297 a comparison of transduction efficiency and long term repopulating capacity of lentiviral transduced SLAM versus KSL enriched HSC using an abbreviated transduction protocol

297 A Comparison of Transduction Efficiency and Long Term Repopulating Capacity of Lentiviral Transduced SLAM Versus KSL Enriched HSC Using an Abbreviated Transduction Protocol findings demonstrate mu[.]

findings demonstrate much more efficient gene transfer to skin

stem cell populations after early gestational IAGT using lentiviral

vector driven by the K5 promoter compared to the CMV promoter

The K5 promoter appears to be superior for potential therapeutic

applications for congenital skin diseases,particularly those,such

as epidermolysisbullosa, that alTect the basal layer

(Figure1)Anti-GFP immunofluorescenceimages of3

weeks-old mouse injected atE8with lentiviral vector containing GFP

transgene drivenbyCMV promoter (Left) andK5promoter

(right)

296 Transient Gene Expression, Random

Chromosomal Integration and Homologous

Recombination in Cynomolgus Monkey

Embryonic Stem Cells with Helper-Dependent

Adenoviral Vectors

Keiichiro Suzuki;Kouichi Hasegawa,'Kaoru Mitsui.' Emi

Aizawa,' Haruka Shiiba,'Hirofumi Suemori,' Norio Nakatsuji,'

Consideringthat a variety of embryonicand adultstem cells have

been isolated and characterized, development ofa general strategy

to obtain efficient gene delivery and homologous recombination

(HR) in various cell types would be ofpar amountimportance for

biological studies and clinical applicationsof stem cells HR also

can be applied to introduce a gene into safe and transcriptionally

active chromosomal sites for gene therapy of inherited diseases

However, its application in a varietyof cell types,especially in

primate embryonic stem (ES) cells, has been hampered by a lack

ofa suitable gene delivery method Toward these goals, we

exam-ined an ability of helper-dependent adenoviral vector (HO AdV)

to transiently and stablyexpress a gene as well as to knockout the

hypoxanthine phosphoribosyl transferase (HPRT) locusvia HR in

male cynomolgus monkey ES cells The efficiencies of transient

gene expressionweremeasuredby

usingincreasingamountsofGFP-encoding HOAdvs, and were -10% at a multiplicities of infection

(Mal) of 10 GFP-transducing units (gtu; measured on 293 cells)

per cell and reached a plateau at -SO% at an Mal of 1000 gtu per

cell There was no significant difference in efficiency between the

HO AdV with the fiber from human adenovirus type 5 (Ad5) and

the Ad35 fiber-pseudotypcdHO AdV The frequency of random

chromosomal integration was measured with a HD AdV encoding

the~-geo marker gene G4lS-resistant colonies were obtained at

frequencies of 1.7x10-5and 5.0 x 10-1 per infected cell after

infec-tion at MOls 000 - 300 and 1000- 3000 gtu per cell, respectively

Thefrequencyof chromosomalintegrationvia HR was measuredby

using a HO AdV encoding a l3-kb homology from introns 3 - S of

HPRT,in which exons 5 and 6 were replaced with theIRES-~-geo

gene A total of 7.2x106ES cells were infected with the vector at

MOls of 100- 300 gtu per cell,and 9 G418-resistant colonies were

SII2

obtained Among them, one was 6-thiogunanine-resistant (HPRT knock-out) This colony expressed the alkaline phosphatase at a levelequivalent to that ofparental ES cells,suggestingmaintenance ofthe undifferentiatedstate after vector infection CUITCntly, we are analyzing the fidelity of HR as well as dilTerentiation potentials of this HPRT-knockout ES cell clone These results suggest that both efficient transient gene expression as well as HR can be achieved

by 1-10AdVs in primate ES cells AdVs can infect almost any cell types efficiently, and therefore, might be a viable option as a tool for chromosomal manipulation ofa variety ofstem cells, including human ES cells

297 A Comparison of Transduction Efficiency and Long-Term Repopulating Capacity of

Lentiviral TransducedSLAMVersus

KSL-Enriched HSC Using an Abbreviated Transduction Protocol

Pablo Lajc,'Nidal Muvarak,'Philip W Zoltick,' Alan W Flake.l

Background: The SLAM-family receptors have recently been

identified as useful markers for the isolation of highly enriched murine hematopoietic stem cells (I-ISC).This HSC enrichment protocol is relatively simple, and results in an enriched I-ISC prod-uct that has been shown to have comparable repopulating capacity

to c-kit'Sca-l'Iirr (KSL) HSC The relative capacity to withstand genetic manipulation and, most importantly, maintain long-term repopulating capacity of these highly enriehed HSC populations has not been tested In this study we compare SLAM versus KSL-e?riched HSC populations for I) transduction efficiency,and 2) in VIVOlong-term repopulating capacity, afterlentiviral transduction usinga highlyefficient, abbreviated,transductionprotocol.Materials and Methods: C57BL6-CD45.1 mice were lethally irradiated and used as recipients Four to eight week old C57BL6-CD45.2 mice were used as donors HSC were isolated on the basis of the KSL

or CD15O+C04S- marker magnetic lineage depletion (AutoMACS Miltenyi) followed by FACS sorting (FACSVantage, BO) Cells were transduced with an HIV-MND2.I-GFP lentiviral vector at an Mal of 100, for a total of 14-16 hours,with no prestimulation, in the presenceof only stem cell factorand thrombopoietin(I OOnglml),

in Rctronectin®-coatedwells Irradiated mice were transplanted with 5x102or 5x 103or 5xIO~ transduced cells,along with 2 x lOs radioprotective syngeneicBM MNCs With every transduction.an aliquot ofcells were washed twice to removeall viral particles,ilOd plated in clonogenic media for 72hours to evaluate the transduction efficiency Mice were analyzed for chimerism at 4, S, 12,and 16 weeks after transplantation The last time point included lineage analysis as well as an evaluation ofchimerism in all hemolymphoid organs Results: We observed consistent transduction effieiency among the multiple experiments,ranging from 60 to 70% after 14

to 16 hours of vector exposure and 72 hours in vector-free culture

with no significant difference between the SLAM-HSC and the

KSL-HSC groups The was no significant dilTerences in the ability

oflransduced cells to repopulate theirradiated micebetween both groups, at all time pointsand cell doses analyzed with chimerism, ranging from 2 to 5%, II to 31% and 30 to 53% GFP-positive

PBMC at 16 weeks for the animals transplanted with 500 I 5,000 I

50,000 cellsrespectively,Multilineage reconstitution was observed for both I-ISC types, and the bone marrow, thymus andspleens contained similar percentages of GFP-positive cells in both HSC groups Conclusions: Our results demonstratethat murine HSCcan

be efficientlytransduced by lentiviralvector usinga simple protocol that involves minimal in vitro manipulation and no pre-stimulation

In addition,we have shown that SLAM enriched HSC are equal

to KSL enriched HSC populations with respect to efficiency of

Molecul arThe 4l pyVolume 15 Supp lement t• • \br 2007

Co pyright © " 111e Ameri can S OI;ic;ty o f Gen e Therapy

transduction, and maintenance of long-term repopulating capacity

after transductionusing thisabbreviatedprotocol.These resultswill

be valuable to those interested in genetic manipulation of highly

enriched HSC populations

298 Self-Renewal and Differentiation of Human

Mesenchymal Stem Cells from Single Cell Clonal

Assays

C Chang I Lee; Joanne Huang,' Jared E Christensen,' Christine

I Mall,' Alyssa C Leapley,'Mervin C Yoder.? Alice

F.Taran-tal.'-'

California, Davis, CA; lHerman B Wells Center/or Pediatric

Anatomy, University ofCalifornia, Davis CA.

Bone marrow-derivedmesenchymalstem cells (MSC) from

hu-manand nonhumanprimateshave beenshown to self-renewbeyond

theHayflicklimitand

differentiatetowardmultiplelineagesofmes-enchymalorigin However,it is clear that MSCcultureconsists of a

heterogeneouspopulationof cells that may include"true" MSCand

lineage-committed progenitors In this study,we sought to identify

humanMSC (hMSC)withself-renewaland differentiation potential

at a single celllevcl hMSC obtained from bone marrow of healthy

donors (N=3) were transduced with an HIV-I-derived lentiviral

vector expressing EGFP under the control of the EFIa promoter

Single EGFP-positivehMSCweresortedas single cells into24-well

plates with and without basement membrane extracellular matrix

(ECM) and containingnon-transducedirradiatedautologoushMSC

(5xIQ3cells/em') as feeders in a-MEM supplemented with 20%

FBS for 12days Only wells containingsingleEGFP-positivccells

were assessed and counted every three days; wells containing z 2

~GFP-positive cells were excluded from the study After 12 days

IIIculture, hMSC colonies derived from single sorted cells were

trypsinized and expanded EGFP-positive cells were then sorted

based on EGFPexpression for further experiments To assess

dif-ferentiation potential ofhMSC expanded from single cell colonies,

cells were incubated in adipogenic,chondrogenic,andosteogenic

inductionmedium for 21 days followingestablished protocols

Cells wereanalyzed for gene expression by real-time RT-PCR

(Bglap,COMP,LUM,PPARy2, LPL,Lep,BGN,CBFAI, COLIA1,

ALPL,SpARC, iBSp, SPPJ) specific for tri-lineagedifferentiation

Resultsindicatedthat 5% of single cells platedon plastic underwent

;:::\0population doublings during the initial 12·day culture period,

whereas 20% of single cells plated on ECM showed comparable

growthcharacteristics, In addition,cellsplated on plasticshowed a

significant reductioningrowth rateand differentiated morphologyin

subsequentcultures when comparedto cells plated on ECM When

differentiation was assessed, 2% of all single cells plated on plastic

showed gene expression profiles suggesting multi-differentiation

towards all three lineages However,cells plated on ECM showed

more robustgrowthand withnormalhMSC morphology The

multi-lineagedifferentiationpotential of cells plated on ECM is currently

under investigation Current findings suggest: (I) the existence

of "true"hMSC that can bothself-renewat a single cell level and

differentiate toward multiple lineages of mesenchymal origin; (2)

the frequencyof such hMSC in routine culture is very low; and (3)

that culturinghMSCwith ECMsignificantly enhancedproliferation

of cel.ls derived from single cells Further studies in progress are

focu~mg on karyotype,gene expressionprofiles,surfacephenotype,

andlineagemapping of individualcolonies ofhMSC

Molecular Therapy V olume 15.Suppl em ent I M ;'l" 2007

Coprri ght ©The American Socie tyo f G eneTh erapy

299 Hepatocyte Propagation In Vivo for Liver Tissue Engineering

Kazuo Ohashi,' KoheiTatsumi.' MihoKataoka,'ChiseTatcno,'

KatsutoshiYoshizato,'Akira Yoshioka.' Teruo Okano,'Yoshiyuki Nakajima.'

Background: Recent success in hepatocyte transplantation to-ward the treatment of liver diseases, including metabolic defects has encouraged further investigation into bioengineering hepatic tissues as a cell-based therapy Our group has recently developed

an approachto engineerstable livertissuesunder the kidneycapsule whileretainingfullhepatocyte functionality Consideringthe serious donor livershortagefor hepatocyte isolation,itwouldbe importantto establisha newsystemto expandfunctionalhepatocytes Research-ers, includingour group, have recentlyshown that allogenicas well

asxenogenic hepatocytes could be propagated in the liver of small animals if the host livers had been placed under certain conditions Herewe demonstratea novel strategyto engineerlivertissues under the kidney capsule using hepatocytes that have been propagated

in mouse livers Methods: Donor hepatocytes were isolated from humanalpha-I antitrypsin (A1AT) transgenic mice (provided by

Dr.Bumgardner,Ohio State Univ.)with the FVBINgenetic back-ground Hcpatocyteswere separated from non-parenchymal cells using low-speed centrifugation (hepatocyte purity >99%) Viable

hepatocytes(5 x lOS) were transplanted into the liver of urokinase-type plasminogen activator transgenic SCID (uPA/SCID) mice The viability and the propagation status of the transplanted AIAT hepatocyteswithin the uPA/SCID Iivcrswere determinedby serum AIAT levels and histological examination of the liver tissues The propagatedA IAThepatocyteswere subsequentlyisolated from the livers of the recipient upA/SCID mouse Liver tissue engineering was performed by transplanting the propagatedA1AThepatocytes

under the kidney capsule space of wildtype of rVBIN mice Re-sults: Serum AIAT levels of recipient uPA/SCID mouse showed rapid increase and attained the original donor AIAT mouse levels within 8 weeks, which demonstrated the progressive proliferation and propagationof the donor hepatoytcs Histologicalexamination showedpropagatedA lAT hcpatocytes occupied more than 98% of the uPNSCID livers Then we performed liver tissue engineering methodsin the kidneycapsuleusingAtAThepatocytes that had been

propagatedwithin upA/SCID livers The engineered liver tissues stablypersistedforover 10weeksand functional livercharacteristics were confirmedby histologicalanalyses,Conclusions: The present cell-based strategy using propagated hepatocytes to engineer new liver tissue in vivo could represent a novel therapeutic method in the treatment ofliver diseases,

300 Development of Megakaryocyte-Specific Lentiviral Vectors for Gene Therapy of Platelet Disorders

Liesbeth De Waele,1KathleenPreson,'VeerleGillijns,'Chantal Thys.!Chris VanGeet.!Desire Collen,IThierry VandenDriess-che,' Marinee Chuah.'

'Center for Transgene Technology and Gene Therapy,

Introduction: The prevalenceof congenitalplateletdisordershas not beenestablished, butfor some life-threatening bleedingdisorders

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