271 Expression of IGF1 Receptor Ribozyme and IGFBP3 To Inhibit Rat Glioma Cell Growth 270 Gene Transfer of an Engineered Transcription Factor Promoting Expression of VEGF A Accelerates Rehabilitation[.]
Trang 1270 Gene Transfer of an Engineered
Transcription Factor Promoting Expression of
VEGF-A Accelerates Rehabilitation of the Crushed
Recurrent Laryngeal Nerve
S Brett Heavener,'KevinFung.!S KayeSpratt,'CaseyC Case,'
Laurel E Roberts,' Norman D Hogikyan,' EvaL.Feldman.'
'Medical Center; University ofMichigan, Ann Arbor, M/;
20tolar-yngology; University0/Western Ontario, London, ON, Canada;
'Development Sangamo Biosciences, Richmond CA.
Vocalfold motion impairmentmay result from recurrentlaryngeal
nerve (RLN) injury or degeneration and is a frequent yet unsolved
problem faced by theotolaryngologist, In addition to dysphonia,
RLN injury may lead to aspiration, and airway compromise is
269 Flow Cytometric Analysis of DNA Binding
and Cleavage by Cell Surface-Displayed Homing
Endonucleases
PetraVolna.l-'Jordan Jarjour,2.3SarahBaxter,'BarryL Stoddard,'
Raymond J Monnat,Jr,' Andrew M Scharenberg.P?
'Pediatrics University of Washington , Seattle WA; 2Immllllolog);
ChildrensHospital and Regional Medical Center; Seattle , IVA;
'Immunology; University ofWa shington, Seattle WA; 'Division
0/Basic Sciences Fred Hutchinson Cancer Research Center;
Seattle /I'iI; :;Pathology University of Washington, Seattle./I'iI
LAGLtDADG homing endonucleases (LHE)s cleave 18-24base
pair DNA sequences and are promising enzymes for applications
requiring sequence specific DNA cleavage amongst genome-sized
backgrounds We developed a method for cell surface display of
LHEs which facilitates analysis oftheir DNA binding and cleavage
properties using flow cytometry.Cells expressing surface Lt-IEs can
be stained withfluorescently
conjugateddouble-strandedoligonucle-otides (dsOligos) containing their respective target sequences The
signal isabsolutelysequencespecificand undetectablewithdsOligos
carrying single base pair substitutions
[Figure I].LHE-dsOligo interactions facilitate rapid enrichment
and viable recovery ofrare LHE expressing cells by both FACSand
MACS.Additionally,dsOligos conjugated with unique fluorophores
at opposite termini can be tethered to the cell surface and used to
de-tect DNAcleavage.As recapitulation of DNA binding and cleavage
by surface displayed Ll-lEsprovides a high-throughput approach to
library screening, we have explored using LI-IE surface expression
in combination with somatic hypermutation as a means for iterative
selection of variants with novel sequence recognition specificities
Data from this approach and future directions will be discussed
Staining ofeell Surf ace HEs willi dsOligos:
I·AnII express ing clones H -Ore I express ing done
common in cases of bilateral dysfunction Current management in-cludes: voice therapy,injectionlaryngoplasty,laryngeal framework surgeryand/or laryngeal reinnervation However, these methods typically result in incomplete restoration of function The present study sought to ameliorate RLN injury usingin vivo gene transfer
of an engineered zinc finger protein transcription factor (ZFP TF) activator of vascular endothelial growth factor (VEGF-A) RLN crush injury was performed with subsequent intraneural injection
of an adenoviral vector encoding the VEGF-ZFP TF activator or
an empty adenovirus as control proximalto the site of theinjury, Rats were euthanized at 3,7 and 14 days following RLNcrush and injection and percent nerve-endplate contact in the thyroarytenoid muscle determined.Functional recovery was assessed with direct laryngoscopy, When the RLN was injectedwith the VEGF-ZFP
TF activator construct, the rats displayed a higher percent nerve-endplate contact at the 7 and 14-day time points than control rats Only the 7-day values were significantlydifferent (p<0.05) when compared to the controls Functional recovery was improved at 7 days over the control animals We conclude that activation of the endogenous VEGF-A gene mediated via gene transfer appears to ameliorate damage and/or possibly enhance recovery after RLN crush injury,
271 Expression of IGF1 Receptor Ribozyme and IGFBP3 To Inhibit Rat Glioma Cell Growth Leah R Villegas,IMaria Grant,'Scan M Sullivan.'
'Pharmaceutics University0/Florida, Gainesville, FL.
Introduction The insulin-like growth factor (IGF) system is composed of ligands IGFI and IGF2,receptors IGFIR and IGF2R and the IGF binding proteins (IGFBPs) Binding ofIGFI to IGFI R initiates a cascade of downstream signaling,activating PI3K1Aktl TOR and Ras/Raf/MAPK pathways,resulting in cell proliferation and cell survival The roleof the IGF system in cancer has been studied extensively In glioma tissue versus normal brain tissue, expression of IGFI, IGFI R, IGFBPs and protease activity are upregulated Since IOF has a higher binding affinity for IOFBPs than for itsreceptor,IGFBPs generally inhibit IOFI signaling But because IOFBPs alsoincreasethe circulationhalf-lifeof IOF and are cleaved by tumor specific proteases at the cell surface, they can also potentiate IGFI signaling.Objectives: (I)investigatethe role of IGPI signalingin ratglioblastoma(RG2) cells by IGFIR knockdown via (a) inducible expression ofan IGFI R-ribozyme (IGFI R-Rz) in
a stable cell line (b) therapeutic application ofthe ribozyme in lipid mediated transient transfections (2) inhibit IOFI signaling by lipid mediated gene deliveryof an IOFBP3expressionvector (3) develop protease resistant IGFBP3 to increase inhibition of IGF ligand-re-ceptor interactions.IGFI signaling plays an important role in rat glioma cell growth Induced expression ofan IGFI R-Rz was used
to eliminate IGFI/IGFIR interactions, which showed arrested cell growth up to 96 hours after induction Western analysis confirmed
an 89% decrease of the IGFIR after induction IGFIR-Ribozyme and IGFBP3 expression inhibits RG2 growth in lipid-mediated transient transfections To investigatea more therapeuticapproach, R02 cellsweretransfected with the IOFIR-ribozyme expression plasmid A 20% inhibition ofcellgrowth was still observed, which correlated directly with the transfcction efficiency, Since IGFBP3
is a secreted protein, it has the potential to affect surrounding cells, thus reducing the direct dependence of efficacy on transfection ef-ficiency Transient transfections ofRG2 cells with IGFBP3 yielded
a 20% inhibition of cell growth after 72 hours Brain tumor cells secreteseveraldifferent metalloprotcinases such as MMP2,which specifically cleaves IOFBP3 Immunoprecipitation and ELISA studies showed that there is Icss IGFI bound to IGFBP3 whcn MMP2 is present This may be one reason whyagreater percent inhibition of cell growth was not observed IGFBP3 mutations
Molecular Therapy Volum e '5 Supplement I , \ by 2<)0 7
Trang 2at MMP2 cleavage site show increased efficacy Because wild
was mutated to increase the percent inhibition of the tumor cell
growth rate Five mutations at this site were tested for inhibition of
cell growth resistance to MMP2 proteolysis and binding of IGFI
Immunoprecipitationand ELISAstudies showed a 75% increase in
IGFI binding compared to wild type IGFBP3 and 30% reduction in
tumor cell growth rate compared to untreated control The y99_LIOO
binding and greatest decrease in growth rate Future studies include
quantifying the reduction in phosphorylaiton ofsignal transduction
proteins and testing the effect of mutant IGFBP3 expression in an
IGFI negative background using IGFI siRNA
272 Incorporation of a Prostate-Specific
Amplification Loop with FasL and TRAIL
Expression To Boost Prostate Cancer Killing
IMicrobiologyand Immunology, Medical UniversityofSouth
Carolina, Charleston,Sc.
While prostate cancer-related deaths have declined since the
of cancer death among men in the United States Thus, there is a
great need for new treatment strategies to improve these outcomes
One such strategy is to specifically eliminate prostate cancer cells
through induction ofthc natural pathway ofprogrammed cell death
known as apoptosis This may be accomplished by introduction
of pro-apoptotic genes, such as Fas ligand (Fasl.) or TNF-related
com-plex recombinant adenovirus (rAd) vector with cell-type specific
transgene expression However, the majority ofcurrently available
expression-regulating systems either deliver only modest levels of
be considered safe We have developed a complex rAd vector that
induces high transgene expression in prostate cancer cells but not
in the cells of other origins Our strategy utilizes a modified
Tet-regulated system that incorporates a positive feedback loop and a
This approach generated significantly higher Icvels of transgene
expression and cytotoxicity than the prostate-specific promoter
demonstrat-ing its potential as an efficient and safe gene therapy for prostate
cancers Incorporation of other tissue-specific promoters and/or
cancer-specific promoters into the system could be translated into
specific therapeutics against a variety of cancers
273 Enhanced Hypoxia Specific Expression
Activity of the Epo Enhancer and SV40 Promoter
by Co-Transfection ofthe HIF-1a Gene
Suyeon Lee; Kyunghwa Kim,' HyunAh Kim; Sung Wan Kim,'
Minhyung Lee.'
I Bioengineering, Hanyang University , Seoul, Korea ;2
Pharma-eel/tics and PharmaceuticalChemistry ; UniversityofUtah , Salt
Lake City.
Gene therapy with the vascularendothelialgrowth factor (VEGF)
unregulated VEGF expression had deleterious effect The tight
regulation of the gene expression is one of the requirements for
severalhypoxiaresponsivesystems have beendeveloped.Our group
developedthe erythropoietin(Epo) enhancer-SV40 promotersystem
for a hypoxia-inducible gcne expression In this system, hypoxia
inducible gene expressions arc mediated by endogenous hypoxia
S104
without compromise of the specificity by the co-transfection of the
of 293 cells by RT-PCR using specific primers pSV.HIFla was
transfection assay for optimization of the ratio between pEpo-SV-Luc and pSV-HIFIa, the luciferase expression of each sample was
pEpo-SV-Luc increased the promoter activity of the Epo enhancer and
was confirmedwith the VEGF expressionplasmid,pEpo-SV-VEGF
increased the VEGF expression under hypoxia condition without compromise ofspecificity In addition, pSV-HIFla may induce the endogenous hypoxia responsive angiogenic genes, which will be
co-transfection ofpSV-HIFla and pEpo-SV-VEGF may be useful for gene therapy for ischemic disease
274 Correction of Albinism Mutations by Targeted Genomic Rearrangements Using Engineered Zinc Finger Endonucleases
I Department ofPediatrics, Univer sity ofArizona, Tucson, AZ; 2Department ofPharmacology and Toxicology, UniversityofAri-zona, Tucson, AZ ; ' Genome Center and Department ofPharma-cology , Univer sity ofCalifornia , Davis , Davis, CA.
We arc working to develop a novel corrective gcne therapy ap-proachfor oculoeutancousalbinism(OCA) OCAis eauscd by a lack
of pigmentation in the eycs of albino individuals and is associated with loss of visual acuity Genetic mutations in the gene OCA2
affecting lout of 10,000African-Americans.There is currently no treatment Wehave engineered zinc fingernucleases that trigger the correction of a mutant allele ofthe OCA2 gcnc homolog in a mouse model.Zinc finger-basednucleases deliver a targeteddouble-strand
cellular process of homologous recombination Me1anoeytes in retinal preparations from affected mice were observed to become
far suggest this approach may lead to an effective therapy for the amelioration ofOCA2
275 Effect of the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE)
on Transgene Expression Controlled by the Human Synapsin 1 Gene Promoter Following Adenoviral-Mediated Gene Delivery
Yongxin Gao,'Hongwei Li.'Nan Jiang,' Rachael Harrison,'
Colin Sumners.I
I Department ofPhysiology and Functional Genomics, University ofFlorida, Gainesville, FL.
The human synapsin-I promoter drives restricted in vivo
substantia nigra, cortex and other regions in CNS following
C opyright © The Am eric m Society {I t Gene Therapy