, we may expect the secondary
responses induced by the vectors to be correspondingly accelerated and
more intense than effects generated in naive animals.
Given these limitations, one might be tempted to give up on
adenovectors entirely and seek a better alternative. This would be a
mistake. In the appropriate setting, and with appropriate modifications, adenovectors have characteristics that make them very
desirable for certain gene therapy applications. To appreciate this
potential, it is necessary to understand how adenovectors produce their
adverse effects and how these may be thwarted. The toxicity of
adenovectors occurs in 3 overlapping phases. During the first few hours
of infection, the adenovector proteins act directly on the host's
defense system, provoking an acute-phase response marked by rapid
release of inflammatory cytokines, including interleukin-6 (IL-6) and
IL-8, and recruitment of host cellular defenses. Indeed, in some
species, this phase of infection may include the activation of mast
cells and basophils, which induce IgE-independent acute
anaphylaxis.8-10 Over the next 24 to 96 hours, most of the
vector-associated toxicity can be attributed to cellular production of
adenoviral late proteins involved in the assembly of the viral coat.
Although the precise mechanisms of adenovector-related toxicity are
unknown, some insights have been gained. Fiber protein, for example,
can disrupt cellular endosomes, thereby disabling or killing the
infected cell.9-12 Subsequently, the immune system
recognizes and destroys adenoviral peptides on the surface of infected
cells, mainly by cytotoxic T lymphocytes (CTL)-mediated
and natural killer (NK) cell-mediated responses, and this
late phase toxicity may be associated with hepatocyte hypertrophy and
hepatic fibrosis.4,5
How might these different toxicities be overcome? Most of the earliest
efforts focused on reducing the production of viral proteins by
infected cells, with the hope of limiting both direct adenoviral
toxicity and the antiadenoviral immune response. First generation
vectors were deleted in the E1 region, which contains genes
whose products regulate transcription of the late adenoviral structural
proteins.6 This type of vector was selected by Lozier et
al2 for their study of gene therapy to correct factor IX deficiency. Unfortunately, as was amply demonstrated by these investigators, E1 deletion by itself is not adequate to block late protein expression and, hence, the toxic effects of the vector.
Subsequent generations of adenovectors lacked more than one set of
adenoviral genes, eg, E1 and E4,13
E2a9 and E4,12 or E1
and E3,10,11 and so on (reviewed in Hitt et
al14). Although showing less immunogenicity and toxicity
and more durable transgene expression in some studies, these modified
vectors produced little or no benefit in
others.10-14 Indeed, it was an
E1/E4-deleted vector that was associated with the death
of the young patient with enzyme deficiency.1 The
quintessential attenuated adenovector is the so-called helper-dependent
or gutless vector, in which virtually all of the adenoviral genes have
been removed and replaced with the gene of interest and its promoter,
together with irrelevant DNA to allow packaging in the viral
envelope.15,16 These vectors can only be made with the
assistance of a helper adenovector, which must then be separated from
the deleted vector.17 Helper-dependent vectors have shown a
much higher therapeutic index than conventional adenovectors in several
different models.14,16,18-20 Importantly, they also seem to
be much less immunogenic, so that transduced postmitotic cells (eg,
muscle or liver) may secrete vector-derived proteins over many
months,8,18 a prime consideration in the treatment of many deficiency disorders. On the other hand, these vectors are proving quite difficult to manufacture in adequate quantities for human trials. They likely remain nonintegrating and
do not replicate as episomes, so that transduction of rapidly dividing
tissues, such as hematopoietic stem cells, becomes feasible only when
short-term gene expression is desired.
Aside from gene removal, modification of cell targeting could increase
the safety and efficiency of adenovectors. Adenoviruses bind to at
least 2 molecules on their target cells: the Coxsackie adenovirus
receptor (CAR) and cell surface integrins (usually  v 3
or v5).14,21-23 Binding is mediated by
domains on the adenoviral knob protein. Because the sequence and
crystal structure of this protein is known, one could select new ligand
sequences and incorporate them into positions that would disrupt
pre-existing patterns of binding and establish new
ones.21-23 In this way, it would be possible to reduce the
initial doses of infused virus, thereby decreasing immediate toxicity,
and target the circulating virus to organs that are more resistant to
the toxicity of adenovectors than are lung, liver, and vascular
endothelium, currently the major tissues affected by this route of administration.
Given the liabilities of adenovectors, how can they be optimally
applied in the clinic? The narrow therapeutic index, the short duration
of expression, and the immunogenicity of E region-deleted vectors, illustrated so clearly by Lozier et al,2 probably mean that they are not going to be suitable for protein replacement therapy in genetic diseases, although continued refinement of late-generation constructs may well change this assessment. In particular, the ability of helper-dependent adenovectors to induce secretion of high levels of the gene product for a prolonged period may
make them well suited to treatment of deficiency disorders. Currently
available adenovectors are well suited to applications in cancer gene
therapy. Because they transduce many different cell types with
relatively high efficiency, they can be relied on to transfer toxic or
immunomodulatory genes to a broad spectrum of tumors, both ex vivo and
locally in vivo. The availability of conditionally
replication-competent adenovectors, which will only divide in malignant
cells with genetic abnormalities, such as a nonfunctioning p53
gene, further increases the utility of such treatment.24
Because the aim of cancer gene therapy is to kill tumor cells, the
limited duration of transgene expression by adenovectors is not
necessarily a problem. Instead, the induction of a destructive immune
response against the transduced cell and the transgene product can be
used to advantage.
Adenovectors have been used to transfer the thymidine kinase gene to a
range of different tumors, including gliomas and prostate or ovarian
carcinomas, where the kinase phosphorylates the pro-drug ganciclovir
into an active agent, resulting in significant antitumor responses.25,26 The utility of this strategy for metastatic disease is limited by the need to inject the vector locally. Success has also been reported from the use of adenovectors encoding a wild-type p53 protein in a range of tumor types; this reagent is now entering phase III clinical trials.27 Similarly, a
conditionally replication-competent adenovirus has produced remarkable
tumor regression in relapsed head and neck tumors and is now also
entering wider clinical testing.28 Finally, the use of
adenovectors to transfer immunostimulatory genes, such as IL-2 and
granulocyte-macrophage colony-stimulating factor
(GM-CSF), to generate a tumor vaccine has been accompanied
by antitumor responses in both adult and pediatric malignant
diseases.29,30 None of these approaches has produced
significant toxicity. Although few, if any, of the patients treated in
this manner are likely cured of their disease, the availability of
cancer therapies that appear to be non-cross-reactive with cytotoxic
drugs or radiation would significantly increase the number of treatment
options. As more is learned about which genes should be transferred to
cancer cells, and with continued improvement in vector technology and
the ability to incorporate gene therapy into other modalities, we can
anticipate substantial contributions from adenovector-driven cancer
treatments, extending to hematologic malignancies. In the longer term,
the maturation of gene therapeutics from early adolescence to adulthood
should see the application of highly modfied adenovectors to a broad range of ailments, which may well include hematologic disorders such as
factor IX deficiency.
| |
FOOTNOTES |
Address reprint requests to Malcolm Brenner, MD, Baylor College of
Medicine, 1102 Bates, Suite C1140, Houston, TX 77030.
| |
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