1174 Comparison of E1a Modifications To Achieve Tumor Cell Selective Viral Replication and Toxicity Molecular Therapy �������� ��� ���� ���������������� �������� ��� ������®������������ �!����� ����"�[.]
Trang 1Molecular Therapy Vol 7, No 5, May 2003, Part 2 of 2 Parts
1172 Minimally Invasive In Vivo Imaging of an
Oncolytic Herpes Simplex Virus Mutant
Expressing Green Fluorescent Protein
Brendon M Stiles,1 Prasad Adusumilli,1 Amit Bhargava,1
Stanziale F Stephen,1 Richard J Wong,1 Valerie W Rusch,1 Fong
Yuman.1
1 Department of Surgery, Memorial Sloan-Kettering Cancer
Center, New York, NY, United States.
Introduction: Herpes simplex virus-one (HSV-1) oncolytic
therapy and viral-based gene therapy are promising treatment
modalities against cancer A replication-competent HSV-1 mutant,
NV1066, carries a transgene for enhanced green fluorescent protein
(EGFP), which is commonly used in vitro as a reporter/marker gene.
While techniques for assessing EGFP expression in cell culture or
animal models are readily available, methods for detecting regional
distribution and activity in man are needed The purpose of this
study was to determine whether: 1) NV1066 is cytotoxic to a lung
cancer cell line in vitro and in vivo, 2) EGFP is a detectable marker
of viral infection, and 3) EGFP expression and viral distribution
could be localized in an animal model of pleural cancer using a
minimally invasive system
Methods: A549 human lung cancer cells were infected in vitro at
multiplicities of infection (MOI: number of viral particles per tumor
cell) of 0.1 and 1.0 Viral replication was determined by plaque
assay, cell kill by LDH release assay, and EGFP expression by flow
cytometry To mimic pleural spread of lung cancer, 1 x 106 A549
cells were injected percutaneously into the pleural cavity of athymic
mice Mice were treated day 1 following tumor implantation with
intrapleural injection of either NV1066 (1 x 107 plaque forming
units) or PBS, then sacrificed at 4 weeks to determine treatment
effect For EGFP localization studies, mice were treated with
NV1066 day 14 after tumor implantation Mice were examined 48
hours later for EGFP expression in tumor deposits, using an Olympus
thoracoscopic/laparoscopic system equipped with the appropriate
fluorescent filters
Results: NV1066 progressively replicated in and killed tumor
cells Peak viral titers demonstrated a 54-fold increase over the initial
infecting dose By day 9, cell kill at MOIs of 0.1 and 1.0 was 83%
and 96% respectively (p < 01, t-test) EGFP expression in infected
cells was initially dose-dependent, but increased over time with
viral replication By day 5, over 95% of all remaining live cells
expressed EGFP at both MOIs (p < 01, t-test) Treatment with
NV1066 decreased pleural disease burden after four weeks Average
chest wall nodule count was 71 in the PBS group compared to 8 in
the NV1066 group (p < 01, t-test) Following intrapleural
administration of NV1066, EGFP was easily visualized in tumor
deposits in the pleural cavity using a thoracoscope with a fluorescent
filter system Expression could be used to identify tumor deposits
as small as 2 mm in diameter EGFP expression and viral localization
to tumor were confirmed by histopathology
Conclusions: NV1066 has significant oncolytic activity against
A549 lung cancer cells in vivo Expression of EGFP in infected cells
can be used to localize the virus and helps to identify tumor foci By
incorporating fluorescent filters into thoracoscopes, laparoscopes,
or other endoscopic systems, a minimally invasive means of detection
and localization of viral therapy may be developed, using the EGFP
marker gene The technique reported here can be applied clinically
in many oncolytic viral or gene therapy trials to monitor viral
distribution and activity in vivo.
1173 Selection and Application of Chronic Lymphocytic Leukemia Binding Ligands from Peptide-Presenting Phage Libraries
Satoshi Takahashi,1 Hoyin Mok,2 Frank C Marini III,3 Michael Andreef,3 Malcolm K Brenner,1 Michael A Barry.1,2
1 Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX; 2 Department of Bioengineering, Rice University, Houston, TX; 3 Department of Molecular Hematology and Therapy, M.D Anderson Cancer Center, Houston, TX.
Previous work with random phage libraries demonstrated that cell binding peptide ligands can be selected from phage libraries by binding directly to cells in culture (Barry et al Nat Med
2(3):299-305 1996) or by binding to vascular cells in vivo (Pasqualini et al Nature 380: 364-366) One goal is to identify ligands that bind directly to human cancer cells to use these with gene therapy vectors
to mediate gain of function transduction in vitro or for the direct targeting in vivo To test this application, we have selected peptide-presenting phage libraries against human prostate and breast cancer cell lines and against primary patient chronic lymphocytic leukemia (CLL) cells In this work, libraries with large peptides have always out-competed libraries with smaller peptides and the build up of consensus peptide motifs is unusual To provide cell-binding ligands for ex vivo gene therapy and CLL-targeting ligands for in vivo drug and gene therapy, we have selected 44 20-mer peptides from peptide-presenting phage libraries by panning against patient primary CLL cancer cells 29 of the selected peptides were assayed for cell binding Fourteen of the selected peptides bound CLL, B, T, and monocyte cells, six bound only CLL and B cells, and one peptide bound only
B cells However, eight of the selected peptides were CLL specific The degree if specificity of the peptides was highly dependent on the target cell used for selection, with some patient’s cells producing highly specific peptides while other’s generating largely promiscuous peptides When peptides were tested out of the context of phage, synthetic peptide 1-5 was able to functionally re-target adenoviral vectors for increased ex vivo gene delivery to primary CLL cells These data demonstrate the ability to identify cancer-targeting peptides by selection of phage libraries against primary human cancers This work also emphasizes the importance of patient to patient heterogeneity in cancer cells for the identification of targeting peptides and their functional application for gene therapy vector targeting
1174 Comparison of E1a Modifications To Achieve Tumor Cell Selective Viral Replication and Toxicity
Harald Sauthoff,1 Teona Pipiya,1 Sheila Heitner,1 William Chang,1 Shu Chen,1 William N Rom,1 John G Hay.1
1 Medicine, NYU School of Medicine, New York, NY, United States.
Replicating adenoviral vectors have the capability of multiplying
up to a thousand fold in the target cell and may have a tremendous potential for cancer therapy However, restricting viral replication
to cancer cells is of great importance to limit toxicity The adenoviral E1a protein binds to and modifies Rb and p300 functions, forcing resting cells into S-phase and creating an environment suitable for viral replication It has been proposed that modifications of the E1a protein that impair Rb- or/and p300-binding prevent S-phase induction in normal cells resulting in selective viral replication in tumor cells However, it remains unclear which of several possible E1a modifications would confer the most protection to normal cells without compromising the oncolytic effect of the vector The purpose
of this work was to determine the best E1a modification to target a replicating adenovirus to cancer cells The evaluated vectors contained the following E1a modifications 1 Deletion of the E1a domain that binds to Rb (dl121-128) 2 Deletion of the Rb binding domain plus
Trang 2Molecular Therapy Vol 7, No 5, May 2003, Part 2 of 2 Parts
Copyright ®The American Society of Gene Therapy
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deletion of the N-terminal region that binds to p300
(dl4–25/121-128) 3 Deletion of the E1a domain that binds to the transcriptional
adapter motif (TRAM) in p300 (dl65-68) 4 Deletion of the Rb
binding domain plus deletion of the domain that binds to TRAM in
p300 (dl65-68/121-128) 5 Deletion of the E1a core binding domain
to Rb plus deletion of an adjacent region which functions
independently of Rb binding in the prevention of cell-cycle arrest
(dl121-138)
Results: Deletion of the domain that binds to Rb plus deletion of
the region that binds to p300 (dl4–25/121-128) were required to
effectively block S-phase induction in growth arrested normal cells
In three growth arrested normal cell types, a small decrease in new
virus production was seen for all E1a-modified viruses (except
dl121-138) without correlation to S-phase induction All viruses were
equally cytotoxic to cancer cells, as compared to wild-type virus
Only dl65-68 and dl4–25/121-128 showed evidence of decreased
toxicity in at least 2 of 3 normal cell types
Conclusion: Deletion of only the Rb-binding domain of E1a in a
replication-competent adenovirus does not effectively block S-phase
induction or reduce toxicity to normal cells Deletion of the Rb
binding domain plus deletion of the region that binds to p300 (dl4–
25/121-128) or deletion of the E1a domain that binds to the
transcriptional adapter motif (TRAM) in p300 (dl65-68) is
associated with reduced toxicity to some normal cell types, probably
by a mechanism that is independent of the ability of the virus to
induce S-phase
1175 Oncolytic Herpes Simplex Virus-1 G207
Induces Cyclooxygenase-2 Gene Expression by
Activation of the Mitogen Activated Protein
Kinase Pathway
Teresa H Kim,1 Amit Bhargava,1 Brendon M Stiles,1 Richard J
Wong,1 Yuman Fong, 1Andrew J Dannenberg.2
1 Department of Surgery, Memorial Sloan-Kettering Cancer
Center, New York, NY; 2 Department of Medicine, New York
Presbyterian Hospital-Cornell, New York, NY.
Introduction: Oncolytic viral therapy stimulates various
mediators of inflammation Increased cyclooxygenase-2 (COX-2)
levels are found in inflammatory as well as neoplastic states Recent
investigations have found enhanced antitumor effects combining
COX-2 inhibitor with various other cancer treatment modalities
The purpose of this study was to investigate the effects of G207, an
oncolytic herpes simplex virus type-1 (HSV-1) on COX-2 levels
and to investigate the effects of combination therapy
Methods: RAW 264.7, mouse macrophage cells, were treated
with G207 (multiplicity of infection 1.0 ) and harvested at 6, 12,
and 24 hours post infection Total cellular protein and RNA were
isolated for Western and Northern blotting respectively PGE2 levels
were measured using enzyme immunoassay PD98059,
extracellular-regulated protein kinase 1 and 2 (ERK1/2) inhibitor and SB202190,
p38 mitogen-activated protein kinase (MAPK) inhibitor, were added
to G207 treated RAW cells and cellular protein was harvested for
COX-2 Western blotting analysis 24 hours post infection
HTB-147, human pancreatic adenocarcinoma cells, were implanted in the
flanks of nude mice and treated with intratumoral G207 injection (5
x 106plaque forming units) alone or in combination with celecoxib
feed (150 parts per million powder chow)
Results: There was an increase in COX-2 protein levels with
G207 treatment seen by Western blot analysis (Figure 1)
Measurement of PGE2 levels showed a 10-fold increase in production
by cells treated with G207 Northern blot showed an increase in
COX-2 mRNA levels in G207 treated RAW cells PD98059 and
SB202190 both blocked the G207 induced increase in COX-2 protein
level (Figure 2) Pancreatic flank tumor model showed smaller average
tumor volume in the combination group as compared to the each of the single treatment groups 28 days post treatment (combination vs G207, p<0.05; combination vs celecoxib, p<0.02)
Conclusions: Oncolytic HSV-1 therapy stimulates COX-2 and
induction involves the MAPK pathway with increase in COX-2 gene transcription Combined therapy in a pancreatic animal model showed that HSV-1 oncolytic viral therapy and COX-2 inhibitor can be given together to achieve greater antitumor effect
1176 Probasin-Targeted Replication-Competent Retrovirus Vectors Achieve Highly Efficient and Prostate Cancer-Specific Gene Transfer In Vivo
Makoto Ohori,1 Eiji Kikuchi,1 Silvia Menendez,1 Christopher R Logg,2 Gloria Juan,3 Robert Matusik,4 Carlos Cordon-Cardo,3 Noriyuki Kasahara,2 Bernard H Bochner.1
1 Urology, Memorial SLoan-Ketetring Cancer Center, New York, NY; 2 Medicine, UCLA Geffen School of Medicine, Los Angeles, CA; 3 Pathology, Memorial SLoan-Ketetring Cancer Center, New York, NY; 4 Urologic Surgery, Vanderbilt University, Nashville, TN.
Objective: Replication-competent retrovirus (RCR) vectors
derived from MLV are highly selective for rapidly dividing cancer cells, and can achieve replicative spread and gene transfer throughout
entire solid tumors (Logg et al., 2001, Hum Gene Ther 12: 921) To
further improve the efficiency and safety of this system, we have developed a RCR vector that is transcriptionally targeted to prostate cancer (PCa) cells by a synthetic probasin promoter, and we have recently demonstrated its high transduction efficiency and highly
prostate cell-specific replication in vitro (Logg et al 2002, J.Virol., 76: 12783) In the present study, we evaluated the in vivo
transduction efficiency and tissue-specificity of the probasin-RCR vector using a variety of tumor cell types
M&&M: Tumors were established by subcutaneous injection of 2
receptor-neg PCa), NMU (androgen receptor-posi mammary ca) and 5637 (bladder ca) cells into the anterior flanks of 8-week-old nu/
nu BALB/c mice By 3 to 4 weeks later, the tumors had grown to 1.5 cm³, at which time they were injected with 6 x 10³ PFU of probasin-targeted RCR vector encoding GFP marker gene, non-probasin-targeted RCR vector encoding GFP as a positive control, or vehicle control The subcutaneous tumors were surgically removed at various time points and examined by immunohistochemical (IHC) staining for GFP expression
Results: None of 16 control tumors (5637 n=7, NMU n=4,
PC-3 n=PC-3, LNCaP n=2) demonstrated GFP expression following exposure to vehicle Of 33 tumors injected with non-targeted positive control RCR, 27 (82%) demonstrated GFP expression regardless of cell type (5637 8/8, NMU 9/11, PC-3 5/9, LNCaP 5/6), as expected Out of 9 LNCaP tumors injected with probasin-RCR, all were positive for GFP, with up to 70% transduction levels observed in