681 Ad TβRIIδcyt Treatment Decreases Liver Steatosis and Fibrosis by Decreasing mRNA Expression of CB1 Cannabinoid Receptor and TGF β and Increasing MMP 1 Molecular Therapy Volume 19, Supplement 1,[.]
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Copyright © The American Society of Gene & Cell Therapy S261
ADENOVIRUS AND OTHER DNA VIRUS VECTORS III
CONCLUSION: To our knowledge, this is the fi rst study to
demonstrate effi cacy in a viral therapy against TNBC cells Our results
support that NV1066 can infect and kill TNBC As overexpression
of p-MAPK has been correlated with resistance to chemotherapy, the
down-regulation of p-MAPK by NV1066 suggests a potential role for
NV1066 as an adjuvant therapy in chemoresistant TNBC
679 Generation of Complex High-Capacity
Adenoviral Vectors with Modifi ed Capsids for
Applications In Vitro and In Vivo
Martin Hausl,1 Richard Voigtlander,1 Zsolt Ruzsics,1 Anja
Ehrhardt.1
1 Virology, Max von Pettenkofer-Institut / LMU, Munich, Germany.
First-generation adenoviral vectors (FG-AdVs) are easy to
produce and therefore represent the best option to analyse
capsid-modifi cations or new expression systems On the other hand
high-capacity adenoviral vectors (HC-AdVs) lacking all viral coding
sequences are the most attractive option for therapeutic approaches
due to reduced toxicity and immunogenicity and high capacity for
sequences of interest Therefore, there is a great interest in a method
for adoption and combination of vector-modifi cations as well as
expression systems evaluated in FG-AdVs towards HC-AdVs
Recently we developed a novel platform based on homologous
recombination of bacterial artifi cial chromosomes (BACs) allowing
arbitrary modifi cations On the basis of the plasmid pHCA-eGFP
encoding a HC-AdV genome containing an eGFP expression cassette
we showed smooth access to the platform exchanging regular plasmid
backbone with BAC backbone Full potential of BAC technology was
demonstrated utilizing a recombination pipeline based on alternating
resistances for rapid incorporation of 9 different DNA fragments into
one HC-AdV BAC The resulting BAC pBHCA-2indsys encodes
two expression cassettes enabling liver-specifi c and
mifepristone-inducible expression of Renilla luciferase as well as two expression
cassettes allowing doxycycline-inducible and stem cell-specifi c
expression of eGFP-Firefly Luciferase reporter construct For
generation of respective HC-AdV we currently utilize column-purifi ed
unmodifi ed helper-virus and a fi ber-modifi ed variant, constructed by
traceless exchange of the fi ber with fi ber chimera fi b5/35 Generated
HC-AdV-2indsys will be tested for functionality in hepatocytes and an
oct4-expressing cell line For in vivo applications we modifi ed hexons
of the helper-virus to allow repeated administrations Therefore, we
synthesized hexons with precisely exchanged hypervariable regions
(HVRs) with respective sequences of other serotypes (Ad12, Ad41,
Ad44, Ad48, Ad50) Additionally we generated modifi ed hexons by
complete exchanges of domains DE1 and FG1 containing sequences,
which encode the HVRs from Ad4, Ad7, Ad12, Ad13, Ad41 Western
blot analysis demonstrated intact interaction of modifi ed hexons
with adenoviral 100K-protein as well as effi cient nuclear uptake
and trimerization Subsequently we replaced the original hexon of a
helper-virus BAC with an eGFP/Fluc reporter cassette incorporated
in the E3 region with modifi ed hexon sequences Although rescue
of hexon modifi ed adenoviruses is challenging, we could show
successful reconstitution of 4 of the above mentioned hexon-modifi ed
helper-viruses containing precise as well as complete exchanges
of HVRs During amplifi cation we noted a prolonged time until
observing complete cytopathic effect, suggesting impaired virus
assembly Currently, purifi ed viruses are analysed in detail with
respect to titers (viral particles and transducing units), biodistribution
after intravenous injection in mice and potential to escape neutralizing
antibodies in human sera in neutralizing antibody assays
680 Dual-Expression Adenoviral Vectors in Vaccine Development
Xiangyang Zhou,1 Juliana C Small,1 Dongming Zhou,1 Raj K Kurupati,1 Heng Chen,1 Ang Bian,1 Hildegund C Ertl.1
1 The Wistar Institute, Philadelphia, PA.
We have previously reported on a dual-expression chimpanzee adenoviral vector (AdC7) that we developed to express nucleoprotein (NP) of infl uenza A virus in E1 locus under the control of CMV promoter and gag of SIV in E3 locus under the control of chicken beta actin (CB) promoter Both expression cassettes contained the
CMV enhancer as well By using the vectors in vitro and in vivo,
the results showed expression and immunogenicity of the transgene product encoded by the cassette in E1, to be low In order to enhance immunogenicity, the vectors were modifi ed by removing the CMV enhancer from either cassette A vector that contained the CMV enhancer and promoter in front of the NP gene in E1 locus and SIVgag under control of CB promoter without CMV enhancer in E3 locus showed signifi cantly improved immunogenicity for both transgene products The data suggest that duplication of even small sequences such as an enhancer has to be avoided in the design of dual expression adenoviral vectors, which could be very useful in vaccine development
681 Ad-T βRII∆cyt Treatment Decreases Liver Steatosis and Fibrosis by Decreasing mRNA Expression of CB1 Cannabinoid Receptor and TGF- β and Increasing MMP-1
Mayra G Mena,1 Ana S Sandoval,1 Jazmin Santillan,1 Juan Armendariz.1,2
1 Institute of Molecular Biology in Medicine and Gene Therapy, CUCS, Universidad de Guadalajara, Guadalajara, Jalisco, Mexico; 2 O.P.D Hospital Civil de Guadalajara “Juan I
Menchaca”, Guadalajara, Jalisco, Mexico.
Introduction: Transforming growth factor β (TGF-β) is the main profi brogenic cytokine; consequently strategies to block its signal pathway have been developed Gene therapy using a truncated receptor type II for TGF-β (TβRII∆cyt) had showed to reduce hepatic fi brosis; but its effect on the expression of cannabinoid receptors (CB1 and CB2) which participate in the progression of
liver cirrhosis or steatosis has not been elucidated Objective:
Evaluate the effects of Ad-TβRII∆cyt on fi brosis, steatosis and gene expression of profi brogenic molecules and cannabinoid receptors
in rat liver Material and Methods: Cirrhosis was induced in male
rats administrating carbon tetrachloride (CCl4) for 8 weeks Cirrhotic rats were divided into groups (n=6): cirrhotic control, cirrhotic with therapeutic gene (Ad-TβRII∆cyt) and cirrhotic with irrelevant gene (Ad-GFP) At the 4th week of CCl4 intoxication, 2x1010 pi/kg of Ad-TβRII∆cyt or Ad-GFP was administered via iliac vein Sacrifi ce was made at day 2, 3 and 28 after treatment Fibrosis index, steatosis, hydroxiproline content, gene expression of TGF-β, collagen α1 (I), PAI-1, MMP-1, CB1 and CB2 were analyzed, as well as, ALT and
AST serum levels Results: Treatment with Ad-TβRII∆cyt decreased 22%, 28% and 35% of fi brotic tissue; while liver steatosis was reduced 47%, 43% and 42%; at day 2, 3 and 28 respectively (p<0.001)
Ad-TβRII∆cyt suppressed expression of profi brogenic molecules: TGF-β (11-fold at day 28), collagen α1 (I) (11-fold at day 3 and 5-fold at day 28) (p<0.05) and PAI-1 (13-fold at day 2 p<0.05 and 19-fold
at day 3 p<0.01) and increased expression of MMP-1 (13.1-fold p<0.01) compared to cirrhotic rats CB1 gene expression showed a clear tendency to decrease (50% less expression) and CB2 mRNA levels increase to the double compared to cirrhotic controls, even
no statistical signifi cance was achieved Hydroxyproline content decreased 3 fold times (p<0.01) at day 28 after treatment compared
to cirrhotic group Liver function improved signifi cantly in the
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ADENOVIRUS AND OTHER DNA VIRUS VECTORS III
TβRII∆cyt-treated rats compared to cirrhotic group (AST; 490±126.4
versus 180±58.1 p<0.001 and ALT 469±290.2 versus 102±60.4
p<0.05) at day 28 Conclusion: Expression of TGF-β-truncated
receptor II prevents fi brosis progression in the liver and improves
liver function reducing gene expression of pro-fi brogenic molecules
(TGF-β, Col-1, PAI and CB1) Signifi cant reduction in steatosis
was also observed when Ad-TβRII∆cyt therapy was employed and
there was a tendency of CB1 to decrease, which suggest that the two
phenomena might be related
682 Development of a Virus-Associated
RNA-Deleted Adenovirus Vector
Mitsuhiro Machitani,1 Kazufumi Katayama,1 Fuminori Sakurai,1
Hayato Matsui,1 Tomoko Yamaguchi,2 Takayuki Suzuki,2 Kenji
Kawabata,1,2 Hiroyuki Mizuguchi.1,2,3
1 Biochemistry and Molecular Biology, Graduate School of
Pharmaceutical Sciences, Osaka University, Suita, Osaka,
Japan; 2 Laboratory of Stem Cell Regulation, National Institute of
Biomedical Innovation, Ibaraki, Osaka, Japan; 3 The Center for
Advanced Medical Engineering and Informatics, Osaka University,
Suita, Osaka, Japan.
A major limitation of the use of adenovirus (Ad) vectors is the innate
immune response, which causes infl ammatory cytokine production
and tissue damage To overcome this limitation, it is necessary to
develop safer Ad vectors that are less likely to induce this response
The adenoviral genome encodes two noncoding small RNAs,
virus-associated (VA)-RNA I and VA-RNA II, which are transcribed by
RNA polymerase III and promote adenovirus amplifi cation Recently,
we reported that VA-RNAs are produced in the cells transduced with
conventional fi rst-generation (E1-deleted) Ad vector (FG-Ad) and
trigger innate immune responses through intracellular nucleic acid
sensors In this study, we have developed a VA-RNA-deleted Ad
(Ad∆VR) vector from which the transcriptional control elements
of the VA-RNA-expression were deleted First, pAd∆VR-EGFP, a
plasmid encoding the PacI-fl anked Ad∆VR vector genome with an
EGFP-expression cassette in place of the E1 region, was digested
with PacI and transfected into 293 cells according to the conventional
method for preparing Ad vectors However, no propagation of the
Ad vector was observed Next, we prepared 293 cells inducibly
expressing VA-RNA I under the control of a tetracycline-dependent
H1 promoter (VR293 cells) VR293 cells with appropriate induction
of VA-RNA I-expression allowed the propagation of the Ad
∆VR-EGFP vector The Ad∆VR-EGFP vector showed effi cient transduction
in the cultured cells, comparable to that of the conventional FG-Ad
vector expressing EGFP Furthermore, although the FG-Ad vector
shows E1-independent replication in some cultured cells, the Ad∆VR
vector exhibited signifi cantly lower levels of viral replication than
the FG-Ad vector Given these results, the Ad∆VR vector may be a
safer alternative to the FG-Ad vector
683 Pre-Clinical Characterization of Endothelial
Cells as Carriers of Oncolytic Adenoviruses
Bart Thaci,1 Ilya V Ulasov,1 Maciej S Lesniak.1
1 Neurosurgery, The University of Chicago, Chicago, IL.
Background Cancer remains the second most common cause of
death in the developed world New targeted therapies are being tested
in clinical trials Oncolytic adenoviruses are one of the promising
new agents, but delivery to tumors is still unsatisfactory Systemic
delivery of oncolytic adenoviruses to tumors via cell based carriers
is very attractive and needs to be optimized In this work, we show
that endothelial cells can deliver fi ber modifi ed, survivin driven
adenovirus to glioma cells Material and methods Human umbilical
vein endothelial cells were screened for surface receptors via fl ow
cytometry Transduction effi cacy of fi ber modifi ed viruses (Ad
Luc-Pk7; Ad.Luc-RGD; Ad.Luc-5/3) was compared to wild-type
fi ber adenovirus by measuring luciferase expression Survivin and CXCR4 activity was analyzed via qRT-PCR Adenoviral replication was quantifi ed by measuring E1A region copy numbers and progeny titer after infection with replication competent CRAd.Survivin.5/3
To show that endothelial cells can deliver adenovirus to tumor cells: endothelial cells were infected with CRAd.Survivin.5/3 and washed thoroughly before plated in different ratios with U87 glioma cells
Viability was visualized via Crystal Violet assay Results Fiber
modifi cations increased adenoviral transduction 2.5-3.5 folds Longer incubation time (4h vs 1 h) during infection enhanced transduction four folds Survivin expression in endothelial cells was 20 fold higher than CXCR4 and was dependent on the presence of growth factors It dropped four fold when using starved media instead of growth factor replenished media The same did progeny titer levels, while E1A copy numbers were affected even more (up to 10 fold) Endothelial cells were able to deliver oncolytic adenovirus and effectively kill glioma
cells after co-culture Conclusion Endothelial cells can be used as cell
carriers for oncolytic adenoviruses Increasing expression of survivin
in endothelial cell can enhance adenovirus delivery
684 miR122 Targets Insertion in Wild-Type Serotype 5 Adenovirus To Ablate Liver Replication
Sergio Lavilla-Alonso,1 Erkko Ylösmäki,1 Kalle Saksela.1
1 Virology, Haartman Institute - University of Helsinki, Helsinki, Finland.
Adenovirus -based oncolytic therapies present important limitations regarding tissue selectivity and toxicity, an issue well-documented for the commonly used adenovirus serotype 5 (Ad5) Basic research has shown a specifi c micro-RNA (miR) pattern for many tissues, like the virtually exclusive expression of miR122 by liver We have shown that this feature can be utilized to selectively ablate adenovirus replication
in cells of hepatic origin by genomic insertion of miR122 targets to different reduce E1A gene expression In this study we have examined
in more detail the optimal number of miR122 targets, studied the cell type-specifi c inhibitory potential of miR122 via careful cell viability analyses, and extended these studies to more relevant experimental systems These results will provide a rational basis for proceeding
towards ex vivo and in vivo studies to test the applicability of this
system for clinical gene therapy purposes
685 TRAIL-Expressing Oncolytic Viral DNA/ Liposome Hybrid Vector for Lung Cancer Gene Therapy
Oh-Joon Kwon,1 Eunah Kang,1 Jaerim Kim,1 Kyungmin Nam,1
Sung Wan Kim,2,3 Chae-Ok Yun.1
1 Brain Korea 21 Project for Medical Sciences, Institute for Cancer Research, Yonsei Cancer Center, Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul, Republic
of Korea; 2 2Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, UT; 3 Department
of Bioengineering, College of Engineering, Hanyang University, Seoul, Republic of Korea.
Adenovirus (Ad) as cancer molecular therapeutics has been extensively exploited with modifi cation of Ad genetic information,
and administration methods in vivo for effective delivery However, delivery effi cacy in vivo has been limited due to Ad envelopment by
pre-existing neutralizing antibody, the liver uptake, and hepatotoxicity
of Ad In this study, as an alternative approach of cancer virotherapy, oncolytic viral DNA delivery via lipid envelopment was investigated for orthotopic lung cancer gene therapy For synergistic therapeutic effect, multifunctional oncolytic Ad DNA expressing TRAIL as a model of therapeutic protein was generated Lipid hybrid vectors encapsulating oncolytic Ad DNA, DOTAP:DOPE/H5mT-Rd19/