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Gene Therapy for Ovarian Cancer

Gene Therapy for Ovarian Cancer

ABSTRACT: Advances in molecular virology and biotechnology have led to the engineering of vectors that can efficiently transfer genes to target cells. Gene therapy strategies were developed along two lines: Cytotoxic approaches involve the transfer of genes that encode enzymes, which convert inactive prodrugs into cytotoxic drugs. Corrective gene therapy approaches aim to repair specific molecular alterations in signal transduction mechanisms that control the cell cycle or induce apoptosis. Clinical evidence suggests that gene therapies are best suited for patients with minimal residual disease. Multimodality approaches with conventional strategies and novel therapeutic tools in various combinations will most likely prove advantageous, compared to single-modality treatments. However, clinical trials will need to test these hypotheses. [ONCOLOGY 15:1197-1208, 2001]

The recent advances in cell and molecular biology of cancer
have elucidated some of the mechanisms underlying malignant transformation,
tumor cell migration, metastasis, angiogenesis, and cell response to
chemotherapy. Advances in molecular virology and biotechnology have allowed for
the engineering of vectors that can efficiently transfer genes to target cells.
As a result, gene therapy strategies for the treatment of cancer were developed
along two fundamental directions. Cytotoxic or suicide gene therapy approaches
entail the transfer of genes that encode enzymes able to convert inactive
prodrugs into cytotoxic drugs. Only transfected cancer cells expressing the
specific enzyme become susceptible to killing. Corrective gene therapy
strategies aim at repairing specific molecular alterations occurring in signal
transduction mechanisms that control the cell cycle or induce apoptosis.

Gene Therapy Vectors

The Recombinant DNA Advisory Committee has approved hundreds of gene therapy
protocols for the treatment of genetic diseases or cancer. Although gene therapy
strategies have produced highly promising results in preclinical in vitro and
animal models, their application in clinical practice has proven to be much more
difficult. A fundamental problem concerns the efficiency of gene transfer. Viral
vectors remain the vectors of choice, mainly because of their high efficiency of
transfection. After all, viruses have evolved successful strategies for
introducing their genome into eukaryotic cells and using the host cell
biochemical machinery.

The vast majority of phase I trials employ viral vectors, but some use DNA
protein complexes, DNA particles, ribozymes, or lipid-based vehicles. To
minimize side effects, viral vectors are rendered replication-incompetent. To
date, several vectors have been developed that are associated with significant
limitations related to low efficacy, inability to penetrate deeply in tumor
nodules, inactivation by the immune system, and undesired side effects (Table
1
). To enhance efficacy, inserted genes (transgenes) are positioned under the
control of a strong exogenous promoter. A reporter gene, such as the Escherichia
coli
LacZ gene encoding beta-galactosidase, is also inserted to assess the
efficacy of gene transduction.

Adenovirus

Adenoviral vectors are currently the vectors of choice in gene therapy,
including cancer gene therapy.[1] Adenoviral vectors are remarkably efficient,
but yield only transient expression of therapeutic genes and are generally
administered repeatedly to lengthen the duration of gene expression.
Adenoviruses infect dividing and nondividing cells, and can be produced
relatively easily on a large scale. They are stable, may be manufactured without
contamination by replication-competent adenovirus (RCA),[2] and can accommodate
up to 7.5-kb transcripts. Backbones have been derived from Ad2 and Ad5 serotypes,
both of which belong to the wild-type-C adenovirus subgroup.

The vitronectin receptor alpha(v) beta(3) integrin and the coxsackie/adenovirus
receptor mediate virus entry into human cells through chlathrin-coated
vesicles.[3] The viral genome remains extrachromosomal in the nucleus.
Recombinant adenovirus is produced on appropriate packaging cell lines
complementing the missing genes. The early genes—specifically, E1—were first
targeted because they control viral replication and regulate the expression of
late genes. Substitution of E1A and E1B genes by a designated transgene led to
the creation of the first generation of adenoviral vectors.

Due to the high likelihood of recombination events occurring during
manufacturing in vitro, engineering of strains lacking only one gene were
characterized by a prohibitive degree of replication-competent adenovirus (RCA)
contamination. This translated to high toxicity, particularly hepatotoxicity,
considering the elevated liver tropism of the wild-type virus. A
second-generation virus was then produced by adding an additional mutation in
the E2A or E3 regions.[4] This improved the toxicity profile of the vectors by
dramatically decreasing RCA contamination.

Administration of adenovirus is followed by an intense inflammatory and
immune response. An early innate response consists of the release of
inflammatory cytokines such as interferon (IFN)-gamma, interleukin (IL)-1, and
IL-6 as well as the recruitment of an acute inflammatory infiltrate followed by
a specific neutralizing antibody and T-cell response.[5] Both the transgene and
viral genes expressed by transfected cells are cross-presented on major
histocompatibility complex (MHC) class I or II sites, triggering specific CD8-
and CD4-positive T cells. An intense inflammatory reaction in proximity to the
tumor may enhance tumor immune recognition. However, immune-mediated vector
neutralization may pose marked limitations on the transduction efficacy of the
vector.

We and others have recently demonstrated the presence of neutralizing
antibodies in the serum and peritoneal fluid of patients with epithelial ovarian
cancer that significantly decrease the efficacy of adenoviral vectors in vitro.
However, the presence of antiadenoviral antibody titers did not appear to
compromise the efficiency of gene transfer in a phase I trial of
adenoviral-based gene therapy for pleural mesothelioma conducted at our
institution.[6] Nevertheless, there is still considerable concern about the
limitations posed by the immune system, particularly if repeated administrations
of vectors are planned. A third-generation adenoviral vector with somewhat
decreased immunogenicity was recently produced by the deletion of the E4 region
(in addition to E1 region) and preservation of the E3 region. The protein
product of E3 inhibits MHC I transport to the cell surface, thereby preventing
immune recognition of adenovirus-infected cells.

To address the concerns of RCA contamination and immune-mediated response,
"gutted" or "gutless" adenovirus vectors that contain no
viral genes have been generated.[7] These vectors accept up to 38 kb of foreign
DNA. Gutless adenovirus vectors are replication-deficient and require helper
viruses for propagation. One advantage of gutless vectors is that they are less
immunogenic and, by using helper viruses derived from different adenovirus
serotypes, production of host antibodies can be directed away from the
therapeutic gutless vector. Gutless adenovirus vectors are showing promise in
gene therapy. In mice, sustained expression of transgene was seen after a single
injection, with no serious virus-associated toxicity.[8]

Epithelial ovarian cancer cells are susceptible to infection by adenoviral
vectors.[9] We recently demonstrated that an E1/E4-deleted adenovirus was
similarly efficacious to an E1/E3-deleted one in several epithelial ovarian
cancer cell lines (unpublished observation). Current investigation is focusing
on improving the tissue selectivity of adenoviral vectors. Crosslinking of
adenoviral particles to basic fibroblast growth factor has proven to
significantly increase the binding of adenovirus on epithelial ovarian cancer
cells (SKOV3.ip), resulting in a 10-fold enhancement of its efficacy in
vitro.[10] Furthermore, the incorporation of an Arg-Gly-Asp (RGD)-containing
peptide to the HI loop of the adenovirus fiber knob through genetic engineering
directs the virus away from coxsackie/adenovirus receptor binding sites and
enhances its binding to the ovarian cancer cell surface.[11]

Adeno-Associated Virus

Adeno-associated virus has a 4.7-kb single-stranded DNA genome surrounded by a protein coat. Adeno-associated
virus is not autonomous; in the absence of a helper virus, adeno-associated
virus enters a latent state of infection, while coinfection by an adenovirus or
herpesvirus enables adeno-associated virus to replicate.[12] Its genome has at
least six transcripts with three internal promoters and two inverted terminal
repeats. Construction of recombinant adeno-associated virus vectors can be
accomplished by stripping off the gene sequences encoding viral structural
proteins and generating a backbone with the two inverted-terminal repeats
surrounding the inserted transgene. A packaging cell line is necessary for viral
replication and production.

Wild-type virus integrates at a specific site on human chromosome 19. Adeno-associated
virus vectors also integrate into host DNA for sustained gene expression. Adeno-associated
virus proteins are not toxic to cells. Although 80% of the population is
seropositive, adeno-associated virus is not associated with any known disease in
humans, and an existing immune response does not impair the efficiency of
transfection. Moreover, adeno-associated virus infection is not accompanied by
inflammation or generation of a strong recall immune response.[13]

Adeno-associated virus shows promise in the setting of gene therapy because
it infects a wide variety of cells. Its ability to infect both dividing and
nondividing cells may be a major advantage in targeting tumors with low
S phase. Sustained expression from adeno-associated virus vectors has been
observed in several tissue types. Epithelial ovarian cancer cells harvested
directly from patients are susceptible to infection by adeno-associated virus,
making adeno-associated virus a promising vector for further testing in cancer
gene and immune gene therapy of this disease in particular.

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