Molecular Therapy Volume 13, Supplement 1, May 2006CANCER-APOPTOSIS AND SUICIDE Combined intratumoral injection of TRAIL plasmid and survivin antisense ODN significantly supressed the gr
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CANCER-APOPTOSIS AND SUICIDE
Combined intratumoral injection of TRAIL plasmid and survivin
antisense ODN significantly supressed the growth of tumor
xenografts in nude mice as compared to TRAIL plamid alone
(0.31±0.05 vs 2.35 ±0.38 cm3, P <0.01) during a 4-week of
observation Conclusions: The findings indicate that survivin may
play a role in tumor cell resistance to TRAIL-induced apoptosis, at
least in part, through cell cycle regulation Manipulation of survivin
expression may sensitizes HCC to TRAIL-induced apoptosis This
approach may offer a novel approach in molecular therapy against
HCC for which no effective treatment is available for an advanced
stage
945 Combination Treatment of Adenovirus
Mediated HSV-Thymidine Kinase Suicide Gene
Therapy and Docetaxel in Bladder Cancer
Hideyuki Yamashita,1 Weiguo Jian,1 April Gillbert,1 Seth P
Lerner.1
1 Scott Depertment of Urology, Baylor College of Medicine,
Houston, TX.
Purpose: The objective of this study was to evaluate the combined
effect of adenovirus vector encoding Herpes simplex thymidine
kinase plus ganciclovir (GCV) suicide gene therapy and Docetaxel
(DTX) in human bladder cancer cell lines
Material & Methods: IC50 was determined for both Ad5F35TK
plus ganciclovir (GCV) or DTX monotherapy at 48, 72 and 96 h
after exposure to a serial log-fold dosing of Ad5F35TK or DTX in a
coxsackievirus-adenovirus receptor (CAR) positive human bladder
cancer cell line (5637) and a CAR negative cell line (TCC-SUP) Cell
growth inhibition was then assessed at 72 h after treatment with
Ad5F35TK+GCV or Ad5F35TK/GCV/DTX by MTT assay
Ad5F35 and DTX were given within 3, 6, 9, 12, and 24 hours of
each other then MTT assay was performed at 72 h The viability of
control cells was set as 100%, and viability in other groups was
calculated by comparing the optical density (OD) readings with the
control
Results: The inhibitory concentration of docetaxel to achieve 50%
cell death in 5637 cell line ranged from 0.002 to 0.008 ug/ml and
from 0.002 to 0.02 in TCC-SUP cell line at 48, 72 and 96 hours
Adding docetexal chemotherapy to Ad5F35TK gene therapy
resulted in 57% benefit in 5637 cell line (p<0.0001) and 39% benefit
in TCC-SUP cell line (p<0.005)
Conclusion: Combination of Ad5F35TK+GCV suicide gene
therapy with DTX in human bladder cancer cells was considerably
more active in vitro than with monotherapy This result suggests
that combination treatment of Ad5F35TK+GCV suicide gene
therapy with DTX has the potential to enhance clinical activity as
compared to monotherapy in human bladder cancer
946 Effect of Androgen Receptor Suppression
Using Dominant Negative Inhibition on
Castration-Resistant Prostate Cancer
Brian J Zeithaml,1 Mark Titus,2 Karin Haack,1 Adam Cockrell,1
Angela Ponguta,2 Elizabeth Wilson,2 James Mohler,3 Tal Kafri.1
1 Gene Therapy Center, University of North Carolina at Chapell
Hill, Chapel Hill, NC; 2 Linberger Comprehensive Cancer Center,
University of North Carolina Chape Hill, Chapel Hill, NC;
3 Roswell Park Cancer Institute, Buffalo, NY.
An American dies from CaP every 17 minutes Advanced prostate
cancer almost always responds to androgen deprivation therapy
but inevitably recurs as castration-resistant prostate cancer Recent
evidence suggests that the growth of most cases of castration-resistant
prostate cancer depends upon androgen receptor (AR) activation1
and that AR activation may be due to a combination of AR
hypersensitization2-6 and low levels of dihydrotestosterone and/or
testosterone (DHT/T), 7,8 intracellular testicular androgens produced by intracrine metabolism from cholesterol or adrenal androgens The role of AR in castration-resistant prostate cancer can be studied using an AR dominant negative mutant, ∆TR (Wilson unpublished) and CWR-R1, a castration-resistant prostate cancer cell line9 ∆TR contains a deletion in the NH2-terminal region that results in loss of the AR transactivating domain and inhibits wild type AR possibly due to dimerization and loss of binding of one or more coactivators Activation of ∆TR appears to require binding of DHT and/or T for interaction with and transactivational inhibition
of endogeneous AR Past evidence reveals that CWR-R1 cells express high levels of AR, grow in the absence of DHT/T when supplemented with EGF, and increase cell proliferation when supplied with DHT9 Suppressing AR using ∆TR delivered via lentiviral vectors tested the hypothesis that CaP recurrence can be delayed or prevented by interfering with AR function Lentiviral vectors achieved high level
AR-regulated gene product) expression, decreased plasmid luciferase expression from the MMTV promoter (an AR target promoter), decreased CWR-R1 cell proliferation in culture, and inhibited xenografted CWR-R1 tumor growth in castrated mice with DHT pellet implants (all unpublished) ∆TR must require higher amounts
of DHT or other cofactors than AR for activation since in vivo CWR-R1 tumor growth rates were slowed but not eliminated To address this question, ligand-independent shRNA will be used to
dimerization and effect CWR-R1 proliferation and in vivo tumor growth of the ∆TR-transduced and AR-targeted shRNA-transduced cells will further enlarge our understanding of the role of AR in castration-resistant prostate cancer
1Debes JD and Tindall DJ (2004) N Engl J Med 315, 1488-90 2Tapin ME et al (1995) N Engl J Med 332, 1392-8
3Shi X et al (2002) Cancer Res 62, 1496-1502
4Culig Z et al (1993) Mol Endocrinol 7, 1541-50
5Peterziel H et al (1995) Int J Cancer 63, 544-50
6Tan JA et al (1997) Mol Endocrinol 11, 450-9
7Mohler JL et al (2004) Clin Cancer Res 10, 440-48
8Titus MA et al (2005) Clin Cancer Res 11, 4365-71 9Gregory CW et al (2001) Cancer Res 61, 2892-8
947 Gene Directed Enzyme/Prodrug Therapy
of Human Glioma Xenografts Using Mutant Escherichia coli Cytosine Deaminase
Sergey A Kaliberov,1 Valentina Krendelchtchikova,1 Debbie Della Manna,1 Jeffrey C Sellers,1 Lyudmila N Kaliberova,1 Margaret
E Black,2 Donald J Buchsbaum.1
1 Department of Radiation Oncology, University of Alabama at Birmingham, Birmingham, AL; 2 Department of Pharmaceutical Sciences and the School of Molecular Biosciences, Washington State University, Pullman, WA.
Combined treatment using suicide gene therapy and radiation therapy has the potential to become a powerful new method of cancer therapy We have developed a non-replicative adenoviral vector encoding a mutant bacterial cytosine deaminase (bCD) gene harboring substitution of an alanine (A) for the aspartic acid (D) at position 314 in the CD protein (AdCD-D314A) which has a higher affinity for 5-fluorocytosine (5-FC) than wild type bCD (CDwt)
The purpose of this study was to evaluate cytotoxicity in vitro and therapeutic efficacy in vivo of the combination of AdCD-D314A
with the prodrug 5-FC and radiation treatment (RT) against human glioma The results of CD enzyme activity assays showed that the conversion of 3H-5-FC to 3H-5-FU was elevated 183.3, 205.6 and 102.2-fold in D54MG, U87MG and U251MG human glioma cells, respectively, for cells infected with 2 multiplicity of infection (MOI)
of AdCD-D314A compared to AdCDwt AdCD-D314A infection
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CANCER-TARGETED GENE THERAPY: OTHER VIRUSES AND NEW APPROACHES
also resulted in increased 5-FC-mediated cell killing The 50%
inhibitory concentration produced by 5-FC decreased by 7.8-fold
for D54MG, 32.9-fold for U87MG, and 8.1-fold for U251MG cells
infected with 10 MOI of AdCD-D341A in comparison with
AdCDwt Animal studies showed significant inhibition of tumor
growth of D54MG glioma subcutaneous xenografts by the
combination of AdCD-D314A/5-FC with RT as compared with
either agent alone, or with AdCDwt/5-FC plus RT The results
demonstrate that the combination of AdCD-D314A/5-FC with
radiation produces markedly increased cytotoxicity in cancer cells
in vitro and in vivo compared to AdCDwt These data indicate that
combined treatment with this novel mutant enzyme/prodrug therapy
and radiotherapy provides a promising approach for glioma therapy
CANCER-TARGETED GENE THERAPY: OTHER VIRUSES
AND NEW APPROACHES
948 Rapid Production of Specific Artificial
Meganucleases for Gene Correction in Patients
with Inherited Disease
Philippe Duchateau, Patrick Chames, Jean-Charles Epinat,
Arnould Sylvain, Julie Smith, Agnes Gouble, Christophe Perez,
Sylvestre Grizot, Frederic Paques
1 Research, Cellectis SA, Romainville, France.
Meganucleases are sequence-specific endonucleases recognizing
large (>14 bp) sequences Because of this unusual specificity,
meganuclease have emerged as powerful tools for genome engineering:
creation of double-strand breaks by meganucleases at specific loci
can be used to enhance homologous gene targeting by several orders
of magnitude in mammalian and plant cells Recently, such proteins
could be used to induce gene repair in mouse hepatocytes, paving
the way for a novel strategy for inherited diseases therapy based on
gene correction instead of complementation Thus, the generation of
meganucleases with tailored specificities is under intense
investigation A major issue is whether it is possible to obtain novel
dedicated endonuclease which levels of specificity compatible with
the high requirements of therapeutic applications
Homing endonucleases (HEs) are natural meganucleases encoded
by mobile genetic elements such as class I introns and inteins Their
exquisite specificity identifies them as ideal scaffolds to derive novel
meganucleases with engineered DNA binding domains HEs from
the LAGLIDADG family, the largest and most widespread family
of HEs, display two independent domains or units Moreover,
despite the lack of apparent additional modularity at the structural
level, we have identified separable functional subdomains within a
same LAGLIDADG domain binding distinct parts of the DNA
target Using a semi-rational approach, we have used a two steps
strategy to produce meganucleases cleaving natural genes In a first
step, we have derived hundreds of novel meganucleases with novel
specificities from I-CreI, a natural homodimeric homing
endonuclease In a second step, a combinatorial approach allows us
to assemble these I-CreI variants into novel meganucleases with
predictable cleavage site Importantly, these proteins have retained
high levels of specificity Examples of meganucleases with potential
therapeutic applications will be disclosed
949 Oncolytic Autonomous Parvovirus (rH1-yCD) Targeting with P4/Carcinoembryonic Antigen Chimeric Promoter for Virotherapy of Pancreatic Cancer
Soukaina Rejiba, Marc Aprahamian, Amor Hajri
1 Tumor Biology & Gene Therapy, IRCAD - INSERM U701, Strasbourg, France; 2 Tumor Biology & Gene Therapy, IRCAD -INSERM U701, Strasbourg, Finland; 3 Tumor Biology & Gene Therapy, IRCAD - INSERM U701, Strasbourg, France.
Background: Successful gene therapy depends on the
development of safe and efficient vectors for gene expression targeting We investigated the use of a rH1CEA-yCD recombinant parvoviral vector, bearing the yeast cytosine deaminase (yCD) suicide gene The parvoviral P4 promoter was modified by the addition of
a 319 bp Carcinoembryonic Antigen (CEA) tissue-specific promoter sequence, to improve the oncotropic and oncolytic intrinsic properties of the autonomous H-1 parvovirus
Material && Methods: To this end, we explored in vitro, the
ability of this rH1CEA-yCD virus to infect CEA-producing cell line (BxPc3) and CEA-nonproducing cell lines (HA-HPC and HeLa) The suicide gene expression was evaluated by RT-PCR and western
blotting The cytotoxicity effect was measured by MTT test For in
vivo experiments, we evaluated the oncolytic efficacy of this new
recombinant parvovirus on tumor growth upon subcutaneous/ peritoneal pancreatic carcinomatosis models in nude mice
Results: Our data indicate that rH1CEA-yCD infect efficiently
the tumor cell lines in the same manner as its parental rH1-yCD RT-PCR and western blot analyses showed a down-regulation of the expression of both the NS1 (viral gene) and the yCD (suicide gene) brought by rH1CEA-yCD in HA-HPC and HeLa cell lines, when compared with rH1-yCD With regard to its cytotoxic effect, rH1CEA-yCD/ 5-FC treatment was incapable to induce cell death
in those cell lines, but was as efficient as rH1-yCD to kill BxPc3 cell
line In vivo, infection with rH1CEA-yCD led to significant
retardation of BxPc3 tumor growth both in subcutaneous and intraperitoneal disseminated pancreatic models The survival rate of animals bearing CEA-producing cell lines (BxPc3) was improved (p<0.001) However, there was no effect on HA-HPC tumor models
Conclusion: In summary, we have developed a new targeted
recombinant parvovirus where the parvoviral oncolytic effect and the suicide gene expression are tightly restricted to tumor cells with CEA tumor marker promoter signaling
Cancer
Maria Rajecki,1,2 Lotta Kangasniemi,1,2 Mikko Tenhunen,2 Anna Kanerva,1,2,3 Renee A Desmond,4 Ulf-Hakan Stenman,5 Martti Y Ala-Opas,6 Akseli Hemminki.1,2
1 Rational Drug Design Program, University of Helsinki, Helsinki, Finland; 2 Department of Oncology, HUCH, Helsinki, Finland;
3 Department of Obstetrics and Gynecology, HUCH, Helsinki, Finland; 4 Comprehensive Cancer Center, Biostatistics and Bioinformatics Unit, University of Alabama at Birmingham, Birmingham, AL; 5 Department of Clinical Chemistry, HUCH, Helsinki, Finland; 6 Department of Urology, HUCH, Helsinki, Finland.
Background: Hormone refractory metastatic prostate cancer
(PrCa) remains incurable Conditionally replicating adenoviruses (CRAds) are attractive therapeutic agents for cancer due to their innate capacity for oncolysis Transduction efficacy of native