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

fragments, or heat shock proteins complexed to antigenic
peptides and then process the tumor antigens for
presentation; IL-3 stimulated antigen-presenting cells
(APCs), which are macrophage-like, within the tumor
leading to generation of cytotoxic T lymphocytes (CTLs).
This constitutes a plausible pathway for enhancement in
tumor rejection by IL-3 stimulation (Pulaski et al, 1996).
IL-3 signaling proceeding either via the JAK-STAT or
the Ras-Raf pathways, stimulates a number of genes such
as the DUB-1 encoding a deubiquitinating enzyme the
overexpression of which leads to G1 arrest (Zhu et al,
1996); deubiquitination might be an additional mechanism
to couple extracellular signaling to cell growth. IL-3
signaling leads to stimulation in myeloid cell proliferation.
F. Cancer immunotherapy with the IL-7
gene
Primary cell cultures from 45 patients with malignant
melanoma were transfected via electroporation with the
gene encoding for human interleukin-7 (IL-7) resulting in
the production of biologically active IL-7 without altering
the expression of HLA class I and II, ICAM-1, and of a
melanoma-associated antigen. Irradiation of the
transfected cells with 10,000 cGy, which arrested tumor
cell growth in vitro, did not affect the ability of the cells to
secrete IL-7 in the culture medium; this approach, which
does not use retroviruses, could be applicable in
vaccination protocols for melanoma patients (Finke et al,
1997).
Transfer of the IL-7 cDNA for cancer immunotherapy
is being used in a human clinical trial (protocol 70,
Appendix 1).
G. Cancer immunotherapy with the IL-12
gene
IL-12 gene therapy is one of the more novel and
promising approaches in cancer therapy. IL-12 is a
heterodimeric cytokine composed of two subunits, p40
and p35, that requires the simultaneous expression of both
the p35 and p40 chain genes from the same cell for
production of biologically active IL-12. Coordinate
expression of the IL-12 p40 and p35 genes in several solid
tumor models has been found to induce strong and specific
antitumor immune responses. A variety of biological
functions have been attributed to IL-12 including the
induction of IFN-γ and the promotion of predominantly
Th1-type immune responses to antigens (Tahara et al,
1996).
The local secretion of IL-12 achieved by gene
transduction suppressed tumor growth and promoted the
acquisition of specific antitumor immunity in mice. This
was shown by intradermal inoculation of mice with
NIH3T3 cells transduced with expression plasmids or a
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retroviral vector expressing the murine IL-12 gene
admixed with murine melanoma BL-6 cells; CD4 + and
CD8 + T cells, as well as NK cells, were responsible for the
observed antitumor effects resulting from IL-12 paracrine
secretion. Transduction of tumor cells with B7.1 gene
enhanced the antitumor immune response (Tahara et al,
1996).
The antitumor effect of several transgene expression
plasmids encoding the cytokines IL-2, IL-4, IL-6, IL-12,
IFN-γ, TNF-α, and GM-CSF was tested using the gene
gun-mediated DNA delivery into the epidermis overlying
an established intradermal murine tumor; this study
showed that IL-12 gene therapy was much more effective
than treatment with any other tested cytokine gene for
induction of tumor regression as determined from the
increased CD8 + T cell-mediated cytolytic activity in the
draining lymph nodes of tumor-bearing mice; treated
animals were able to eradicate not only the treated but also
the untreated solid tumors at distant sites; elevated
systemic levels of IFN-γ, were found after IL-12 gene
therapy. This approach is providing a safer alternative to
IL-12 protein therapy for clinical treatment of cancers
(Rakhmilevich et al, 1997).
Lieu et al (1997) have evaluated three IL-12 retroviral
vector designs for their level of IL-12 expression in
leukemia/lymphoma cells; these retroviral vectors were
based on the murine stem cell virus (MSCV) which
efficiently transduces functional genes into normal
hematopoietic cells. MSCVpac-mlL-12 and MIPV-mIL-
12 contained an encephalomyocarditis virus internal
ribosome entry site for internal translation of bicistronic
mRNA transcripts, while MDCVpac-mIL-12 carried an
expression cassette in the U3 region of the 3' LTR. The
MSCVpac-mIL-12 vector was more efficient and directed
robust expression of both p40 and p35 IL-12 genes in
several murine tumor cell lines of hematopoietic origin,
including a T-cell lymphoma, a B-cell lymphoma, and a
plasmacytoma/myeloma.
Adenoviral delivery of the IL-12 gene was effective
against breast tumors (Bramson et al, 1996) or metastatic
colon carcinoma (Caruso et al, 1996) in animal models:
mice bearing breast tumors, injected intratumorally with a
single dose of an adenovirus expressing IL-12 showed
regressions in greater than 75% of the treated tumors; this
effect was accompanied with a maximum expression of
IL-12 within the tumor between 24 and 72 hr postinjection
which lasted for 9 days and an elevation in IFN-γ
within the tumor; local production of IL-12 also stimulated
IFN-γ production in tumor-draining lymph node cells
(Bramson et al, 1996). Whereas intratumoral adenoviral
transfer of the HSV-tk and the murine IL-2 genes resulted
in substantial hepatic tumor regression, induced an
effective systemic antitumoral immunity in the host and
prolonged the median survival time of the treated animals
from 22 to 35 days a recombinant adenovirus expressing
the murine IL-12 gene was much more effective: