682 A New Experimental Model for HIV 1 Lesions in the Brain Based on SV40 Vectors Expressing Envelope Glycoprotein gp120 680 Alcohol Drinker Rats Lower Their Ethanol Intake for One Month after a Singl[.]
Trang 1680 Alcohol Drinker Rats Lower Their Ethanol
Intake for One Month after a Single Injection of an
Adenoviral Vector Carrying an Antisense Gene for
Aldehyde Dehydrogenase
Paula Ocaranza,' Maria Elena Quintanilla.? LutskeTampier,'
EduardoKarahanian,'Amalia Sapag,'Yedylsrael.P:'
'Gene Pharmacotherapy Laboratory Department0/
Pharmaco-logical and ToxicoPharmaco-logical Chemistry Faculty ofChemical and
Pharmaceutical Sciences, Universidad de Chile, Santiago, Chile;
2Program0/Molecular and Clinical Pharmacology, Faculty0/
Medicine, Universidad de Chile, Santiago, Chile; "Faculty0/
Health Sciences, Universidad Diego Portales, Santiago , Chile;
"Department0/Pathology, Anatomy and Cell Biology , Thomas
Jefferson University; Philadelphia, PA.
Nature has developed a proteetive mechanism against alcohol
abuse and alcoholism In mostindividuals aldehyde
dehydrogenase-2 (ALDHdehydrogenase-2) readily oxidizes acetaldehyde, a product of ethanol
metabolism, allowing excessive alcohol consumption In contrast,
subjects who carry a point mutation in the ALDH2 gene which
lowers aldehyde dehydrogenase activity are strongly protected
against alcohol abuse and alcoholism by 66%to 100%because
of the aversion triggered by the elevation of blood acetaldehyde
upon alcohol drinking The aim of this study was to mimic such
an aversion to ethanol in rats by inhibiting the expression of the
Aldh? gene Rats bred as heavy alcohol drinkers (UChB) from the
Wistar strain were allowed free access to both 10 % ethanol (v/v)
and water for 45-60 days (ethanol intake 6-7 g/kg/day) After this
time animals were administered, by a single tail vein injection, an
adenovirus (lx101 particles per kg) carrying an antisense gene for
the entire Aldh2 mRNA under the control of a CMV promoter and
were subsequently deprived of ethanol for 3 days Afer the
with-drawal period animals were allowed access to both 10%ethanol
and water for one hour each day On the first day of re-access to
ethanol both the control and the antisense treated animals consumed
equal amounts ofethanol (1-1.2 g/kg/hour), Aversion to ethanol was
markedly evident on subsequent days after the animals had
expe-rienced the effects ofthe antisense RNA, with an overall reduction
in alcohol intake of 50 %for 34 days (ANOVA p<O.OOI) versus
control Ievcls Liver ALDH2 activity was reduced by 80% (p<0.002)
which allowed bloodacetaldehyde buildup No toxicity or interferon
response was observed Overall,high acetaldehyde levels and an
aversion to ethanol,similar to those experienced by subjects who
carry an inactivating mutation ofALDI-I2,was achieved in rats by
the administration of an Aldh2 antisense gene These studies open
the possibility of developing specific and long-lasting gene based
therapies against alcoholism Supported by FONDECYT 1040555,
ICM P99-03IF, ICM P05-001F and NIAAAAAOl542 I
681 Improvement of Survival of Skin Flaps by
Combined Gene Transfer of Hepatocyte Growth
Factor and Prostacyclin Synthase
AyaNakagawa.PMotokuni Aoki,' Takashi Miyake,ISuguru
Shi-raya,' Toshikazu Nakamura,'Toshio Ogihara,'Yoshihiro Kimata.'
Ryuichi Morishita.'
'Clinical Gene Therapy Osaka University Graduated School
ofMedicine , Suita Osaka, Japan ; 2 Plastic and Reconstructive
Surgery', Okayama University Graduated School ofMedicine
and Dentistry, Okayama, Japan ; JGeriatric Medicine, Osaka
University Graduated School a/Medicine , Suita , Osaka, Japan;
'Diviston ofMolecular Regenerative Medicine, Department0/
Biochemistry and Molecular; Osaka University Graduated School
ofMedicine , Suita, Osaka , Japan.
Background: Increasing the local blood flow is a critical factor
for long-term survival of skin flaps Thus, a molecular therapy to
Molecular Therapy volum e 15.Supp lement I ~I 2 007
Coprri ght @ The American &)oC: f)' of Gene Therapy
increase the blood flow by means of an angiogenic factor is con-sidered to be a useful strategy to improve skin flap survival In this study, we focused on a combined strategy to stimulate not only angiogenesis, but also vasodilation of local microvessels,using co-transfection ofthe hepatocyte growthfactor (HGF) and prostacyclin synthase (PGIS) genes to enhance the survival of random-pattern skin flaps Methods & Results: Human HGF or human PGIS cDNA was inserted in the PVAX expression vector, which utilizes the cy-tomegalovirus promoter/enhancer.In this study,12-week-old male rats were used to make a 2 x 8 em full thickness cranialpedicled random-pattern flap (McFarlane musculocutaneous flap) At 3 days before operation, 400 Jlg HGFandlorPGIS naked plasmid DNA or control plasmid was transfected into the flaps by needle-lessinjection using a Shima Jet Injection of naked plasmid by ShimaJct resulted
in successful expression of human HGF and PGIS in the skin flaps Transfection of both genes into the distal half ofskin flaps at 3 days prior to operation significantly increased the survival rate of skin flaps,while transfection into all over the flaps did not In addition, transfection prior to operation was more effective than simultaneous treatment Moreover,co-transfection of these genes improved the survival area ofskin flaps, accompanied by an increase in blood flow ofskin flaps,even in a diabetic model that showed a significant delay
in wound healing Conclusions: Overall,these results indicate that combination treatment with HGF and PGIS genes by Shima Jet could
be an effective strategyto improve skin flap survival Genetically modified skin flaps might have therapeutic value for the treatment
of severe ischemic uleers such as diabetic ulcers
682 A New Experimental Model for HIV-1 Lesions in the Brain Based on SV40 Vectors Expressing Envelope Glycoprotein gp120 Jean-Pierre Louboutin,ILokesh Agrawal,' BeverlyA.S Reyes,' Elisabeth J van Bockstaele,' David S Strayer.'
I Pathology, Thomas Jefferson University Philadelphia , PA;
2Nel/-rosurgery; Thomas Jefferson University Philadelphia, PA.
HIV-I infection is the most common cause ofdementia in adults under40 years ofage HIV-I envelope (Env) glycoprotein, gp 120, is thought to mediate neurotoxicity,since it causes apoptosis in cultured neurons Model systems in which recombinant gpl20 is injected directly into the striatum allow animal studies of Env-induced neurotoxicity in vivo, but the toxic effect is too acute to be useful for studying chronic tissue injury or for testing most therapeutic interventions The lesions of HIV dementia reflect chronic injury caused by repeatedproduction of gpl20 and other substances by HIV- I-infected cells We have developed an experimental model
of chronie HIV-I Env-induced neurotoxicity It uses injection
of rSV40 vectors carrying gpl20 into the brain SV(gpI20) is a recombinant, Tag-deleted SV40-derived vector that expresses HIV-INL4-3 gp I20 under the control ofthe cytomegalovirus immediate early promoter (CMV-IEP) To test the effectiveness oftransduction with SV(gp 120) in establishing chronic gp 120-related brain injury, the vector (or an unrelated rSV40 control vector) was injected stereotaxically in the rat caudate-putamen (CP) on one side The opposite side was untreated Brains were then examined at various
timepoints thereafter for the chronicity and extent of injury, as well
as its characteristics Examination of brains 7,14 and 28 days after the injection showed a partly hemorrhagic lesion in thc CP with loss
of neurons We tested for apoptosis by TUNELassay Apoptotic cells,mostly neurons (immunostained using Neurotrace, NT), were found at thedifferenttime points after the injection gp 120 protein produced as a result oftransduction was immunolocalized mainly in neurons within the first 2 weeks after injection Some ofthe apoptotic cells colocalized with gp120; some did not Microglial cell prolif-cration was seen at the several time points The contralateral side, and brains injected with control vectors,were unremarkable One
S261
Trang 2month after SV(gp120) injection into the Cp, gp120-positive cells
were still present near the site ofinjection Some were NT-negative
with the appearance of microvessels or microglial cells Wetested
for oxidative damage following SV(gp120)injection,as such injury
peroxidation,was measuredby calorimetricmalonaldehyde(MDA)
assay and production of 4-hydroxy-nonenal (HNE) SV(gpI20)
elicited more MDA than did the control rSV40 HNE-positive
neurons were seen in SV(gpI20)-injected CPs, and HNE-positive
-IIV-I, SV(gpI20) neuron damage is associated with oxidative stress
These data indicate that in vivo transduction with SV(gp120) in the
least for a month after transduction SV(gp120) transduction may
therefore be a promising animal model for chronic HIV-I-related
Hematopoietic Stem Cells after Transduction with
Helper-Dependent Ad5/35 Vector
Hongjie Wang,Andre Lieber
Weare interestedin
targetingadenovirusvectorintegrationtospe-cific sites in the genome of human hematopoietic stem cells (HSC)
by transient expression of proteins with site-specific endonuclease
activity, such as the AAV Rep78 protein Such vectors should lack
endonuclease expression in producer (293) cells and allow for
induciblelregulated expression in HSC We constructed a set of
helper-dependent adenovirus vectors with Ad serotype 35 fibers
(HD-Ad5/35) which can efficiently transduce human CD34+ cells,
HD-Ad5/35 vector contained the human globin LCR and the beta globin
promoterto drivetransgeneexpression.Backgroundexpressionfrom
thisvectorin 293 cells was low, which allowed for the production
ofHD-Ad5/35.Rep78 vectors Although the globin LCRIpromoter
not confer transgene expression to CD34+ HSC Wetherefore
con-structed three HD-Ad5/35vectors with Tet-induciblesystems using
OFP as a reporter gene In all three vectors the expression cassette
avoid unspecific interaction with enhancers and promoters present
in the Ad ITRIpackaging signal and stuffer DNA HD-Ad5/35
(KRAB)domainand the tetracyclinerepressor(tetR) In this system,
tTRKRAB-mediated repression of a OFP promoter juxtaposed to
tet operator sequences can be reversibly controlled by doxycycline
(Dox) HD.Ad5/35.Tet-2 used an autoregulated rtTA (activator)
(human CD34+ erythroleukemia cells) were lower for I-ID-Ad5/35
after infections at MOIs of>400 genomes per cell After induction
with Dox, OFP expression levels increased 5-fold and 1000fold in
OPPexpressionlevelsincreased2-foldand 200-foldfor I-ID-Ad5/35
Tet-land HD-Ad5.35-Tet2 after induction respectively.After
In an attempt to combine the advantages of both HD-Ad5/35.Tet
vectorswe combined the two systems (rtTAand tTRKRAB) in one
vector (HD-Ad5/35.Tet-3).Preliminary data in 293 cells show that
S262
Tet-3 in M07e cells and primary CD34+ cells will be presented Future efforts are focused on the construction of HD-Ad5/35-Tet3 vectors for inducible expression of Rep78, Zinc-finger nucleases, and meganucleases (I-Ceul)
684 Expression of Foxp3 in a Subset of Rat Dendritic Cells (DCs), Plasmacytoid DC, Generated from a Long-Term Culture of Bone Marrow Cells (BMC) Driven by FLt3-Ligand and IL-6
/Research Surgery, Division ofCollaborative Research, National Research Insitute for Child Health & Development, Tokyo, Japan;
Co" Ltd, Tokyo, Japan
a forkhead/winged helix motif at the carboxy terminus, a C2H2 zinc finger domain, and a 3-heptad Zip motif Heretofore,
and currently is it representing the most definitive marker for this
study that specializedantigen presentingcells (APC), dendriticcells
from rat DCsgenerated from long term culture driven by FlO ligand
forms of2 and 3, Real time RT-PCR analysis revealed that the level
same as or higher than that ofCD4+CD25+ T cells Although west-ern blotting analysis revealed that isoform I was indeed expressed
Incontrastto this, DCs generated from a short-term culture driven
lyrnphoproliferativedisorder of scurfy mice and a human X-linked
of CD4+CD25+ Treg cells rather it may represent a more broader function ofthe gene regulationresponsiblefora subsetofspecialized antigen presenting cells such as DCs and macrophages (M<I»
Molecular Therapy Volume 15 , Supplemen t I• \b r 2007
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