744 Improvement of the TK/GCV Strategy for the Treatment of Gliomas by 5 Fluorouracil and TK30, a More Potent TK Enzyme Molecular Therapy �������� ��� ���� ���������������� �������� ��� ������© ������[.]
Trang 1Molecular Therapy Vol 7, No 5, May 2003, Part 2 of 2 Parts
ONCOLYTIC VIRUS AND SUICIDE GENE THERAPY FOR CANCER isogenic tk vector (TOZ.1) in the presence or absence of GCV
bioactivity to inhibit the NF-κB translocation to the nucleus was
examined by electrophoretic motility shift assay In human
already been translocated in the nucleus without receptor mediated
the translocation of NF-κB was significantly inhibited, and the
caspase 3 activity was found to be significantly upregulated in
infected cells At an MOI of 0.1, the cytotoxicity against U-87MG
killed 85% of U-87MG cells, and in the presence of GCV, all the
translocated to the nucleus, cells had become apoptotic by infection
enhanced the cytotoxicity by the bystander effects of HSV-TK/
GCV system
Apoptosis in Lung Cancer Cells
Katherine M Finan,1 Greg Hodge,2 Ann M Reynolds,1 Sandra
Hodge,1 Mark D Holmes,1 Andrew H Baker,3 Paul N
Reynolds.1
1 Department of Thoracic Medicine, Royal Adelaide Hospital,
Adelaide, South Australia, Australia; 2 Department of
Haematology, Women’s and Children’s Hospital, Adelaide, South
Australia, Australia; 3 Division of Cardiovascular and Medical
Sciences, University of Glasgow, Glasgow, Scotland, United
Kingdom.
New options are needed for the treatment of advanced lung cancer
Tumour invasion and spread involves a complex interplay between
tumour, stromal and inflammatory cells with the extracellular matrix
(ECM) The matrix metalloproteinases (MMPs) are a family of
enzymes involved in ECM maintenance and tissue inhibitors of
matrix metalloproteinases (TIMPs) are important factors in the
regulation of the activity of MMPs Gene delivery of TIMP 1 and
2 has been shown to inhibit tumour growth in animal models There
is evidence that TIMP 3 may also be therapeutic in this way, and in
contrast to TIMP 1 and 2, may directly induce apoptosis in tumour
cells, although this has not yet been evaluated in lung cancer
Hypothesis: Overexpression of TIMP 3 delivered by adenoviral
(Ad) vectors to lung cancer cells causes apoptosis and increased cell
death Aims: To compare the effect of TIMP 1, 2 and 3 gene delivery
on lung cancer cell lines Methods: A549, 1299, H1466 and 522
cells were plated at a known concentration, then infected with Ad
vectors carrying the genes for TIMP 1, 2 and 3 Following infection
the cells were examined for changes consistent with apoptosis and
cell death Analysis by flow cytometry using Propidium Iodide and
Anexin V staining was used to establish the presence of apoptosis
Cell counts were used to evaluate any reduction in cell number
Results: For all lines maximal reduction in cell number was seen
with TIMP 3 overexpression and this was dose dependent Flow
cytometry confirmed the induction of apoptosis by TIMP 3 (eg for
A549 cells 32.5 ± 6.3% at 72 hours vs 2.2 ± 2.1% for uninfected
cells) Rates of apoptosis with TIMP 1 and 2 infected cells were
similar to controls Conclusion: TIMP 3 induces a reduction in cell
numbers and causes increased apoptosis in lung cancer cell lines
Further study using animal models of lung cancer is thus warranted
and is currently being pursued
in Combination with Cell Cycle-Altering Drugs
Tiina Pasanen,1 Anne Karppinen,1 Leena Alhonen,1 Juhani Jänne,1 Jarmo Wahlfors.1
1 Department of Biotechnology and Molecular Medicine, A.I Virtanen Institute for Molecular Scieces, Kuopio, Finland.
Suicide gene therapy with HSV-TK and ganciclovir (GCV) has been widely used against malignant cells and its efficacy has been demonstrated in a variety of different in vitro and in vivo models However this gene therapy regimen needs to be enhanced for a true clinical success We studied whether polyamine biosynthesis inhibition by 2-difluoromethylornithine (DFMO), a clinically tested and well-tolerated chemotherapeutic drug, can increase the cytotoxicity of HSV-TK/GCV in 9L rat glioma cells Polyamines putrescine, spermidine and spermine are cationic molecules that are indispensable to cell growth and differentiation The hypothesis was that prolonged S-phase caused by polyamine biosynthesis inhibition, would lead to accumulation of cells in this particular cell cycle phase When the cells are released from this block, there would
be higher proportion of cells in the middle of DNA-synthesis phase Because HSV-TK/GCV cytotoxicity is killing cells only after their DNA has been replicated, the preceding DFMO treatment would make the cell destruction more efficient When the cells were exposed
to GCV 3 or 4 days after removal of DFMO from growth medium, the cytotoxicity was increased up to 2.5-fold We also verified whether cell cycle blockage per se could yield similar effect as DFMO Our results from serum deprivation experiments showed that, despite of apparent cell growth and cell cycle phase distribution effects, serum starvation was weaker enhancer of HSV-TK/GCV cytotoxicity than DFMO Finally, the general utility of HSV-TK/ GCV DFMO combination was tested in another tumor cell type, human prostate carcinoma cell line DU-145 DFMO sensitized these cells to HSV-TK/ GCV cytotoxicity, but the effect was less prominent than in 9L cells In conclusion, we have demonstrated that a correctly timed induction of DFMO-mediated polyamine biosynthesis inhibition can enhance the efficiency of HSV-TK/GCV gene therapy in vitro The observed synergistic effect is potentially useful in clinical trials because, as opposed to use of other cell cycle-altering drugs, DFMO has already been tested in the treatment of human tumors and used as chemo preventive regimen with excellent tolerability We are currently testing whether other cell cycle-altering drugs can enhance ganciclovir-mediated cytotoxicity Furthermore, DFMO treatment in combination with HSVtk/GCV therapy will be tested in vivo
the Treatment of Gliomas by 5-Fluorouracil and TK30, a More Potent TK Enzyme
Gaelle V Roullin,1 Emmanuelle F Germain,1 Manuel P Caruso.1
1 Cancer Research Center, Laval University, Quebec, QC, Canada.
BACKGROUND: Gene transfer of the Herpes Simplex virus thymidine kinase (TK) associated with gangiclovir (GCV) administration represents a promising approach to treat malignant gliomas TK phosphorylates GCV into GCV-monophosphate, which
is further converted into the di- and triphosphate forms by cellular enzymes GCV-triphosphate is incorporated into elongating DNA, which leads to cell death Complete tumor eradication has been obtained in animal experiments with only a small proportion of cells expressing TK (TK cells) This phenomenon, called the bystander effect (BE), is mainly due to the transfer of phosphorylated GCV from TK cells to neighbouring TK negative cells In clinical trials, the low level of gene transfer and inhomogeneous distribution of the vector within the tumor, have been highlighted Moreover, an effective
Trang 2Molecular Therapy Vol 7, No 5, May 2003, Part 2 of 2 Parts
Copyright © The American Society of Gene Therapy
S288
ONCOLYTIC VIRUS AND SUICIDE GENE THERAPY FOR CANCER
drug concentration is difficult to obtain in the vicinity of the tumor
since GCV does not cross readily the blood brain barrier We have
previously reported a major improvement by using TK30, a much
more potent mutant of TK: TK30-expressing cells (TK30 cells)
were killed more efficiently than TK cells (10- to 100-fold) and the
BE was strongly increased too The use of 5-fluorouracil (FU) has
been shown by others to enhance the efficacy of the TK/GCV
strategy FU is a thymidylate synthase inhibitor which acts by
decreasing the pools of thymidine, the major competitor for GCV
phosphorylation In this study, we have investigated the potential
of FU to interact with GCV on four different human (SKI1, SKMG4,
U87 and U251) and three rat (C6, F98 and 9L) TK- or TK30 glioma
cells
METHODS: The various cell lines were retrovirally infected to
stably express the mutant TK30 or the wild-type TK Drug
sensitivities were determined by their median inhibitory
concentration (IC50) using conventional MTT proliferation assays
BE experiments with either TK30 or TK were realized by mixing
ratios of infected and non-infected cells The multiple drug-effect
analysis of Chou-Talalay was used to qualify the interaction between
GCV and FU
RESULTS: As calculated by the Chou-Talalay method, at least
an additive effect of the drugs was noted in proliferation experiments
with all glioma TK30 cells In BE experiments carried out with SKI1
and SKMG4 cells, potentiation and/or synergism were observed
and the synergism was stronger in TK- than in TK30 cells
Nevertheless, the IC50 (GCV) was still much lower with TK30- vs
TK cells in the presence of FU (by a factor of 35 and 126 for SKI1
and SKMG4, respectively) Interestingly, except for U251 cells, a
strong synergistic effect was also found when non-infected cells
alone were treated with GCV + FU, a phenomenon unreported until
now The mechanisms of this antitumor effect as well as in vivo
studies are presently under investigation
CONCLUSION: Based on these data, the local delivery of GCV
+ FU associated with TK30 gene transfer should be considered to
optimize the TK/GCV strategy by efficiently eliminating both
infected and more importantly non-infected glioma cells In vivo
experiments will address the antitumoral efficacy of this system
and also the possible loco-regional toxic side effects of the drug
combination
CANCER TARGETED GENE THERAPY II
Chemotherapy for Cancer Treatment
Ziqun Han,1 Jennifer Hu,2 Binlei Liu,1 Michael Robinson,1 Ruth
Branston,1 Charles Coombes,2 Robert Coffin.1
1 BioVex Ltd, Abingdon, Oxon, United Kingdom; 2 Cancer Cell
Biology, Hammermsith Hospital Campus, Imperial College of
Science and Medicine, London, United Kingdom.
OncoVEX is an oncolytic version of HSV with several
improvements as compared to oncolytic versions of HSV previously
tested in the clinic These improvements are (i) OncoVEX is derived
from a clinical isolate of HSV with enhanced anti-tumour potency
as compared to previously used laboratory strains (ii) tumour
selective replication is enhanced via increased expression of US11 in
addition to deletion of ICP34.5 (iii) ICP47, which usually blocks
antigen presentation in HSV infected cells, is deleted A version of
this virus additionally encoding the gene for GM-CSF is currently
in a Phase I clinical trial in a number of tumour types This virus is
designed to provide a direct anti-tumour effect by tumour selective
virus replication and also to induce an anti-tumour immune response
enhanced by the expression of GM-CSF It is anticipated that in
routine clinical practice oncolytic viruses are likely to be combined
with standard therapies such as chemo- or radiotherapy The work
reported here assesses the anti-tumour effects of OncoVEX in combination with a number of first line chemotherapeutic agents
(e.g taxol and cisplatin) in in vitro models Work in vivo is underway.
Results so far demonstrate that OncoVEX treatment of tumour cells either prior to, concurrently with, or after addition of the drug provides enhanced drug sensitivity and that the chemotherapeutic agents tested do not reduce the effectiveness of the oncolytic effect Initial conclusions are therefore that combination of OncoVEX with anti-cancer drug therapy may be beneficial for cancer treatment
Cancer with an Oncolytic Herpes Simplex Virus Incorporating Two Membrane-Fusion Mechanisms
Mikihito Nakamori,1 Xinping Fu,1 Feng Meng,1 Aiwu Jin,1 Lihua Tao,1 Robert C Bast, Jr.,2 Xiaoliu Zhang.1
1 Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX; 2 M.D.Anderson Cancer Center, The University of Texas, Houston, TX.
Replication-competent herpes simplex virus (oncolytic HSV) holds considerable promise for treating malignant solid tumors including ovarian cancer However, the potency of the virus needs improvement if its full clinical potential is to be realized In our previous studies, we showed that incorporation of membrane fusion capability into an oncolytic HSV, either by screening for a syncytial HSV mutant after random mutagenesis or by inserting a hyperfusogenic glycoprotein from gibbon ape leukemia virus (GALV.fus) into the viral genome, can significantly increase the antitumor capability of the virus We recently constructed a newer version of fusogenic oncolytic HSV by incorporating both membrane fusion mechanisms into a single virus In vitro characterization of this doubly fusogenic oncolytic HSV (Synco-2D) showed that this virus produces a distinctive syncytial phenotype, leading to a significantly increased tumor cell killing ability, compared with that
of a nonfusogenic virus When injected directly into the abdominal cavity of mice bearing human ovarian cancer xenografts, Synco-2D eradicated all tumor masses in 75% of the animals, whereas no animals in the conventional oncolytic HSV treated group was tumor-free Our results indicate that this newly generated fusogenic oncolytic HSV is a promising candidate for clinical testing against advanced ovarian cancer
Oligonucleotides and siRNAs Strategies in Human Pancreatic Cancer
Amor Hajri, Hazare Nouari, Severine Wack, Marc Aprahamian
1 Tumor Biology and Gene Therapy Dept., IRCAD-INSERM U375, Strasbourg, France.
Human pancreatic cancer is an extremely aggressive and incurable carcinoma because of its poor sensitivity to chemotherapy and/or radiotherapy Point mutations of the K-ras gene are detected in > 90% of human pancreatic cancers and may play an important role in tumorigenesis Antisense therapeutics with phosphorothioate oligonucleotides (PS ODN) and short interfering RNAs (siRNA) targeting specific gene expression represent a promising strategy for pancreatic cancer treatment The aim of the present work was to address a comparative study concerning the antitumor activity of synthetic PS-ODN and siRNA antisense targeting specific k-ras
mutation (k-Ras mut12) The siRNA was delivered as a synthetic
19-22 bases and as a recombinant vector for stable expression The
antitumor activity was evaluated (i) in vitro, in cell culture with
different human pancreatic cancer cell lines (BxPc3, Panc1, Capan1) The endogenous Ras expression was determined by RT-PCR and Western-blotting Antiproliferative and cytotoxic effects were
evaluated by [3H] thymidine incorporation and MTT tests (ii) In