313 Liver Transduction and Brain Safety of an Antifibrogenic shRNA for Cannabinoid Receptor CB1 in a Model of Liver Cirrhosis Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The Americ[.]
Trang 1Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
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ADENOVIRUS VECTORS AND OTHER DNA VIRUS VECTORS II
Transposase Hybrid-Vector System To Treat von
Willebrand Disease
Manish Solanki,1 Wenli Zhang,1 Zsuzsanna Izsvak,2 Simon F De
Meyer,3 Karen Vanhoorelbeke,3 Anja Ehrhardt.1
1 Institute of Virology and Microbiology, Private University
Witten-Herdecke, Witten, North Rhine-Westphalia, Germany;
2 Max Delbrück Center for Molecular Medicine, Berlin, Germany;
3 Laboratory for Thrombosis Research, KU Leuven Kulak, Kortrijk,
Belgium.
Von Willebrand factor (VWF), a multimeric plasma protein, has a
crucial role in initiating primary hemostasis It binds to the damaged
vessel wall and attracts platelets and platelet adhesion is followed by
platelet aggregation which eventually leads to cessation of bleeding
Genetic defects in the VWF gene results into the bleeding disorder
von Willebrand disease (VWD) While administration of
plasma-derived VWF/FVIII is an effective therapy for treatment of severe
type 3 VWD, it has clear limitations such as a short term effect, high
cost and risks of contamination
Monogenetic diseases, like VWD, often present an ideal target
for gene therapy treatment potentially leading to a lifelong cure As
the VWF cDNA is of up to 8.4kb in size, high-capacity adenovirus
(HCAdV) is a desirable choice of vector because of its large transgene
carrying capacity up to 36kb To achieve lifelong treatment, we
have developed a hybrid-vector system which enables transgene
delivery and its subsequent integration into the host genome using
hyperactive Sleeping Beauty (SB) transposases HSB5 and SB100X
In this study we aimed at developing HCAdV encoding VWF under
the control of a liver-specifi c promoter fl anked by SB inverted repeats
and Flpe recombinase recognition sites to achieve VWF cDNA host
chromosomes integration
To clone the VWF in the HCAdV genome, a recombineering
technology based on a bacterial artifi cial chromosome (BAC) carrying
the HCAdV genome (B-HCAdV) was applied in combination with
counter selection marker galK (galactokinase) Initially, the counter
selection marker, fused with kanamycin (galK-Kan), was fl anked
with both ends of the VWF transgene using overlap PCR In fi rst
recombineering step the overlap PCR product was inserted into
B-HCAdV followed by counter selection which replaced
galK-Kan with VWF transgene The fi nal clone pB-HCAdV-VWF was
confi rmed by diagnostic restriction enzyme digests and sequencing
For HCAdV production the viral genome from pB-HCAdV-VWF
was linearized and transfected into 116 producer cell line for HCAdV
Reconstitution and amplifi cation of HCAdV encoding VWF
(HCAdV-VWF) was successful which was monitored by eGFP expression, also
expressed from HCAdV-VWF By using a similar strategy, HCAdV
carrying VWF fl anked by FRT sites for FLP recombination and SB
transposase inverted repeats (pB-HCAdV-VWF-IR-FRT) was cloned
In summary, we have successfully produced HCAdV carrying
VWF gene (HCA-VWF) and we are reconstituting
HCA-VWF-IR-FRT that will be used for VWF genomic integration using a second
vector encoding hyperactive transposase SB100X We are currently
testing whether VWF protein is expressed from the vector
HCAdV-VWF Moreover, we are evaluating FLP mediated circularization of
the VWF transgenes and transposition from HCAdV-VWF-IR-FRT
in the presence of a vector encoding Flp recombinase and SB100X
In the near future we will explore our novel therapeutic approach in
a mouse model for curing VWD
Increase HDAd-Mediated Hepatocyte Transduction Through Inhibition of Vector Uptake By Kupffer and Sinusoidal Endothelial Cells
Pasquale Piccolo,1 Pratibha Mithbaokar,1 Patrizia Annunziata,1 Nicola Brunetti-Pierri.1,2
1 Telethon Institute of Genetics and Medicine, Naples, Italy;
2 Translational Medicine, Federico II University, Naples, Italy.
Helper-dependent adenoviral (HDAd) vectors can mediate long-term, high-level transgene expression from transduced hepatocytes with no chronic toxicity However, the vector therapeutic index is narrow because of an acute toxic response with potentially lethal consequences elicited by high vector doses Kupffer cells and liver sinusoidal endothelial cells (LSECs) are major barriers to effi cient hepatocyte transduction We recently identifi ed scavenger receptor type A (SR-A) and scavenger receptor expressed on endothelial cells type I (SREC-I) as HDAd receptors on both Kupffer cells and LSECs in vivo By phage display, we have now developed two small peptides that block SR-A (PP1) and SREC-I (PP2) mediated vector uptake and result in increased effi ciency of hepatocyte transduction
by HDAd Pre-incubation of J774 macrophages with either PP1 or PP2 before HDAd infection signifi cantly reduced viral vector uptake Fluorocrome conjugated PP1 and PP2 injected intravenously into C57BL/6 mice co-localized with both CD68 and CD31 suggesting that both peptides bind SR-A and SREC-I on Kupffer cells and LSECs Compared to saline pre-treated animals, intravenous administrations
in C57BL/6 mice of PP1 and PP2 peptides prior to the injection
of an HDAd bearing the alpha-fetoprotein reporter gene under the control of a liver-specifi c promoter resulted in a signifi cant 3.7- and 2.9-fold increase of hepatic transgene expression, respectively The enhancement of transgene expression was confi rmed by increased number of beta-galactosidase positive liver cells in mice injected with HDAd-CMV-LacZ after PP1 and PP2 pre-treatment Serum IL-6 was moderately increased in mice pre-injected with both peptides prior
to HDAd compared to control mice pre-treated with saline and was not associated with increased Kupffer cell mortality, as shown by hepatic CD68 staining In summary, we developed small peptides that increase signifi cantly hepatocyte transduction effi ciency, improve the HDAd therapeutic index, and have potential for clinical applications
Antifi brogenic shRNA for Cannabinoid Receptor CB1 in a Model of Liver Cirrhosis
Adriana Díaz-Rivera,1 Juan Armendáriz-Borunda,1 Leonel García-Benavides,2 Ana Sandoval-Rodríguez.1
1 Molecular Biology and Gene Therapy Institute, Guadalajara, Mexico; 2 Institute of Experimental and Clinical Therapeutics, Guadalajara, Mexico.
Introduction: Pharmacological antagonist of CB1 has demonstrated anti-fi brogenic effects in different models of liver cirrhosis but, with important adverse side effects Then, gene therapy with a shRNA for CB1 delivered by hidrodinamic transfection or adenovirus may have the advantage of hepatic tropism, reducing possible side effects and increasing effi ciency of transduction Also, in liver fi brosis levels of TGF-b1, Col I and α-SMA produced by activated Hepatic Stellate Cells are increased
Objective: Demonstrate antifi brogenic effects of liver expression
of a shRNA against CB1 and generate a replication incompetent adenovirus that transiently expresses this shRNA-CB1
Materials and methods: An shRNAs against CB1receptor was designed to align in position 1232 of the mRNA of CB1 using Block
iT RNAi Designer software (Invitrogen) This shRNA was cloned into the expression plasmid pENTR™/U6 to generate pshRNA-CB1 The effectiveness of the shRNAs was evaluated by inhibition of mRNA
Trang 2Molecular Therapy Volume 22, Supplement 1, May 2014
Copyright © The American Society of Gene & Cell Therapy S121
ADENOVIRUS VECTORS AND OTHER DNA VIRUS VECTORS II
and protein level of CB1 in an experimental model of liver cirrhosis
induced by CCl4 Safety was evaluated monitoring CB1 expression
in brain tissue
3 mg/kg of pshRNA-CB1 were administrated via iliac vein using
hydrodynamic injection to cirrhotic rats mRNA of TGF-b1, Col 1
and α-SMA; AST and ALT serum levels and percentage of fi brotic
tissue were evaluated Then, this sequence was used to generated
an Adenovirus backbone by homologous recombination with pAd/
BLOCK-iT ™ DEST
Results: Hydrodynamics-based transfection of shRNA-CB1 via
iliac vein in CCl4-cirrhotic rats allows effi cient delivery to the liver;
silencing CB1 mRNA (76%; p>0.05) and protein (56%; p>0.05)
On the other hand, brain expression of CB1 mRNA presents no
difference versus control, indicating a safe administration Using this
treatment, fi brogenic molecules like TGF-b1, Col I, α-SMA reduced
(p<0.05) 60%, 47% and 77%; respectively Also, fi brosis analyzed
by morphometric analysis diminishes 49% (p<0.05) compared
to untreated controls Backbone of denovector was generated by
homologous recombination between the attL and attR regions of
pshRNA-CB1-B and pAd / BLOCK-iT ™ DEST mediated by the
Excisionase
Conclusions: The pshRNACB1-B that aligns in position 1232 of
the CB1-mRNA demonstrates effi cient gene and protein silencing
of CB1; showing no collateral effects in brain expression This
treatment reduces RNA levels of key molecules involved in hepatic
fi brosis, and diminishes fi brotic tissue Homologous recombination
between pshRNA-CB1-B and pAd / BLOCK-iT ™ DEST allows
the generation of an adenovirus backbone, a vector with elevated
hepatic tropism when administrated systemically Also, Adenovector
production allows high titers and relatively safety to be used in clinical
scenarios of liver cirrhosis
Among Species C Adenovirus Vectors
Jie Tian,1 Rituparna Moitra,1 Zhili Xu,1 Qi Qiu,1 Donna J Palmer,2
Philip Ng,2 Andrew P Byrnes.1
1 Division of Cellular and Gene Therapies, Center for Biologics
Evaluation and Research, Food and Drug Administration,
Bethesda, MD; 2 Molecular and Human Genetics, Baylor College
of Medicine, Houston, TX.
Most studies of adenoviral gene therapy have used vectors based
on human adenovirus type 5, and these Ad5 vectors transduce liver
relatively effi ciently after i.v injection in mice Ad5 liver transduction
depends on the ability of Ad5 to interact with coagulation factor X
(FX), which shields Ad5 from attack by the classical complement
pathway Ad5 has a high degree of homology to the three other Ad
serotypes in species C: Ad1, Ad2 and Ad6 Studying gene therapy
vectors derived from these four related serotypes may give general
insight into how Ad vectors transduce the liver and how liver
transduction is affected when virions are opsonized by plasma proteins
such as coagulation factors, natural antibodies and complement In
the current study, liver transduction was compared in various mouse
strains using helper-dependent vectors based on Ad1, Ad2, Ad5
and Ad6 In addition, we examined the ability of these serotypes
to resist neutralization by naive mouse serum in vitro Results: In
Balb/c mice, liver transduction was highest with the Ad6 vector
However, in C57BL/6 mice liver transduction was highest with
the Ad5 vector Interestingly, liver transduction with Ad1 and Ad2
vectors was relatively poor in both strains of mice, especially for Ad2
Experiments with antibody-defi cient and complement-defi cient mice
suggested that some of the differences among serotypes are related
to different abilities to resist natural antibodies and complement
Experiments where Ad vectors were mixed with nạve mouse serum
confi rmed that FX protects Ad5 from neutralization, as previously
reported Ad6 was relatively resistant to neutralization by mouse
serum, and interestingly this resistance did not depend on FX Ad2,
on the other hand, was susceptible to neutralization by mouse serum
regardless of the presence or absence of FX Conclusions: Even
though species C adenoviruses Ad1, Ad2, Ad5 and Ad6 are closely related, vectors based on these four serotypes show signifi cant differences in liver transduction after i.v injection in mice In vitro and in vivo experiments indicate that at least some of these differences are due to serotype-dependent interactions with plasma opsonins
Stellate Cells by Anti-EGF-Receptor scFv-sTRAIL Fusion Protein
Mohammad Arabpour,1 Klaas Poelstra,2 Wijnand Helfrich,3 Edwin Bremer,4 Hidde J Haisma.1
1 Pharmaceutical Gene Modulation, University of Groningen, Groningen, Netherlands; 2 Drug Targeting and Toxicology, University of Groningen, Groningen, Netherlands; 3 Surgery, University Medical Center Groningen, Groningen, Netherlands.
Background: Progressive liver fi brosis is the result of chronic liver
injury and characterized by excessive accumulation of extracellular matrix that may result in liver failure Activated Hepatic Stellate Cells are known to play a central role in this process and their elimination
is a crucial step towards the resolution and reversion of liver fi brosis Here we investigated the potential application of an anti-Epidermal Growth Factor receptor scFv antibody-Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (scFv425-sTRAIL) fusion protein
in the targeted elimination of activated Hepatic Stellate Cells
Methods: Activated hepatic stellate cells (LX2 cells) were treated
by Adenovirus-derived scFv425-sTRAIL to evaluate its effect on the viability and extracellular matrix production of this type of cells
Results: In vitro treatment of activated hepatic stellate cells
with scFv425-sTRAIL induced a signifi cant reduction in viability (up to 100% reduction) and ECM production (60% reduction), yet
no signifi cant effect was observed on hepatic parenchymal cells Blockage of the Epidermal Growth Factor Receptor by a monoclonal antibody signifi cantly reduced the effectiveness of scFv425-sTRAIL
in activated hepatic stellate cells, while a reduced effectivity was also observed after inhibition of the caspase pathway
Conclusion: Evidence is presented for the successful application
of the scFv425-sTRAIL fusion protein in the targeted elimination of activated HSCs via EGF-R and simultanous activation of the caspase pathway scFv425-sTRAIL may thus represent a new therapeutic compound against liver fi brosis
Adenovirus Variants That Incorporate Human Metallothionein Into Protein IX for Analysis of Biodistribution
Lei Liu,1 Buck E Rogers,2 Bing CHeng,1 Natalia Aladyshkina,1 David T Curiel,2 J Michael Mathis.1
1 Gene Therapy Program, Dept of Cellular Biology and Anatomy, LSU Health Sciences Center, Shreveport, LA; 2 Dept of Radiation Oncology, Washington University School of Medicine, St Louis, MO.
As the limits of existing cancer treatments are recognized, it has become clear that novel approaches must be considered, and adenovirus (Ad) based therapies are a promising strategy Despite encouraging results, major limitations have been defi ned using serotype 5 Ads such as high prevalence of neutralizing antibodies, down regulation of the CAR receptor, and liver sequestration In addition, current clinical biodistribution tracking of Ad vectors in vivo is limited to invasive procedures such as biopsies, which are sampling-error prone, cannot be repetitively performed, and do not give a full representation of the pharmacokinetics involved Several