01. Gene therapy Boulikas.pdf - Gene therapy & Molecular Biology

HIV-1 envelope DNA construct developed anti-HIV
envelope immune responses (Wang et al, 1993);
intramuscular injection of plasmid DNA expression
vectors encoding the three envelope proteins of the
hepatitis B virus (HBV) induced humoral responses in
C57BL/6 mice specific to several antigenic determinants
of the viral envelope (Michel et al, 1995). Immunization
of mice with plasmid DNA constructs encoding one of the
secreted components of Mycobacterium tuberculosis,
antigen 85 gene induced substantial humoral and cellmediated
immune responses (Huygen et al, 1996).
Because immunization of cancer patients with tumor
antigen proteins is a very promising approach used
extensively in cancer therapy (e.g. Karanikas et al, 1997)
many of these approaches could be transferred to the DNA
level using the gene encoding the tumor antigen.
As an extension, this method could find application
using human tumor antigen genes rather than
bacterial/viral antigen genes, that is genes encoding for
proteins expressed in tumor but not in normal cells leading
to development of tumor vaccines (Graham et al, 1996;
Okamoto et al, 1997); this method mimics the infection of
the cell in the host by a pathogenic virus resulting in the
intracellular processing of the viral proteins and their
presentation on the cell surface. Human tumor antigens
are, however, weakly immunogenic compared to microbial
antigens a problem connected with polymorphism in the
major histocompatibility complex proteins of the host and
in antigen presentation.
Development of a fusigenic viral liposome vector was
made possible using the HVJ (hemagglutinating virus of
Japan, a Sendai virus) renowned for its cell fusion ability;
plasmid DNA containing the human tumor antigen genes
MAGE-1 and MAGE-3 was mixed with HMG-1
nonhistone protein (to increase nuclear import and
expression of the plasmid after transfection) and was
encapsulated into anionic liposomes (phosphatidylserine,
phosphatidylcholine, cholesterol) followed by the addition
of inactivated HVJ; intramuscular injection into mice
resulted in production of MAGE-1 and -3 IgG antibodies
(Okamoto et al, 1997).
XVI. Gene therapy of cancer and
candidate genes
A. Mechanisms of carcinogenesis
Whereas for inborn errors of metabolism transfer of a
single gene can correct the disorder, cancer is a complex
disease involving mutations in a number of protooncogenes
and tumor suppressor genes as well as an
imbalance and disarray in phosphorylation events and
regulatory circuits of the cell cycle. As a result of
transformation, tumor cells acquire a proliferation
advantage compared with normal cells, most of which are
quiescent in the adult organism; cancer cells acquire
Gene Therapy and Molecular Biology Vol 1, page 51
51
partial independence from regulatory signals from
neighboring cells for restricted cell growth. A crucial step
in cancer development is the nonelimination of pre-cancer
cells by apoptosis (usually a subsequence of a mutation in
the p53 gene); such cells acquire a number of unrepaired
damage in their DNA, such as strand breaks, which induce
chromosomal translocations and result in clonal expansion
of this cell population.
Tumor cells are able to survive after DNA damage,
and display an increase in mutation rate; cancer cell
populations are heterogenous with respect to
translocations, loss of heterozygosity, point mutations and
transpositions in various genes. Whenever the mutated cell
acquires an advantage for rapid growth over other cells in
the tumor mass, escaping cell cycle checkpoints, it may
replace the original population, a phenomenon known as
tumor progression; this may lead to appearance of a more
malignant phenotype. As a result, tumor cells are of
different genotypes and clones obtained from the same
solid tumor may differ in the level of malignancy.
A number of candidate genes, when become mutated
or overexpressed, may lead to tumor phenotype: p53, RB,
and p21 appear to be the most important. The deregulation
of other genes is connected to tumor progression whereas
different groups of genes are associated with tumor cell
metastasis. These facts make a single gene transfer
approach to tumor cell mass to inhibit its growth or change
its phenotype from malignant to normal very challenging.
B. Human clinical trials
The genes used for cancer gene therapy in human
clinical trials include a number of tumor suppressor genes
(p53, RB, BRCA1, E1A), antisense oncogenes (antisense
c-fos, c-myc, K-ras), suicide genes (HSV-tk, in
combination with ganciclovir, cytosine deaminase in
combination with 5-fluorocytosine) which have been very
effective in eradicating solid tumors in animals. Also the
cytokine genes (IL-2, IL-7, IFN-γ, GM-CSF) are being
used for the ex vivo treatment of cancer cells isolated from
human patients and are able to elicit an immunologic
regression especially on immunoresponsive malignancies
(melanomas, colorectal carcinomas, renal cell carcinomas)
(Culver, 1996). Future directions might be toward use of
genes involved in the control of tumor progression and
metastasis. Discovery of new genes which are over- or
under-expressed during transformation and metastasis is a
promising approach for the identification of novel gene
targets in cancer gene therapy (Georgiev et al, 1998, this
volume).
Diseases amenable to therapy with gene transfer in
clinical trials (Appendix 1 and Table 4 in Martin and
Boulikas, this volume) include cancer (melanoma, breast,
lymphoma, head and neck, ovarian, colon, prostate, brain,
chronic myelogenous leukemia, non-small cell lung, lung
adenocarcinoma, colorectal, neuroblastoma, glioma,