247 long term efficacy and safety of hematopoietic stem cell gene therapy in the murine model of wiskott aldrich syndrome

247 Long Term Efficacy and Safety of Hematopoietic Stem Cell Gene Therapy in the Murine Model of Wiskott Aldrich Syndrome spleen of injectedanimals 30 35% offemoral BM cells expressed AUI at 2w and 56[.]

spleen of injected animals 30-35% offemoral BM cells expressed

AUI at 2w and 56w after receiving intramarrow SV(RNAiR51

RevM10) Percentages of marrow cells expressing AUI in other

bones that were not injected with the vector (humerous, tibia and

iliac crest) varied: in the humorus II % were positive for AUI; in

the tibia,31%; and in the iliac crest,45% The greatest percentages

of AUI-positive cells were in granulocyte series and in cells that

expressed stem cell antigen (SCA) Thus, 73.6% ofSCA+ cells in

the femur expressedAU I 56 weeks after intramarrowinjection At

56 weeks after intrafemoral injection with SV(RNAiR5/RevMIO),

the percentagesofSCA-positive cells in the various bonesthat were

also AUI-positive were: in the humerus, about 32%; in the tibia,

61.5%; and in the iliac crest 78.4% Other organs were studied for

AU1 expression 56 weeks after BM injection Of note, about 50%

of spleen cells, includingT lymphocytesof all lineages, B cells and

cells bearing CDI4 macrophage/monocyte marker Thus, direct

intramarrow injection ofrSV40 vectors in vivo is highly effective

in transducing HSC and preserving transgene expression in their

differentiated progeny, both in the blood and in the tissues The

success of this approach lies in the high transduction efficiency

of rSV40s for resting HSC and continued transgene expression in

daughter cells for extended periods of time

246 Very High Levels of Expression of the06

Methylguanine-DNA-Methyltransferase P140K

Mutant Results in an In Vivo Engraftment Defect

Michael D Milsom,' Chad E Harris,' Axel Schambach,' Jeff

Bai-ley,' Emily Broun; ChristopherBaum.PGeoffrey P.Margison,'

Ina Rattmann,' MoranJerabek-Willemsen;'Thomas Moritz.l-'

Jurgen Thomale,' Elke Grassman; David A Williams.'

I Department ofExperimental Hematology, Cincinnati Childrens

Hospital Medical Center, Cincinnati; lDepartment

ofExperimen-tal Hematology Hannover Medical School Hannover, Germany;

JCarcinogenesis Group, Paterson Institute for Cancer Research.

Manchester; United Kingdom; 'lnternal Medicine (Cancer

Research), University ofDuisburg-Essen Medical School, Essen,

Germany; Jlnstitute ofCell Biology University ofDuisburg-Essen

Medical School, Essen, Germany.

Thedrug rcsistaneegene~mcthyguaninc-DNA-methyltransfer

ase (MGMT) is of particular importancein the fieldof gene therapy

due to its proposed use in hematopoietic stem cell (I-ISC)

chemo-protective/chemoselectivestrategies OverexpressionofMGMT in

HSC confers protection against~ alkylating agents to HSC and

their matureprogeny Sinceone MGMTmoleculecan only detoxify

a single~alkylguanine adduct,it has been previouslyassumedthat

elevatedexpressionofMGMT (or mutantversionsofMGMT which

are resistantto pseudosubstrateinhibitorssuch as~benzylguanine

(6BG), e.g P140K) is beneficialfor HSC protection Weattempted

to assess whetherthe magnitudeofMGMT(P 140K)overexpression

hadany bearinguponHSCfate.Self inactivatingy-retroviral vectors

wereconstructedwhichcontainedthe MGMT(P140K)cDNAdriven

by either the spleen focus forming virus promoter (SF-MGMT) or

the weaker human cellular promoters: elongation factor-In short

form (EFS-MGMT) or phosphoglycerate kinase (PGK-MGMT)

In vitroassays using 32D cells reproducibly demonstrated a

pro-liferation defect in SF-MGMT transduced cells compared to EFS

or PGK-MGMTtransduced cells In primal)' murine bone marrow

cells (BMC) the level of MGMT activity was 2169, 287 and 330

fmol/mg DNA for cells transduced with SF,EFS and PGK-MGMT

respectively, compared to no detectable activity in control

trans-duced BMC We found that the approximate 7-fold lower level of

MGMT activity mediated by the EFS and PGK driven vectors was

still sufficient to protect progenitor cells against treatment with

6BG/temozolomide, detoxify~-methylguanine adducts formed

in vivo, and importantly also facilitatedin vivoselection of gene

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modified HSC In competitive repopulation assays in which there was no 6BG/temozoiomide treatment, cells harboring SF-MGMT demonstrated an approximate 50% reduction in the production of gene markedperipheral bloodcells over the course of6 months(sec table) In contrast, cells expressing EFS or PGK-MGMTgenerated stable grafts over the same time period We propose that the high level of MGMT(PI40K) expression driven by the SF promoter has

a subtle yet reproducibledeleteriouseffect upon hematopoiesisand that the use of weaker promoter elements should be considered for clinical applications

Compctetive en graftment of MGMT transducedcells ~ ~ _ ~ Days po st-trans plan t ( 5 :~ 7 II " d 1 42d _ p Id ;

Chimerism expressed as percentageof cells in peripheralblood at 35d post-trans-plant

247 Long-Term Efficacy and Safety of Hematopoietic Stem Cell Gene Therapy in the Murine Model of Wiskott-Aldrich Syndrome

Francesco Marangoni.P Marita Bosticardo.!Samantha Scar-arnuzza,' Cristina Panaroni,' Sara Trifari,' Michela Locci,' Elena Draghici,IAnneGaly,'LuigiNaldini,1.2AlessandroAiuti,ILore Dupre,1.4AnnaVilla.l-' Maria-GraziaRoncarolo.'-'

'San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), San Raffaele Scientific Institute, Milan, Italy; lVita-Salute San Raffaele University San Raffaele Scientific Institute, Milan, Italy; JUMR CNRS 8115, Genethon, Evry, France; 4INSERM U563, Purpan University Hospital, Toulouse, France; JHuman Genome Department, Istituto di Tecnologie Biomediche (CNR-ITB), Seg-rate, Milan, Italy.

Wiskott-Aldrichsyndrome(WAS)is a severe X-linked immuno-deficiencycharacterizedby recurrentinfections,thrombocytopenia, eczema and increased risk of autoimmune disorders and lympho-mas Hematopoieticstem cell (HSC) transplantation is the current therapy, however, it is not available tor all patients tor the lack of HLA-matched donors Transplantation of genetically corrected autologous HSC could represent an alternative treatment In a mu-rine model of WAS(lJ'IIS"),we recently demonstrated correction

of the T cell defect 4 months after lentiviral vector-mediated gene therapy [Dupre, Marangoni etal.,Hum Gene Ther 2006, 17] The aim of the present study was to investigate the long-term efficacy

and safety of our gene therapy approach in WAS' mice

Transduc-tion ofJE4S·HSC was performedwith a Ientiviralvectorencoding human WASPunder the control ofa 1.6Kb fragmentof the human

WAS autologouspromoter.TransducedHSC were transplanted into

sub-lethally irradiated11'1105" mice Mice were sacrificed 7 and 12 months after gene therapy Donor engraftment in the bone marrow and splenic T cells was >80% WASP expression was detected in 30% of bone marrow CD45+ cells, and in similar percentages of peripheral myeloid cells In the spleen, 75% ofT cells and 50% of

B cells expressed WASP, suggesting a selective advantage for gene corrected cells in these lineages Importantly, functional correction

of splenicT lymphocytes after gene therapy was documented by the complete restoration ofTCR-driven proliferation and cytokine production.Safety of gene therapywas alsodemonstrated.No over-expression of WASPwas observed in bone marrow and splenic T cells, regardless of the lentiviral vector copy number Absence of leukemias or lymphomas was demonstrated by blood count and FACS analysisperformedon blood andspleen, Histopathologic analysis of thymus,spleen,lymph nodes and bone marrow did not show any abnormalities Long-term survival of the gene therapy treated mice was comparable to that of control mice transplanted

with untransducedWAS'-HSC In conclusion, we provideevidence

of engraftment of WASP-expressing cells and restoration ofTCR-driven T cell proliferation without toxicity, up to 12 months after

Molecular Therapy Volume 15 SupplementI•.\by 20m

HSC gene therapy.Experiments aimed at investigating whether II"lS

gene therapy can correct the defectsofplatelets,B cells and dendritic

cells,and restore normalin vivoimmune responseto antigens are

ongoing Results from these studies will contribute to the design of

a clinical trial for Wiskott-Aldrich syndrome

248 Evidence of Globin Lentiviral

Vector-Induced Genotoxicity in a Murine Model of Sickle

Cell Disease

Pestina I Pestina,' Hargrove W.Hargrove,'Huifen Zhao; Kumar

Srivastava,'Gray T.John,IDerek A Persons.'

1 Hematology St Jude ChildrensRe search Hospital, Memphis ,

TN ; 2Department ofBiostatistic s, St Jude Children's Research

Hospital, Memphis TN.

Previously, we evaluated the therapeutic efficacy of a 3'd/4~'

generation HIV-bascd, SIN lentiviral vector encoding the human

y-globin gene driven by a~-globin promoter and 3 kb of elements

from the ~-globin LCR in murine ~-thalassemia (Hanawa et aI.,

Blood 104:2281-2290; 2004) This vector corrected the thalassemic

phenotype at relatively low average vector copy number (1.3) Here,

we describe testing ofthis vector containing the 250 bp x 2 HS4 core

insulator fragment from the chicken~.globin Locus Control Region

in a"double copy" LTR format in the BERK sickle cell disease

(SCD) mouse model This vector and a control vector containing

an MSCV LTR-driven YFP marker gene were used to transduce

lineage negative SCD bone marrow (BM).Thirteen of 16 CFU-S

clones derived from cells transduced with the globin vector were

positive for the vector genome by Southern blot analysis, consistent

with high efficiency gene transfer into primitive cells Lethally

ir-radiated wild-type recipients (n=10)of the same transduced SCD

cell population expressed human y-globin with HbF'"cells ranging

from 1-23% at 12 weeks post-transplant.Although the overall Hb

level in these mice was notyet significantly improved,relative to the

YFP transplanted control animals,the number of irreversibly sickle

cells (ISCs) per high powered field (PHPF) was significantly reduced

in the globin vector animals relative to YFP control animals (mean

of2.2±0.4 ISCslPHPF vs.6.6±0.9;p<0.009).Further molecular

analysis of the above globin vector-transduced CFU-S with

indc-pendent Southern blot analyses using different restriction enzymes

showed an identical banding pattern in all 13 independently derived

clones,consistent with 2 integrated copies of the vector Assuming

equal growth kinetics ofall transduced CPU-S, the probability ofthe

same clone being identified 13 times is 1.8 x10-34•This suggested

that a significant growth advantage was endowed on this particular

transduced,CFU-S-forming cell _LM-PCRanalysis of several of

the isolates ofthis clone, coupled with Blast sequence analysis using

the NCB! mouse genome database,independently confirmed that the

same genomic DNA-vector junction fragments were present in all,

with one ofthevector insertions being an intergenie integrantlocated

in a gene dense region on chromosome 9 Some ofthe genes nearby

the vector insertion included Myo Ie,a Rho GTPase which functions

in intracellular signaling (70 kb away),Ccnb2 (cyclin B2),a

cyclin-dependent kinase important in controlling cell cycle progression (180

kb away),and Bnip2,a Bcl-2 interacting protein(230 kb away).The

250 bpx2 HS4 core insulator fragmentwas completed deleted in

the CFU-S clones as well as in independently transduced NIH3T3

cells.These results suggest that the 2 x 250 bp configuration of the

insulator element isunstable.The globin LCR elementsappear to

have potential genotoxicity in primitive hematopoietic cells leading

to the emergence of clonal dominance during in vitro and/or in vivo

cell division while forming spleen colonies

Molecular Therapy Volume15.Suppkmcnll M ,')' 2 007

C op)'rihht © The American Scciery of Gene " Il ler-Ill) '

249 Gene Therapy in Adenosine Deaminase-Deficient Children with Autologous Bone Marrow Cells Transduced with Two Retroviral Vectors after Withdrawal of Enzyme Replacement Treatment and Low-Dose Chemotherapy

Robert A Sokol ic,IGreg Podsakoff,' Linda Muu!,IBarbera Engel,' Elizabeth K Garabedian; DeniseCarbonaro.'Joanna Ireland.' Laura M.Tuschong,?Michael S.Hershfield,'Donald B Kohn,' Fabio Candotti.'

J Disorders0/Immunity Section , Genetics and Molecular Biology Branch National Human G enome R esearch Institute, Bethesda

MD ; lDivision ofResearch Immunology /Bon e Marrow Trans-plant, The Saban Research Institute ofChildrens Hospital Los Angeles Department0/Pediatrics, Los Angeles, CA; "Expertmen

-tal Transplantation and Immunology Branch, National Cancer In-stitute Bethesda, MD; 'Department ofMedicine, Duke University Medical Center, Durham, NC.

Adenosine deaminase (ADA) deficiency can result in severe combined immunodeficiency (SCID),a life-threatening pediatric emergency Enzyme replacement treatment with polyethylene gly-col-conjugated bovine ADA (PEG-ADA) is life-saving, but it usu-ally does not restore normal immunity,its efficacy may decline with time and its costs are often prohibitive outside the USA.Allogeneic hematopoietic cell transplantation is curative,but a matched related donor is available for only a minority of patients,and alternative donor transplants carry a high rate oftoxicity For these reasons,gene therapy has long been proposed as an alternative form of treatment for this disease and has recently proven beneficial in Europe and Japan In November,2005 we began a clinical gene therapy trial based on infusion of genetically corrected autologous bone mar-row cells after withdrawal of PEG-ADAand after treatment with rnyclosupprcssivechemotherapy The first treated patient (ADA-302N) suffered from an unexpected prolonged marrow aplasia due

to pre-existing trisomy 8 abnormalities of her bone marrow cells

No engrafiment ofgene corrected cellswas documented,as reported (Blood 2007; 109:503) Our second treated patient (ADA-30IN)

is a 2-year-old boy who was admitted to the NIH Clinical Center in January, 2007 Bone marrow harvest yielded 92.5x 10"6CD34+ cells that,after pre-stimulation with SCF, MGDF and Flt3-L,were divided into two aliquots and separately transduced with the MND-ADA and GCsapM-ADA retroviral vectors.Two days before infusion of gene corrected cells, the patient received busulfan (37.5 mg/m2 IV

x 2) Areas under the curve for the two doses were 1451 and 1583 mcM/minute, respectively On day 0,the patient received 5.lx I0"6 CD34+ cell/kg ADA activity in the GCsapM-ADA and MND-ADA transduced cells was >200 and 43 nmol/min/10"8cells,respectively (ADA activity in healthy control CD34+cells=-70 nmol/minll 0"8 cells) Chemotherapy was well tolerated.The absolute neutrophil count fell below 50D/mcLon day 12,at which time the platelet count wasstill within normal limits It is hoped that this protocol will allow

us to follow the engraftrnent of cells corrected by each vector and determine if either one ultimately contributes more strongly to the recovery of lymphoidimmunity

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