305 surface epitopes involved in cell motility and tumor progression are expressed in low density cultures but not in confluent cultures of human multipotent stromal cells (hMSCs)

305 Surface Epitopes Involved in Cell Motility and Tumor Progression Are Expressed in Low Density Cultures but Not in Confluent Cultures of Human Multipotent Stromal Cells (hMSCs) treated as well as c[.]

treated as well as control mice The remaining cells were grown

in either methycellulose or in 96 well plates Second generation

recipient mice were sacrificed 14 days after transplant and the sp

and nsp cells isolated and grown in either methycellulose or in 96

well plates Tissue culture grown colonies were stained for donor

(LacZ+) cells,expression ofvimentin,endothelin,or F4/80

(macro-phages) In the first generation recipient mice,those pre-treated with

MnSOO-PL had increased expression ofLacZ+ sp and nsp cells per

esophagus (38.4±4.2% and 21.8±5.5%,respectively) compared

to the control irradiated mice (21.4±4.4%, p=0.0105,and 5.2±

2.4%,p=0.0277, respectively) Second generation MnSOD treated

recipient mice, transplanted with SPcells from MnSOD-PL treated

mice had more LacZ+ SP cells per esophagus (47.9 + 3.5%) than

that detected in mice injected with NSP cells from the same donors

(22.3±2.4%,p < 0.0001), control irradiation only SP cells (22.3

±2.0,p= 0.000 I) or control irradiation only NSP (9.6±2.5%, p <

0.000 I) Explanted second generation LacZ+ cell derived colonies

were stained with vimentin,endothelin, or F4/80 For all SP and

NSP subpopulation,colonies formed in vitro contained vimentin

and/or endothelin positive with few F4/80 positive cells In both

the first and second generation recipient mice,more multi-lineage

LacZ+ colony forming cells were recovered from mice pretreated

with MnSOO-PL Therefore,esophageal irradiation induced ROS

can be reduced by swallowed MnSOO-PL treatment improved

engraftment of esophageal stem cells

Repopulating Hematopoietic Stem Cells from

Clinically Relevant Sources

Martina Cesani,'«Alessia Capotondo.PEugenio Montini,' Laura

Tononi,' Manfred Schmidt,' Fabrizio Benedicenti,' Anna

Zin-gale; Francesca Santoni de Sio,' Samantha Scaramuzza,' Christof

Von Kalle,5 Luigi Naldinj,l,3Alessandra BiffiY

[San Raffaele Telethon Institute for Gene Therapy, San Raffaele

Scientific Institute, Milano, Italy ; 2Experimental Neurology

Insti-IIIte, San Raffaele Scientific Institute , Milano, Italy; J/1ta-Salute

University , San Raffaele Scientific Institute , Milano , Italy;

"De-partment ofBiomedical Science and Biotechnology University

ofBrescia , Brescia, Italy; 5National Center for Tumor Diseases,

National C enter for Tumor Diseases, Heidelberg, Germany,

In the perspective ofclinical application oflentiviral vectors (LV)

in hematopoietic stem cells (HSC) gene therapy for Metachromatic

Leukodystrophy, we optimized LVtransduction ofhuman HSC from

clinically relevant sources, such as bone marrow and mobilized

peripheral blood (BMIMPB) In order to confirm transduction of

long-term repopulating, multi-lineage differentiating hHSC by LV,

we monitored the engraftment and differentiation oftransduced cells

in RAG2-/-IL2R-gamma chain-/- mice This xenograft model is

characterized by a long life-span and the ability to sustain T cell

dif-ferentiation and thymic maturation oftransplanted human cells We

first demonstrated efficient LV transduction of cord-blood derived

HSC and reached high transgene expression long-term in the

multi-lineage human graft of transplanted mice We then tested different

transduction protocols on MPB/BM cells using LV encoding either

the therapeutic arylsulfatasc A (ARSA) or GFP,tuning vector dose,

number of transduction hits and total time of in vitro culture We

reached efficient and reproducible gene marking of HSC (ranging

from 40% to 70% transduction in the various conditions),assayed

both in liquid culture and c1onogenic assays,with a mean of 1.5-4

vector copy number (VCN) per genome Sustained transgene

ex-pression was observed, MPB HSC,transduced with a single vector

hit at multiplicity of infection (MOl) of 100,efficiently repopulated

recipient mice long-term upon transplantation We detected human

cells engraftmcnt in all hematopoietic organs up to 18 weeks after

transplantation,and we observed multi-lineage differentiation of

Molecular Therapy Volum e 15,Suppl ement I ~, 2007

Co prright © TheAmerican Socie ty o f G ene Th erapy

transduced cells Interestingly, thymic maturation of human lym-phoid progenitors was documented.When isolatedfrom chimeric mice 18 weeks post-transplant, human cells retained transgene expression at comparable levels to the in vitro samples Moreover, LAM PCR demonstrated a polyclonal pattern of LV integrations in both pre-transplant samples and tissues retrieved from repopulated mice,without apparent indication of in vivo skewing.Overall these data demonstrate in a stringent in vivo model the capability of LV

to efficiently transduce human MPB/BM HSC without impairing their long-term repopulation and differentiation

from Long Term Cultured Mesenchymal Stem Cells Has No Functional Effect after Transplantation into the Injured Heart

Nan Ma,l Wenzhong Li; Oario Furlani,' Lee-Lee Ong,' Karola

Lutzow, 'Christian Klopsch,IAndreas Liebold,IAndreas Lend-lein,'Gustav Steinhoff.'

[Department ofCardiac Surgery; FKGO, Rostock, Germany; 2GKSS Forschungszentrum Geesthacht GmbH, Institut Fiir

Poly-merforschung, Teltow, Germany.

Mesenchymal stem cells are characterized by their self renewal and differentiation potential They have been evaluated widely both

as a cell source tor various therapeutic applications and as a platform for ex vivo genetic manipulation In this study, mesenchymal stem cells were isolated from normal Lewis rat bone marrow underwent

spontaneous transformation following long term (3-4 months) in vitroculture The cells were noted to appear distinctly different from typical MSCs They were morphologically spherical and cuboidal

to short spindle in shape In terms ofcell surface characteristics, the cells were C045', C029'0", C09010" and CO 117' They exhibited accelerated proliferation and lost of contact inhibition Six weeks after transplantation into the rat infarcted myocardium,encapsulated structure and calcifications were found in the infarcted area or/and border zone.Furthermore,there wasno significant improvement on cardiac function by pressure-volume loop measurement after cell transplantation Our study highlights the need for thorough biosafety investigations of long-term cultivation ofmesenchymal stem cell to achieve the full clinical therapeutic benefits

and Tumor Progression Are Expressed in Low Density Cultures but Not in Confluent Cultures of Human Multipotent Stromal Cells (hMSCs)

Ryang Hwa Lee*, Min Jeong Seo",Andrey A Pulin,Carl Gregory, Joni Ylostalo,Darwin J Prockop

'Center for Gene Therapy , Tulane University Health Sciences

Center; New Orleans, LA

*R.H.L and MJ.$ contributed equally to the work Human rnul-tipotent stromal cells (hMSCs) from bone marrow can be cloned

as single-cell derived colonies, but during proliferation the cells undergo phenotypic changes In cultures ofhMSCs that were plated

at low density, the changes include progression from small, rapidly proliferating cells to large, nat cells that are poised for differentiation

In parallel with these changes,there are dramatic alterations in the profiles of expressed genes Because of these phenotypic changes

as the cells expand,it has been difficult to obtain reliable markers

to characterize hMSCs by surface epitopes.Here, we have identi-fied a series of surface epitopes that early passage hMSCs express

in low density cultures but not inconfluentcultures: hepatocyte growth factor receptor (HGFR), podocalyxin-Iike protein (POOXL), integrin-a6, integrin-a4, the receptor for stem cell derived factor-I (CXCR4) and the receptor for fractal kine (CX3RI) Interestingly, siRNA-mediated downregulated POOXL cause the cells to

aggre-SI15

gate into immobile clusters The results suggestthat the epitopes

CELL PROCESSINGVECTOR PRODUCTION

306 Testing Large-Scale Lentiviral Vector

Preparations for Replication Competent

Lentivirus

LisaDuffy,' Sue Koop,' lingYao,' RobertGetty,' Scott Cross,'

'Medical and Molecular Genetics /National Gene Vector

Labora-to/yo Indiana University School ofMedicine, Indianapolis IN.

screen forreplication competent lentivirus (RCL) will be an

im-portant step in certifying vector supernatant We have previously

Therapy 8:830-9, 2003) and now report our initial experience with

RCL screening In brief, the two-phase assay utilizes an

amplifica-tion phase where the T cell line C8166 is exposed to the test article

After 4 hour incubation the cells are passaged for 3 weeks and cell

free media is used to inoculate naive C8166 cells which are then

passagedfor I week (indicatorphase), RCLis presentwhen p24-gag

detected in indicator phase cells.An attenuated HIV-I virus (R8.71)

is used as a positive control.The assay was validated by performing

infection of C8166 cells according to the protocol using R8.71 at

the TCID50 (0.5 infectious unit/culture).Three cultures were tested

per assay and three independent assays were performed Seven of

nine cultures tested positive at the indicator phase for both p24

antigen and psi-gag recombinants, which validated the assay and

confirmed the limits of detection was approximately I infectious

three positive controls were inoculated at the estimated TCID50 at

through the indicatorphase In addition,fiveculturesof naive C8166

were inoculated with virus at the TCID50 at the start ofthe indicator

phase In reviewing the positive control portion ofthe amplification

27 of27 had detectable virus by p24 and by psi-gag recombination

for psi-gag recombinants at the end of the week of culture These

findings suggest the 3 week amplification does increase sensitivity

compared to a 1 week amplification In terms of test articles, 7 of

the RCLassays were used to screen 4 largescale vector productions

that had a combined pre-concentration volume of over 100 liters,

post-productioncells were also screened from each of the 4

produc-tions and 2 assays were performed on cells transduced with clinical

refine-ment of the RCL assay is warranted to maximize sensitivity while

streamlining the methodology

S1I6

307 Generation of Lentivirus Vectors Using Recombinant Baculoviruses

1.Mahonen,':'Kari 1.Airenne,ISeppoYla-Herttuala.v-'

I Department ofBiotechnology and Molecular Medicine A.I Vlr-tanen Institute University ofKuopio, Kuopio, Finland;2 Depart-ment ofMedicine and Gene Therapy Unit A./ Virtanen Institute University ofKuopio, Kuopio, Finland;jKuopio University Hos-pital Kuopio University HosHos-pital Kuopto, Finland; "Department ofBiological and E nvironmental Science NanoScience Center; University of'Jyvaskyla Jyviiskyld , Finland ; sArk Therapeutics

qgArk Therap euticsqg Kuopio, Finland

clini-cal sclini-cale is challenging The four plasmid method by conventional transienttransfectionto producethirdgeneration lentiviralvectorsis

toxicity of several viral proteins prohibits constitutive expression Baculovirus technology offers an attractive possibility to a scalable virus production as a result of ease production and concentration

ofbaculoviruses,efficient transduction of suspension mammalian cells in serum free conditions and safety of the baculoviruses,As a firststep towards scalable lentiviralproductionsystem we

BAC-gag-pol, BAC-VSVg and BAC-rcv,expressingall c1cmcnts required for

Different baculovirusconcentrationswere used to findoptimal

the conventional four plasmid method Lentivirus transduced Hela

exprcs-sion Our results show for the first time that baculoviruses can be

308 Titer and Long Term Stability of Retroviral Titers Produced in Murine Based Producer Cell Lines

'Medical and Molecular Genetics /National Gene l-ector Labora-tory Indiana University School ofMedicine Indianapolis IN.

While the spectrum of integratingvectors under considerationfor

retrovi-ruses continue to beutilized and improved.Tobetter understand

ofdeletions in the transgene portion of the GcSAM vector (kindly provided by Richard Morgan, NIH) that contains non-expressing

Eight constructs were evaluated with genome sizes ranging from

3258 to 6970 bascpairs Titer was assessed using real-time PCR for

within the packaging sequence Titer for vector genornes between

4559 and 6346 basepairs had similar titers, with a decrease outside

evaluated Data from 27 PG13and 5 GP+envAM12derived Master

titer of vector produced at 32 or 37°C and harvest intervals of 8,

Molecul ar The 4lpy Volume 15.Suppl ement t• • \br 2007

Co pyright © " 111e Ameri can SOI;ic;ty o f Gene