245 Hematopoietic Stem Cell Gene Delivery High Efficiency Gene Transfer to HSC In Vivo with Long Term Expression of Anti HIV Transgenes after Intramarrow Injection SV40 Derived Vectors 243 Insertion S[.]
Trang 1243 Insertion Site Analysis Following
Gammaretroviral Gene Therapy for Canine
Leukocyte Adhesion Deficiency Reveals Lack of
Genotoxicity
Mehreen Hai,' Thomas R Bauer, Jr,' Laura M Tuschong,'
Yuchen Gu,' Xiaolin WU,2 Dennis D Hickstein.'
'Experimental Transplant and Immunology Branch, National
Cancer Institute, National Institutes ofHealth, Bethesda, MD;
2LaboratoJY0/Molecular Technology, SAIC - Frederick, Inc,
NCI at Frederick, Frederick, MD.
Recent successful hematopoietic stem cell gene therapy and
gene marking studies using gammaretroviral vectors have typically
involved a selective growth advantage for the transduced cells
provided by either the therapeutic transgene or a myeloablative
conditioning regimen Genotoxicity has occurred in both
situa-tions: three patients in thc French X-SCID clinical trial developed
leukemia associated with retroviral insertions in the LM02 gene,
and a rhesus macaque developed a granulocytic sarcoma
accompa-nied by retroviral insertions in the BCL2-A I gene Providing the
transduced cells with a growth advantage may have contributed
to the genotoxicity However, in a recent non-myeloablative gene
therapy trial for chronic granulomatous disease (CGD), where there
was no selective advantage provided by the transduced gene, both
patients developed progressive oligoclonality of retroviral marked
cells accompanied by multiple retroviral insertions in EVII-MDS I,
PROMI6 and SETBPI The development of clonal dominance
de-spite a lack ofa selective growth advantage for the transduced cells
in the CGD trial suggests that the vector design and/or transduction
conditions may be important for the emergence ofclonal dominance
To compare our results with those in the CGD trial, we analyzed
the genotoxicity in our study of six dogs with canine leukocyte
adhesion deficiency (CLAD), who were successfully treated using
ex-vivo retroviral-mediated CDI8 gene transfer into autologous
C034+ cells preceded bynon-myeloablativeconditioning Similar
to the transgene in the CGO clinical trial, the therapeutic COl8
transgene does not confer a selective advantage upon the corrected
leukocytes Polyclonality oftransduced cells was demonstrated by
linear amplification-mediated PCR (LAM-PCR) using peripheral
blood leukocyte DNA from all six dogs up to twoyearsfollowing
infusion of gene-corrected cells; no predominant clone emerged
over time Analysis of retroviraIintegration sites (RIS) using
liga-tion-mediated PCR (LM-PCR) identified 398 unique RIS, 53% of
which were located within genes (compared to 39% expected from
analysis of 10,000 randomly generated insertion sites, p<O.OOI)
Analysis of genes within 50 kb of the RIS revealed the absence
of LM02, EVII-MDSI, PRDMI6 and SETBPI, although 16%
of the RIS were within 50 kb of a cancer-related gene (expected:
7%, p<O.OOI) In addition, 14% of the RIS were within 50 kb of
genes associated with RIS in the CGO clinical trial (expected: 10%,
p<O.OI), indicating significant similarities between the RIS found in
our and the CGO study However, despite the parallels between the
two studies, we observed nogenotoxicityin any of the animals in
our study, suggesting that vector design and transduction conditions
may influence the risk for clonal dominance
244 Role of Chromatin Structure in Integration
Site Selection of Helper-Dependent Ad5/35
Vectors Carrying the Globin LCR
Hongjie Wang,' Pavel Sova.! Karol Bomstyk,' QiliangLi,'
George Stamatoyannopolous,' Andre Liebcr.P
'Medical Genetics, University ofWashington, Seattle, filA: 2Pa_
thology, University0/Washington, Seattle, H!;t.
Most approaches for targeted insertion oftransgenes in stem cells
use rctroviral vectors, Wc constructed helper-dcpendentAd5/35
vee-Molecular Therapy Volume15.Supplement I ~Iay 11)Q7
Copyright © 1111; American SocietyorGene Therapy
tors that efficiently transduce human hematopoietic, mesenchymal and embryonic stem cells We found that HO Ad5/35 vectors carry-ing a 28kb fragment of the globin LCR (HD-Ad5135.LCR) prefer-entially integrated into the chromosomal beta-globin LCR ofhuman erythroid M07e cells (which express globin genes indicating activity ofthe globin LCR) A HD-Ad5/35 vector containing X-chromosomal stuffer DNA did not integrate into the globin LCR ofM07e cells
We are currently analyzing the integration pattern of HD-Ad5/35 LCR and control vector in non-erythroid cells (that do not express globin genes) We speculate that the LCR carrying Ad vector co-localizes with the genomic b-globin LCR in a "transcription factory" and that this physical proximity, together with double-strand DNA breaks in "open" chromatin, allows for preferential integration
of our vector into the globin LCR To prove this hypothesis we mapped the chromatin structure ofthe globin LCR using chromatin immunoprecipitation (ChIP) assays These analyses revealed active chromatin within and downstream ofDNAse hypersensitivity region
2 (I-IS2) in erythroid M07e cells but not in non-erythroid cells Other LCR regions (I-ISI, I-IS3, I-IS4,1·IS5) did not show occupation
by "active" chromatin (in both erythroid and non-erythroid cells) Importantly, most HD-Ad5/35.LCR integrations in M07e cells were found within this area of"open" chromatin, indicating a role ofchromatin structure in integration site selection We arc currently tryingto identify complexes between chromosomal globin LCR and vector LCR in erythroid cells infected with HO-Ad5135.LCR us-ing ChIP with antibodies against Terminal Protein, a protein that is covalently linked to the incoming Ad vector genomes Our findings potentially create the basis for a new approach to achieve targeted integration, via chromatin-mediated co-localization of vector and chromosomal DNA Future efforts include the combination of this approach with induction of site-specific DNA breaks
245 Hematopoietic Stem Cell Gene Delivery: High Efficiency Gene Transfer to HSC In Vivo with Long-Term Expression of Anti-HIV Transgenes after Intramarrow Injection SV40-Derived Vectors Alena A Chekrnasova,' Mahender Singh; Jean-Pierre Loubou-tin,1David S Strayer.'
'Pathology, Thomas Jefferson University, Philadelphia, PA.
Gene transfer to hematopoietic stem cells (HSC) could be advan-tageous in treating many human diseases, including infection with HIV-I, providing a high enough percentage ofHIV-susceptible cells can be protected, for an extended period of time Stem cell-based gene therapy of HIV infection aims to inhibit HIV replication and progression of acquired immunodeficiency syndrome (AIDS) by introducing antiviral genes into HSC Ideally, after differentiation into mature blood cells, antiviral genes create a host-cell popula-tion resistant to HIV infecpopula-tion In this study to deliver anti-HIV transgenes into primitive hematopoietic cells of rabbits we used tag-deleted recombinant SV40 vector, SV(RNAiR5/RevM I 0), car-ries CMV-IEP driven anti-Rev (RevMIO) with carboxyl terminus
AU1epitope tag and RNAi R5, an interfering RNA against CCR5 driven by the adenovirus VA1polIIIpromoter Rabbits were injected intrafemorally with SV(RNAiR5/RevMIO) and transgenes expres-sion were tested in bone marrow (BM), peripheral blood and spleen
by flow cytometry (FACS) after intracellular immunostaining using commercial anti-AU I or anti-CCR5 antibodies Simple injection
of SV(RNAiR5/RevM 10) into the femoral bone marrow cavities results in an AU I expression 30-35% peripheral blood mononuclear cells (PBMC) Expression was maintained until the study was terminated at 56 weeks post-injection T-cells (CD3, CD4, C08), Bscells, monocytes (bearing CD 14) and granulocytes all expressed the delivered AUI epitope In addition, levels of cell membrane CCR5 were decreased by 30-50%, compared with mock-transduced animals We also tested expression ofAU I in the bone marrow and
S93
Trang 2spleen 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 the 06
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.
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
S94
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
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