1091 induction of molecular chimerism in rhesus macaques reconstituted with autologous CD34+ cells transduced with the gene encoding the ãžâ±(1 3)galactosyltranferase (αGT)

1091 Induction of Molecular Chimerism in Rhesus Macaques Reconstituted with Autologous CD34+ Cells Transduced with the Gene Encoding the α(1 3)galactosyltranferase (αGT) we assayed response ofpr[.]

we assayed response of primary macrophages from C57BLl6 mice

to Ad capsid components We found that I'ID-Ad DNA together

with penton proteins had the highest stimulatory effect In

conclu-sion, adenoviral vectors activate immunity through TLR-dependent

pathways leading to thc production of pro-inflammatory cytokines

and chemokines This is mediated cooperatively by cell surface and

endosomal receptors, TLR2 and TLR9,respectively Moreove,they

are dependent on a common TLR adaptorMyD88-1- By modulating

signaling via these receptors, we may be able to affect the innate

and adaptive immune responses to HDY in vivo

1089 An In Vitro Model of CD8+ T Cell-Mediated

Killing of AAV -Transduced Human Hepatocytes

Etiena Basner-Tschakarjan,' Daniel J Hui; Shangzhen Zhou;

Federico Mingozzi,' Katherine A High.P

I Hematology , The ChildrensHospital ofPhiladelphia,

Philadel-phia PA; ]Howard Hughes Medical Institute, PhiladelPhiladel-phia PA.

Adeno-associatedvirus (AAY) vectors have been successfully

used as gene therapy vectors, mainly to correct monogenic

disor-ders,where an advantage over other delivery systems is the mild

immune response evoked in animal models,allowing prolonged

expression of the transgene However,in a recent clinical trial of

AAY-mediated hepatic gene transfer for hemophilia B a CD8+ T

cell response to the vector capsid was documented We hypothesize

that this CD8+ T cell response eventually led to the destruction of

transduced hepatocytes with loss of transgene expression and an

accompanying reversible rise in serum transaminases In order to

further elucidate the underlying immune response as well as to try

to identify subjects prone to react to AAY capsid before treatment,

we established an in vitro system by which AAY capsid

epitope-expanded peripheral blood mononuclear cells (PBMC) can be tested

for their cytotoxic capacity towards virus-transduced target cells

We were able to identify several major T cell epitopes from normal

donors and from subjects enrolled in AAY gene therapy trialsusing

ELISpot assays and a peptide library derived from theAAY capsid

YP I protein sequence.Subsequently we used these peptide epitopes

to expand CD8+ T cells in vitro and also used them in a

cytotox-icity (CTL) assay with an HLA-matched hepatocyte cell line (an

immortalised primary human hepatocyte cell line kindly provided

by Arvind H Patel, University of Oxford, Oxford, UK) as target

cells Target cells were transduced with an AAY2 virus prior to the

assay and cytotoxicity could be shown as soon as four hours after

treatment (20% specific lysis at an E:T ratio of 10:I) with higher

levels after 48 hours (30% specific lysis) Peptide loading of the

hepatocytes with the relevant HLA-matched MHC class I epitope

led to constant high levels of cytotoxicity (30-40% specific lysis)

Our data suggest that human hepatocytes transduced in vitro with

AAY2 are able to process capsid antigen and present it in an MHC

I context to CD8+ T cells,recapitulating in vitro what we observed

in vivo in humans.Additional experiments with alternate serotypes

arc currently underway In conclusion we have established an in vitro

system for the expansion of human CD8+ T cells reactive to AAY2

capsid We have also established an in vitro cytotoxicity assay for

AAY2-transduccd human hepatocytes These results clearly

dem-onstrate that capsid-specific CD8+ T cells can lyse HLA-matched

AAY-transduced hepatocytes,providing strong supporting evidence

for our hypothesis that loss ofexpression and transaminitis observed

after hepatic artery infusion of AAY2-F.lX resulted from CD8+ T

cell-mediated lysis oftransdueed hepatoeytes The same system can

be used to test the performance of alternate serotypes This assay

system is particularly valuable since it has so far not been possible

to establish an animal model ofCD8+-mediated T cell destruction

ofAAY-transduced target cells

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GENE EXPRESSION IN ANIMAL MODEL SYSTEMS

1090 Prevention of Type I Diabetes Recurrence Following Islet Transplantation in Diabetic NOD Mice by Gene Therapy

Chaorui Tian,' Mohammed Javeed Ansari,'Jesus Paez-Cortez,' Jessamyn Bagley,' Mohamed Sayegh,' John Iacomini.'

'Transplantation Research Center; Brigham and Womens Hospi-tal and ChildrensHospital Boston, Boston, MA.

Introduction: We have recently shown that induction of mo-lecular chimerism by reconstitution of young NOD mice with autologous bone marrow cells transduced with retrovirus carrying genes encoding MHC class II~ chains from protective haplotypes can prevent occurrence of type I diabetes in NOD mice Here

we show that induction of molecular chimerism in mice that have spontaneously developed diabetes can be used to prevent recurrence

of type I diabetes following islet transplantation Methods: Bone marrow cells were harvested from 4-week old NOD mice treated with 5-fluorouracil (150mglkg) 7 days prior and then transduced with retrovirus carrying genes encoding either a protective MHC class II

~ chain fused to GFP (IAW-GFP) or GFP alone as a control Spon-taneously diabetic NOD mice were lethally irradiated and recon-stituted the following day with eitherIA~d-G FP or GFP transduced bone marrow All mice were maintained normoglycemic with daily subcutaneous insulin injection Four weeks after reconstitution, all recipient mice were transplanted with 1000 NOD.scm islets under the kidney capsule Blood glucose levels were measured daily after islet transplantation Islet grafts were harvested upon hyperglyce-mia or 90 days after islet transplantation Results: All of the mice reconstituted with control GFPtransduccd bone marrow developed diabetes by 9 days after islet transplantation (Median onset time (MOT)=2 days) Immunohistochemical studies showed that there was massive CD3+T cell infiltration and no insulin storage in the islet grafts ofthese mice In contrast, 5 out of6 mice reconstituted with protectiveIA~d-GFP transduced'bone remained normoglycemic for

90 days after islet transplantation (MOT= 90 days, n= 6, p<O.OOI) Histological studies showed that despite peri-islet infiltration of CD3+Tcells,insulin storage remained intact Interestingly,in these mice, we were able to detect significant numbers of Foxp.l" cells in the transplanted islets grafts Only lout of6 mice reconstituted with protective IAW-GFPtransduced bone marrow developed diabetes 42 days after islet transplantation.Conclusion: Induction of molecular chimerism can be used to prevent type 1 diabetes recurrence follow-ing islet transplantation in NOD miec with pre-existfollow-ing diabetes, Foxps" regulatory eclls may be involved in prevention ofdonor islet rejection by auto-reactive T cells in the recipients

1091 Induction of Molecular Chimerism in Rhesus Macaques Reconstituted with Autologous CD34+ Cells Transduced with the Gene Encoding the a(1-3)galactosyltranferase (aGT)

Lorenzo Benatuil,Robert Donahue, Aylin Bonifacino,Cynthia E Dunbar, John lacomini

'Transplantation ResearchCenter:Brigham and Womens Hos-pital and ChildrensHospital Boston , Harvard Medical School, Boston AlA; ]Hemat%gy Branch, NHBLI, NIH Bethesda, MD

Background: We have previously shown that genetic engineer-ing of autologous bone marrow stem cells to induce molecular chimerism can be used to establish B cell tolerance in mice In this study, we examined whether a similar approach could be used to induce B cell tolerance in Rhesus macaques,a preclinical animal model To this end,we investigated whether induction ofmolecular chimerism could be used to affect production of antibodies specific for the carbohydrate epitope Galal-3Gal~I-4GlcNAe-R (aGal)

Molecular Therapy Volume 15 Supplemen t I• \1 ,,)" 2007

C opyri ght © ' 111C A merican Societ y o f G ene Tllt'r.lP) '

Methods: CD34+ progenitors were isolated from the peripheral

blood of Rhesus macaques following mobilization with hG-CSF

and hSCF Autologous CD34+ cells were pre-stimulated with SCF,

simian immunodeficient virus carrying the gene encoding the aGT

enzyme (SIV-aGT) After transduction, the cells were infused into

lethally irradiated recipients All animals (2 experimental and I

All monkeys underwent rapid hematopoietic reconstitution (-18

days) and remained stable throughout the entire procedure Blood

samples were taken before and after reconstitution aGal

expres-sion was detected by flow cytometry and the levels ofaGal specific

antibodies were detected by ELISA Results: Cells expressing the

aOal epitope were readily detectable in the blood of both

approximately 15% of mononuclear cells expressed aOal epitopes

on their surface that was readily detectable by flow cytometry The

frequency ofCD20+, CD 16+ and CD 14+ cells expressing aGal was

express-ing aOal epitopes declined reachexpress-ing a stable level ofapproximately

01'5% ofblood mononuclear cells by day 60 Expression ofaOal on

CD34+ derived autologous cells significantly affected production of

aOal-specific antibodies 30 days following reconstitution, the titers

of aOaI-specific antibodies were approximately 3 times lower than

the titer observed pre-transplant The aGal-specific antibody titer

continued to fall over time, and was 6 times lower at day 60 All

monkeys remained in good health through the experiment and being

that de novo expression ofaOal on primate hematopoietic cells can

be used to affect levels ofpre-existing aOal-specific antibodies Our

results are in sharp contrast to those achieved using other methods to

removed aGal-specific antibodies such as extracorporeal adsorption

in which titers return to normal in 20 to 30 days after the procedure

While further experiments are needed to examine whether molecular

our results suggest that induction ofmolecular chimerism represents

a novel approach to specifically decrease antibody levels long-term

and perhaps avoid their production after tolerance induction

Neural Stem Cells and Their Neuronal Progeny for

MeCP2 Gene Therapy of Rett Syndrome

Mojgan Rastegar,' Peter Pasceri,' Maisam Makarem,' Shauna

disorder that results in mental retardation in females, It is caused

by mutations in the methyl-CpG binding protein-2 (MeCP2) that

participates in gene silencing directed by DNA methylation

Si-lencing of certain target genes is abrogated in RTT and leads to

with 10 fold more E I transcripts than E2 in brain RTr mutations

that reside only in E I suggest it may have an important function

is ex vivo gene therapy ofRTT using retrovirus delivery ofMeCP2

isoforms to transduce cycling Neural Stern Cells (NSC) derived

from McCP2 null mice Delivery of ex vivo transduced NSC into

the mouse brain and MeCP2 vector expression after differentiation

into neurons will be required to correct the R1T phenotype, MeCP2

is known to participate in silencing retrovirus vectors Therefore

we utilized a SIN backbone called HSC-I and a strong ubiquitous

internal EFI a promoter An EGFP vector was created that maintained

Molecular Therapy Volume 15 Supplement 1 ~b , 2007

long-term expression in undifferentiated fetal NSC for over 12

into beta-tubulin 3 positive neurons E I-myc and MeCP2-E2-myc retrovirus vectors were generated without EOFP, to avoid the possibility that MeCP2 expression might silence the vector by recognizing densely methylated CpG sites present in EGFP Both MeCP2 isoform vectors directed protein expression detected by Western blots on infected fetal NSC Immunostaining for myc and MeCP2 showed that the isoform vectors express in >70% of undif-ferentiated neurosphere cells over multiple passages, the isoforms

differentiation into beta-tubulin 3 positive neurons Because MeCP2

is known to cause neurological disease, we created vectors that employ the neuron-specific mouse MeCP2 promoter (McP) In this

undif-ferentiated neurospheres, but detected EOPP or the isoforms after

in NSC without observable silencing The MeCP2-EI-myc and MeCP2-E2-myc vectors will be valuable tools for distinguishing the biological functions of the two MeCP2 isoforms, and the re-stricted expression pattern by the MeP promoter in neurons is well

modified fetal NSC into E I0 mouse brains using ultrasound guided intracerebral injection, and are preparing for ex vivo infections of adult NSC derived from MeCP2 null mice Injected mice will be examined for MeCP2-myc isoforms by immunostaining to demon-strate long-term retrovirus expression after in vivo NSC delivery as

a first step towards RTT gene therapy

for Therapeutic Gene Transfer to Islets for Abrogation of Autoimmunity in Diabetes

ofPittsburgh School ofMedicine, Pittsburgh, PA; ]Division of Molecular Pharmaceutics UNC School ofPharmacy Chapel

University ofPittsburgh School ofMedicine Pittsburgh, PA.

The potential of self-complementary, double-stranded adeno-associated virus (dsAAV) vectors for efficient and long-term gene delivery into pancreatic cells, ineluding J3 cells within islets, has reeently been demonstrated We have compared the efficiency of dsAAV-mcdiatcd gene delivery to endogenous islets among

and express therapeutic transgenes in endogenous J3 cells was then

received adoptively transferred splcnocytes from 22- or 34-wcck-old NOD mice treated with dsAAV-lL-4 Thus, we speculate that J3 cell-specific expression of IL-4 plays an essential role in inhibiting the activation of disease-causing lymphocytes Alternatively, the

cells in the presence ofIL-4 These results demonstrate the utility of using the dsAAV8 vector to transfer irnmuno-rcgulatory agents to endogenous J3 cells in NOD mice to examine their role in preventing the onset of type-l diabetes

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