690 Corneal Gene Therapy Past Experiences, Current Trends and Future Perspectives manufacturing process and its associated release testing can be ac complished in a reasonable timeframe to be clinical[.]
Trang 1manufacturing process and its associated release testing can be
ac-complished in a reasonable timeframe to be clinically relevant We
will present data on the tumor harvest / purification process as well
as the xenograft vaccine quality control testing plan
688 Aiming at Alcoholism with Ribozyme
Genes: Silencing the mRNA for an Alcohol
Detoxifying Enzyme
Lorena Lobes-Gonzalez,ICarlos Munoz-Brauning,IAmalia
Sapag.'
'Gene Pharmacotherapy Laboratory, Department ofPharmaco
-logical and Toxico-logical Chemistry, Faculty ofChemical and
Pharmaceutical Sciences, Universidad de Chile , Santiago , Chile.
Protectionagainst alcoholism is provided by a natural aversion to
alcohol in certain individuals or by pharmacological intervention in
alcoholics Ethanol is detoxified by oxidation to acetaldehyde and
then to acetate by alcohol dehydrogenase and aldehyde
dehydroge-nase-2 (ALDI-I2) Impairment of ALOI·J2results in accumulation
ofacetaldehyde leading to unpleasant physical effects and aversion
to ethanol This condition is observed inindividuals carrying an
inactivating mutation in the ALDH2 gene and in alcoholics treated
with disulfiram,a nonspecifictoxic drug which inactivatesALDH2
by covalent modification Similar protection might be achieved
by diminishing enzyme activity with highly specific ribozymes
designed to silence the mRNA for ALDI-I2 Hairpinand
hammer-head ribozymes targeted to a single region of rat ALOI-12 mRNA
(nucleotides 1551-1571)were tested in rat hepatomacells in culture
Antisense (blockade) effects were assessed with control ribozymes
incapable of cleaving RNA H4-II-E-C3 cells were lipofcctcd with
plasmids carrying ribozymc genes driven by a CMV promoter and
the ALDH2 activity in cell extracts was measured
spectrophoto-metrically.The hairpin ribozyrne provided a 42 % reduction in the
ALDH2 activity, 24 % dueto cleavage and 18 % due to antisense
action.The combined effects of this hairpin ribozyme amount to
-70 % reduction oftheALDH2 activity when protein half life and
lipofection efficiency are taken into account The same hairpin
ribozyme delivered in a plasmid construct which endows it with a
longer RNAtail on its 3' side suppressed 36 % ofthe ALDH2
activ-ity,13% by e1eavage and 23 % by blockade,indicating that overall
ribozymeaction and cleavage efficiency are sensitive to nanking
sequences In contrast, the hammerhead ribozyme provided a
mod-est reduction of 19 % in ALOI-12activity due entirely to antisense
effects In conclusion,a hairpin ribozyme directed to nts 1553-1569
of the ratALDH2 mRNAis a good candidateforin vivoexperiments
geared towards developing genetic drugs for alcoholism Supported
by FONDECYT 1040555, UChile PG 06504,[CM P99-03[F and
[CM P05-001E
689 Targeting Activator Protein-1 To
Investigate Protective Phytocompounds Against
UVB Radiation-Induced Photoaging in Mouse
Vanisree Staniforth,Wen-Ching Huang, Ning-Sun Yang
'Agricultural Biotechnology Research Center; Agricultural
Bio-technology Research Center ; Taipei Taiwan
UV radiation isthe main environmental factorthat causes
prema-ture aging of the skin UVB radiation induces ligand-independent
activation of cell surface growth receptors and cytokine receptors,
which in tum results in the activationof down stream signaling
pathways.These pathwaysconvergeto stimulatetranscriptionfactor
activator protein-I (AP-I) UVB radiation induced AP-I regulates
the expression of matrix-mctalloprotcinascs (MMPs), which have
been shown to play important role in degradation of extra-cellular
matrix and acceleration of skin aging We have initiated a study to
identify the phytocompounds that can abrogate the aging effects
S264
of UVB radiation For this purpose,mouse skin transfected with transgenicpromotercontainingAP-I bindingsites driving luciferase gene expression was irradiated with UVB and treated with various phytocompounds Weobserved that UVB irradiationofmouse skin resultedin significantinductionofAP-I activationand expressionof MMPs Our results indicated that phytocompounds such as caffeic acid,luteolin,ferulie acid and geraniol significantly inhibited UVB radiation induced activation of AP-[ transcription factor.CafTeic acid and ferulic acid showed significant inhibition ofUVB-induced MMP-9 expression Whereas, luteolin and geraniol significantly inhibitedUVB-inducedMMP-2expression.Our resultssuggestthat the above-mentionedphytocompoundsmay have potentialto confer protection against photoaging Further investigation is in progress
to study the effects of the above-identified phytocompounds on morphologicalchanges associated with photoaging and to elucidate the detailed mechanistic basis underlying those effects
690 Corneal Gene Therapy: Past Experiences, Current Trends and Future Perspectives
Eytan A Klausner,IDanPeer,'RobertL.Chapman,IRichard E Multack,? Shridhar V Andurkar,'
'Pharmaceutical Sciences , Midwestern University Chicago College ofPharmacy Downers Grove, IL; lAnesthesia , CBR Institute for Biomedical Research, Boston , MA;JOphthalmolog)~
Midwestern University Chicago College ofOsteopathic Medicine, Downers Grove, IL.
Gcne therapy to the cornea can potentially correct inherited and acquired diseases of the cornea,which arc second only to cataract
as the leading cause of vision loss We have searched the literature
in order to characterize variousaspects ofcorneal gene therapy pre-clinicalstudies; vectors, delivery systems, modes of'administration, the animal species used,and diseases that were treated Experiments
in corneal gene therapy were applied to acquired, but not inherited, corneal diseases DNA vaccination, RNA interference and gene transfer of cytokincs,growth factors and enzymes modulated the cornealmicroenvironment.Positiveresultswereobtainedin preclini-cal studies for prevention and treatment of cornealgraft rejection, neovascularization, haze and herpetic stromalkeratitis Aqueous solution was the most common cornealgene deliverysystem While
in gene therapy, viral vectors are used much more commonly than non-viral vectors or physical techniques, viral vectors were used
in only 45.7% of the corneal gene therapy studies (see Table I) In corneal gene therapy,as in gene therapy in general, viral vectors yielded stronger gene expression that was accompanied by more severe adverse effects, in comparison to non-viral vectors The difficulty in obtaining gene expression following topical ocular administration was circumvented by invasive ocular modes of administration.Thereby, in general,injection into the corneal ante-rior chamber and the stroma Icd to gene expression in the corneal endothelium and stroma,respectively,regardless ofvcctor type or animal species studied Experiments for investigating corneal gene therapy for differentcorneal diseases may require the usc of various animal species and a certain disease might be best studied using a particularanimalspecies For example, almost all (21/23, 91.3%) of the studies involving herpetic stromal keratitis were conducted on mice(see Figure I) Opportunitiesin the fieldof cornealgene therapy lie in expanding the array of corneal diseases investigatedand in the
Molecular Therapy Yofume 15 Supplement I, \b y 2007 Copyright © '111C American Societyof Gene Tllcr.lpr
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692 Regulatory Elements for Improved Gene Therapy Vectors
Amy C Groth; George Stamatoyannopoulos,' David W Emery.'
IDepartment a/Medicine, University0/Washington, Seattle, IVA.
Enhancers and repressor-blocking insulators are important for strong, stable gene expression from gene therapy vectors However, enhancers have the ability to inappropriately activate cellular genes and cause transformation events It is therefore essential to include clements such as enhancer-blocking insulators that will limit the regulatory reach of vector enhancers to the relevantt:~nsgenes We have developed two functional screens,one for positive regulatory elements (enhancers and repressor-blocking insulators), and one for enhancer-blocking insulators To identify new positive regulatory elements we cloned a library ofsize-selected human genomic DNA fragmen;s into the 3' LTR ofthe GFP-expressing gammaretrovirus, MGPN2 Upon reverse transcription and integration, each cloned fragment is copied to the 5'LTR and the GFP gene is flanked by the candidate element We enriched the library for positive regulatory elements by sorting for the top 2-10% of Grp+ cells,expanding, harvesting virus and transducing new packaging cells Enhancers are likely to be present in this population, and repressor-blockers may be over-represented in all fractions due to their anti-silencing effect After three or five rounds of enrichment, we sequenced the inserts and assayed them for enhancement and stabilization ofgen.e expression One element,1-1-11,is likely an enhancer Although It did not exhibit enhancer activity in a transient luciferase assay, there was a 4-fold increase in colony formation in a stable integration promoter-trap assay when compared to the neutral spacer, G6pD
In a mouse bone marrow colony assay, individual 1-1-11 colonies had
a significantly higher mean fluorescence !han the e~pt~ MGPJ;l2 vector (average 115 mfu vs.46 rnfu, p<.O:l),but no significant dif-ference in the percent of positive cells (average 55% for H-Il v~.
43% for MGPN2,p=.19) We identified a second clement,F-b, which may be a repressor-blocking insulator In them~us~ bone marrow colony assay, individual colonies showed no significant increase in mean fluorescence (average 53 mfu for r-15vs 46 mfu for MGpN2, p=.62),but they did show an increase in theperc~nt ~f
positive cells (70% vs 43%,p<.02) This pattern of expressionIS
what one would expect from a repressor-blocking insulator,although more studies arc needed to confirm this classification In addition
to clements that boost transgene expression, we would also like to identify enhancer-blocking insulators, which can be used to increase the safety of gene therapy vectors Toward this goal, we have de-signed a two-color reporter system whereby the activity ofcandid.ate elements cloned into a specific test position can be assessed usmg flow cytometry to measure changes in expression ratios We intend
mental diseases and for the development of prenatal gene therapy for specific disorders
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application ofrecent designs using safer vectors exhibiting reduced
imrnunogenicity and longer duration of gene expression
Table t: The gene transfer method used inill ~'il'O corneall(ene delivery
Adcn ov i rnl vector s 25 , 27 , 2, -.;
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F igure 1 : T tM d i s tributi on of c:o rrntal ge ne d eliv efY pu b li cation s
a ccording 10 the an ima l spec ies an d ex pe ri mental disease mod el
691 Developmental Stage Determines
Distribution of Organ Transduction after
Extracoelomic Cavity Injection of Lentiviral Vector
Masayuki Endo, Tiago H Coelho, Phillip W Zoltick, Antoneta
Radu,Nidal Muvarak,Alan W Flake
IThe Children's Center/or Fetal Research, The Children's
Hospi-tala/Philadelphia Philadelphia, PA,
The development of fetal gene transfer can be applicable both
to fetal gene therapy and developmental biological research:Th~re
are several ways to inject genes directly into the fetus for inVIVO
fetal gene transfer However, extracoelomic cavity gene transfer
(ECGT) has not showed any gene expression in the late gestational
stages We hypothesized that earlygestational administration into
the exracoelomic cavity might result in broader gene transfer to
the mesoderm-derived organs due to developmental accessibility
To test this hypothesis, we injected lentiviral vector carrying the
green fluorescent protein (GFp) reporter gene into thefeta~ murine
extra-coelomic cavity from the late head fold/early somite stage
(E8) to E II Injections were performed under image guidance
us-ing a Visualsonies@ Vev0770 system with a 40 mHzs~anhead. We
observed the injected mice under fluorescence stereomicroscopy at
sequential time points after birth and confirmed G\pe~pression by
immunohistochemistry We injected total 78 fetal mice lrom E8-E II
and total survival rate was 52.6 % In early gestation (E8-EIO),
significant GFPexpression was observed in~ul~iple orga~s (Table)
Remarkably, GFp expression was observed m tissues denved from
endoderm and mesoderm including genital system at E8,whereas
no expression was observed in ectoderm derived tissues After E II,
we could not detect GFP expression.The observed temporal
pat-terns of gene expression correspond to the expected embryologic
accessibility of organ specific cell populations We conclude from
this study that early gestational extracoelomic cavity gene transfer
has higher efficiency and a broader distribution of transduction to
both of mesoderm- and endoderm-derived organs than what has
been previously observed later in gestation This model may be
useful for investigation of mechanisms of genetic and/or
develop-Molecular Therapy Volume15 , Sup plement I May 2 007