852 A Novel Genomic DNA Reporter Gene Model for the Study of Friedreich''''s Ataxia in Neuronal Cells Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy[.]
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OTHER DNA VIRUSES
Furthermore, in vivo mice biodistribution studies exhibited a decrease
in liver sequestration of the hexon modified vectors Utilizing
xenograft tumor models, anti-tumor effi cacy of the hexon-modifi ed
CRAds was investigated There are several potential advantages of
eliminating the Ad liver sequestration observed after intravenous
injection, such as reducing hepatotoxicity and conceivably allowing
more of the injected vectors to be available in the bloodstream to reach
their desired tissue and tumor targets Whether these advantages hold
true will require full clinical translation and evaluation
850 Design of Bi-Specifi c DARPin-Derived
Adapters for Targeting of Adenovirus Vectors
Galina Mikheeva,1 Birgit Dreier,2 Christian Hess,2 Edgar Boczek,2
Natalya Belousova,1 Andreas Plückthun,2 Victor Krasnykh.1
1 Department of Experimental Diagnostic Imaging, University of
Texas M.D Anderson Cancer Center, Houston, TX; 2 Department of
Biochemistry, University of Zurich, Zurich, Switzerland.
Effi cient and target-specifi c gene delivery in vivo is the top priority
in development of genetic interventions for humans Because of
the lack of natural specifi city of viral gene vectors for tumors, the
native tropism of these vectors should be modifi ed This tropism
modifi cation often requires ablation of the virus’ specifi city for its
native receptor(s) and engineering of a novel tropism In this study
we sought to develop bi-specifi c adapter molecules derived from the
so-called designed ankyrin repeat proteins (DARPin) to direct human
adenovirus serotype 5 (Ad5) to Her2 receptor Such adapters would
contain two types of DARPins: the Ad5-specifi c DARPin component
that binds to Ad particles, and the anti-Her2 DARPin ligand that
cross-links the virus to the surface of target cells First, to select for anti-Ad5
DARPins that would bind to Ad virions with ablated natural tropism,
a recombinant Ad5 fi ber knob protein with impaired binding to CAR
receptor was produced Next, this protein was used for the ribosome
display-based selection of a highly diverse library of DARPin genes
followed by affi nity maturation This yielded the DARPin leads that
bind the mutated Ad5 fi ber knob with affi nity in low nanomolar range
Subsequent analysis of the complexes formed by these leads and the
fi ber knob established the stochiometry of this interaction These data
guided the design of multi-valent adapters with improved stability of
the virus-adapter complexes The adapters were made Her2-specifi c
by fusing them with the previously developed anti-Her2 DARPin
ligand and used to direct the CAR-ablated, reporter-expressing Ad5
vector to Her2-expressing cancer cells
851 Infl uence of Factor X on In Vitro and In Vivo
Gene Delivery by Ad5 and Ad35 Vectors
Jenny A Greig,1 Suzanne M K Buckley,2 Simon N Waddington,2
Alan L Parker,1 David Bhella,3 Rebecca Pink,3 Takashi Morita,4
Jerome Custers,5 Jaap Goudsmit,5 Stuart A Nicklin,1 John H
McVey,6 Andrew H Baker.1
1 BHF Glasgow Cardiovascular Research Centre, University
of Glasgow, Glasgow, United Kingdom; 2 Department of
Haematology, Royal Free and University College Medical School,
London, United Kingdom; 3 MRC Virology Unit, University of
Glasgow, Glasgow, United Kingdom; 4 Department of Biochemistry,
Meiji Pharmaceutical University, Tokyo, Japan; 5 Crucell, Leiden,
Netherlands; 6 Haemostasis and Thrombosis, MRC Clinical
Sciences Centre, Imperial College London, London, United
Kingdom.
Recently, the interaction between adenovirus (Ad) 5 and the
blood coagulation factor X (FX) was shown to be pivotal for
liver transduction FX binds directly to the hexon of Ad5 and this
interaction leads to hepatocyte transduction in vivo, an effect mediated
through heparan sulphate proteoglycans Vectors based on the
sub-species B Ads, including Ad35, are in development for gene therapy
as the Ad35 fi ber uses the membrane protein CD46 as a high affi nity cellular receptor instead of the coxsackie and adenovirus receptor, the primary receptor for the Ad5 fi ber Based on this we investigated the Ad35:FX interaction in detail using Ad5, Ad35 and Ad35 chimeras: the pseudotype Ad5/F35, which contains the hexon and penton of Ad5 with the Ad35 fi ber, and Ad5/P35/F35, which has the Ad5 hexon and the penton and fi ber of Ad35 By electron cryomicroscopy at 31Å resolution we observed that FX bound to the Ad35 hexon The ability of the chimeric vectors and Ad35 to bind FX was analysed by BIAcore surface plasmon resonance (SPR) and compared to previous data characterising the Ad5:FX interaction SPR analysis revealed that Ad35 and the chimeric vectors bound to FX with high affi nity:
in particular Ad35 had an equilibrium dissociation constant (KD) of 5.18x10-8M in comparison to Ad5, KD of 1.83x10-9M In CHO-CD46 cells the presence of FX signifi cantly inhibited transduction of vectors containing Ad35 fi bers by 352-, 101- and 188-fold for Ad5/F35, Ad5/ P35/F35 and Ad35, respectively, an effect rescued by the addition
of X-bp To assess targeting in vivo the vectors were injected into
CD46 transgenic mice in the presence and absence of FX binding protein (X-bp) Localisation of the luciferase expressing vectors was visualised by whole-body bioluminescence and vector genomes were quantifi ed by qRT-PCR In the absence of X-bp, Ad5/F35 and Ad5/P35/F35 exhibited high levels of lung vector accumulation with 5.6x105 and 5.7x105 vector genomes (VG)/50ng of total DNA isolated, respectively, whereas Ad5 demonstrated very poor lung targeting at 4.6x104 VG/50ng DNA Pre-administration of X-bp signifi cantly reduced genome accumulation in lung by Ad5/F35 and Ad5/P35/ F35 1.9- and 6.6-fold, respectively However, lung transduction by Ad35 was signifi cantly enhanced by 1.6-fold in the presence of X-bp Liver transduction by all vectors was signifi cantly reduced by X-bp, indicating that FX has an effect on both the Ad5 and Ad35 hexons
Our study shows that FX limits transduction through CD46 in vitro, which can be rescued by the addition of X-bp However, in vivo
ablation of the FX interaction by X-bp enhances CD46-dependent transduction and genome accumulation by Ad35
Other DNA Viruses
852 A Novel Genomic DNA-Reporter Gene Model for the Study of Friedreich’s Ataxia in Neuronal Cells
Michele M P Lufi no,1 Javier Alegre-Abarrategui,1 Filip Lim,2 Richard Wade-Martins.1
1 Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, Oxfordshire, United Kingdom; 2 Departamento
de Biología Molecular, Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM), Universidad Autónoma de Madrid, Madrid, Spain.
Friedreich’s Ataxia (FA), the most common form of inherited ataxias, is characterized by a slow progressive neurodegeneration affecting mainly the central nervous system and the cardiac tissue FA
is caused by an abnormal expansion of GAA repeats in intron 1, which results in a reduction of levels of the mitochondrial protein frataxin (FXN) There is currently no effective treatment able to alleviate the neurological degeneration The lack of suitable neuronal models for the study of both the wild-type and the mutated forms of frataxin has hampered the understanding of its function and the development of therapeutic applications Here we describe the use of the infectious bacterial artifi cial chromosome (iBAC) technology, based on the herpes simplex virus type 1 (HSV-1) amplicon vector, as part of a new improved model for the analysis of frataxin expression in neuronal cells We constructed iBAC-FRDA-Luciferase, a genomic DNA-fusion reporter vector by modifying a BAC containing the entire 135
kb FRDA genomic DNA locus Using homologous recombination, we inserted seamlessly the luciferase sequence at the 3’ of the last exon of
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OTHER DNA VIRUSES
the FRDA gene (exon 5a) Using further homologous recombination
we then subsequently introduced into intron 1 ∼310 GAA repeats,
amplifi ed from FA patient’s DNA iBAC vector delivery into the
neuronal cell line SH-SY5Y was achieved using HSV-1 amplicon
system, which allowed high transduction of this cell line otherwise
resistant to transfection procedures We demonstrated expression
and detection of the fusion protein FXN-luciferase in SH-SY5Y
and, most importantly, we show that the presence of GAA repeats in
iBAC-FRDA-Luciferase induces repression of frataxin expression
by 75%, recapitulating the inhibition of transcription observed in FA
patient cells Further validation to this model came from preliminary
experiments which showed that incubation with histone deacetylase
(HDAC) inhibitors can relieve the repression generated by the
expanded GAA repeats, which are believed to induce heterochromatin
formation Current techniques for frataxin detection are laborious
as they are based on RT-PCR and western blot techniques The new
iBAC-FRDA-Luciferase genomic DNA expression vector described
here represents a novel tool for the study of FA since it allows rapid
quantitative analysis of FXN expression in different conditions in
vitro and in vivo This vector will facilitate the study of frataxin
function and will help unravel the complex mechanism by which
expanded GAA triplets induce suppression of FXN expression The
use of iBAC-FRDA-Luciferase could also provide a platform for
high-throughput screening of drugs able to restore frataxin expression
Finally injection in mouse brain can give insight on FXN delivery
and expression in live animals, thus providing important information
for future gene therapy applications Overall, the work represents a
powerful and unique application for the very high transgene capacity
(∼150 kb) of the HSV-1 amplicon system
853 Recombinant Parvovirus B19 Vectors:
AAV2 Fivefold Channel Swap onto B19 Capsids
Confers Increased Stability and Enhanced
Transduction Effi ciency to Chimeric B19 Vectors
Kirsten A K Weigel-Van Aken,1 Yiwen Xiang,1 Linyuan Chen,1
Lakshmanan Govindasamy,2 Mavis Agbandje-McKenna.2
1 Pediatrics, Molecular Genetics & Microbiology, Powell Gene
Therapy Center, Genetics Institute, Shands Cancer Center,
University of Florida, Gainesville, FL; 2 Biochemistry & Molecular
Biology, University of FLorida, Gainesville, FL.
Parvoviral vectors have a great potential as gene therapy tools if
they keep their promise of effi ciency and safety Adeno-associated
virus (AAV) serotype 2 based vectors are currently being used in
phase I and II clinical trials and some of the recently identifi ed AAV
serotypes bear promise for future use for specifi c indications One
of the major challenges in parvoviral gene therapy remains vector
targeting Parvovirus B19 is a human parvovirus with highly restricted
tropism It replicates exclusively in human hematopoietic progenitor
cells of the erythroid lineage and uses the blood group P antigen
as primary receptor In an effort to exploit the selectivity and the
bone marrow tropism of human parvovirus B19, we have generated
recombinant B19 (rB19) vectors that encapsidate single-stranded
AAV2 genomes into VP2 B19 capsids and have demonstrated that
activated beta1 integrin functions as co-receptor for parvovirus
B19 entry into human cells Due to its restricted tropism and the
fact that the site for P antigen binding on the B19 capsid is known,
allowing targeting peptide insertions, rB19 vectors have the potential
for targeting non-erythroid cell types in the human bone marrow
However, efforts to further develop these rB19-based vectors for gene
therapy applications have been hampered by low packaging effi ciency
which must be overcome Based on previous reports that the fi vefold
channel in the capsid of AAV2 may play a role in Rep mediated
genome encapsidation, we have generated chimeric capsids in which
we replaced B19 fi vefold channel forming residues with those of
AAV2 (a B19-ch5AAV2 vector) with the goal of improving packaging
effi ciency The fi vefold channel swap had no effect on rB19 capsid assembly Wild-type B19 capsids are able to assemble and incorporate partial and full-length self-complementary (sc) AAV2 genomes In contrast, the B19-ch5AAV2 virions incorporated exclusively full-length recombinant genomes suggesting that the presence of the AAV2 fi vefold channel increased the DNA encapsidation effi ciency Co-immunoprecipitation experiments revealed that B19-ch5AAV2 capsids interact with AAV2 Rep proteins, whereas no interaction was observed in B19 wild-type capsids In addition B19-ch5AAV2 vectors with packaged sc-Luc genomes were up to 12-fold more effi cient
in transducing HEK293 cells We also generated P antigen-binding site-deleted B19 capsids that express an EGFR-targeting epitope and demonstrated effi cient targeting of EGFR+ breast cancer cells
in vitro In summary, by swapping the AAV2 fi vefold channel onto B19 capsids we have been successful in increasing the full-length genome encapsidation of pseudo-typed B19 vectors and have gained
a substantial increase in transduction effi ciency These novel rB19 vectors will be useful in exploring viral vector targeting to bone marrow-resident cells, such as EGFR+ breast cancer metastases
854 Methods To Enhance the Spread and Onclytic Activity of HSV Vectors in GBM Therapy
Chang-Sook Hong,1 Ajay Niranjan,1 Bonnie Reinhart,2 Ian Pollack,1 Joseph C Glorioso,2 Paola Grandi.1
1 Department of Neurological Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA; 2 Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA.
Oncolytic HSV (oHSV) vectors have shown promise in the treatment of patients with recurrent glioblastoma However, the great majority of patients show incomplete responses indicating that this treatment modality requires further development Impediments
to effective therapy include limited vector distribution on delivery and ineffi cient vector replication within tumor mass Experiments were carried out to (i) examine novel mutant vector backbones with enhanced oncolytic activity relative to vectors commonly used in clinical trials and (ii) modify the extracullar matrix (ECM) in order
to optimize vector distribution in the tumor mass without encouraging tumor cell migration A new class of oncolytic vector (JD0) deleted for the immediate early gene ICP0 in combination with removal
of the joint sequences was shown to grow specifi cally in a variety
of tumor cells JD0 also possessed a remarkable oncolytic activity following treatment of animal models of human glioma without toxicity for normal brain To enhance viral spread, we investigated the use of the matrix metalloproteinase (MMP9) as means to degrade collagen type IV, a common component of the ECM and basement membranes of the glioblastoma tumor but absent in normal brain tissue Tumor cell lines transduced for expression of MMP9 increased the distribution and infectivity of tumor spheroids in vitro with a consequent improvement in oncolytic activity The over expression
of this protease also decreased the effi ciency of migration of glioma cells in vitro Experiments are underway to determine whether the improved distribution pattern in vitro can be recapitulated in vivo using an MMP9 expressing human tumor model Success in these experiments will encourage the further development of oHSV that express genes encoding additional products to modify the tumor micro-environment in a manner to favor vector spread and interfere with tumor survival pathways