674 Long Term Safety of Clinical Grade LentiGlobin Vectors in a γ Thalassemic and Normal Mice Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Thera[.]
Trang 1Molecular Therapy Volume 21, Supplement 1, May 2013
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672 Transduction of Human CD34+ Cells in
the NOD SCIDγ (NSG) Mouse Model Using
Lenti-hWASP-Vectors Derived from Stable Producer
Clones
Matthew M Wielgosz,1 Yoon S Kim,1 Robert E Throm,1 Gael G
Carney,1 John T Gray,1 Arthur W Nienhuis.1
1 Experimental Hematology, St Jude Children’s Research Hospital,
Memphis, TN.
We have developed several producer clones for a Lenti-human
Wiskott Aldrich Syndrome Protein (hWASP) vector We previously
demonstrated (14th ASGCT Meeting, 2012) that concentrated vector
preparations from these clones transduced human CD34+ cells at
levels generally higher than preparations derived by concentrating
culture media from transfected cells, as assessed by PCR of clones
derived from transduced CFU-Cs Here we demonstrate that vector
preparations from 3 different producer clones transduced CD34+ cells
at levels (23%, 40%, and 46%) comparable to transiently derived GFP
and hWASP vector preparations (26% and 36%, respectively), with
MOIs ranging from 50-100 Following transplantation of human cells
into NSG mice (tail vain injection), harvest of human CD45+ cells
from murine bone marrow (BM) 3 months post transplantation (tibia
and fi bula), and inoculation of 100,000 BM-derived human CD45+
cells into methocult cultures, the percentages of hWASP PCR(+)
or GFP(+) CFU-Cs were higher for producer cell-derived vector
preparations (22%, 37%, and 57%) than those from transiently-derived
preparations (3.3% and 8.4% for hWASP and GFP, respectively),
despite similar levels of human CD45+ engraftment (ranging 11.3
to 20%) We used a vector preparation from one producer clone
(hWASP1) in a second CD34+-NSG transplant experiment, to examine
the effects of several small molecules and varied MOI on CD34+
transduction and engraftment, 3 months post-transplant Nicotinamide
(NAM) and EX527 (both SIRT1 inhibitors) increased human CD45+
engraftment levels (26% and 30%, respectively) over the untreated
control group (16.3%), but yielded decreased transduction levels
(25% and 7%, respectively, vs 37%) Interestingly, the addition of
stromal derived factor 1 alpha (SDF-1) to NAM-treated CD34+ cells
appeared to reverse the NAM-transduction defect (31% vs 17%), and
enhance human CD45+ engraftment levels (23% vs 14%) We also
observed that CD34+ cells that underwent a second transduction had
decreased human CD45+ engraftment levels (2.9%) over the control
group (16%), but had the highest human CD45+ cell transduction
level (55%) In addition, when WPRE mRNA expression levels were
measured by qRT-PCR (a surrogate to estimate hWASP expression
in CD45+ cells sorted from NSG BM), WPRE expression levels
averaged 81% of an RNA control derived from a transduced Jurkat
cell line (MOI 100), whereas the single transduction control group
averaged 44%, normalized to VCN These results confi rm that vector
preparations derived from our lenti-hWASP producer clone transduce
CD34+ cells at levels higher than those obtained from transient
vector preparations as assayed in the NSG mouse transplant model
Importantly, we demonstrate that the evaluation of the transduction
and engraftment of stem repopulating cells (SRCs) in the CD34+
-NSG mouse transplant model can be used to optimize transduction
conditions These fi ndings support our plans to utilize this producer
clone to make vector for our clinical trial
Vector Based Gene Therapy for SCID-X1
Gabrielle M Curinga,1 Iram Khan,1 Swati Singh,1 Joey Pangallo,1
Brian Beard,4,5 Troy Torgerson,1,2 Andrew Scharenberg,1,2 Grant Trobridge,6,7 Hans-Peter Kiem,4,5 David Rawlings.1,2
1 Center for Immunity and Immunotherapies, Seattle Children’s Research Institute, Seattle; 2 Immunology and Pediatrics, University of Washington School of Medicine, Seattle; 3 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle; 4 University of Washington School of Medicine, Seattle;
5 Pharmaceutical Sciences, Washington State University, Pullman, Washington; 6 School of Molecular Biosciences, Pullman.
X-linked severe-combined immune defi ciency (SCID-X1) results from inactivating mutations in the gene encoding the common gamma chain (c), a receptor sub-unit required for lymphoid development and cell function Unless successfully treated by hematopoietic stem cell transplantation, SCID-X1 is fatal within the fi rst years of life While HLA matched allogenic stem cell transplantation boasts survival rates exceeding 90%, such donors are rarely available and use of alternative stem cells sources results in poorer outcomes Thus, gene replacement therapy has emerged as a promising alternative for SCID-X1 patients lacking suitable donors Pioneering c gene replacement clinical trials demonstrated effi cacy, but also the need for improved safety due to adverse events associated with the gamma-retroviral delivery platform Although new self-inactivating (SIN) gamma-retroviral and lentiviral vectors appear to offer improved safety profi les relative to the original gamma-retroviral vector, the lack of native pathogenicity
of foamy viruses and an integration site profi le that is much less focused on either active genes or promoter regions than lentiviruses
or gamma-retroviruses suggest that FV vectors are the safest available platform for c gene replacement therapy We therefore initiated studies to determine whether clinical FV vectors driving transcription of human c will rescue lymphocyte development and function in SCID-X1 mice and in human cells; and whether this can
be achieved without evidence of toxicity Transduction of a human
T cell line lacking c with FV c vectors demonstrated high-level sustained c expression Further, FV transduction restored IL2, IL7,
or IL21 stimulation-dependent phospho-STAT3 or pSTAT5 signals Next, we established protocols resulting in consistent marking of purifi ed lineage depleted (lin-) murine hematopoietic stem cells (HSC) Co-culture of FV-transduced SCID-X1 HSC on a
Notch-ligand expressing stromal cell line, OP9-Delta 1, led to effi cient in
vitro T cell differentiation Thus, transduction with the FV c vector facilitates rescue of c expression and signaling in human cell lines
and in vitro T cell development of murine SCID-X1 HSC In vivo
functional recovery and integration site profi ling is currently being assessed using in SCID-X1 mice transplanted with FV c vector transduced HSC
LentiGlobin Vectors in a β-Thalassemic and Normal Mice
Olivier Negre,1 Cynthia Bartholoma,2 Robert Kutner,3 Beatrix Gillet-Legrand,1 Celine Courne,1 Anais Paulard,1 Byoung Ryu,3
Maria Denaro,3 Christof von Kalle,2 Emmanual Payan,4 Michell Finer,3 Gabor Veres.3
1 Bluebird Bio Inc, MA USA, Fontenay aux Roses, France;
2 DKFZ, National Center for Tumor Diseases (NCT), Heilderberg, Germany; 3 Bluebird Bio Inc, Cambridge; 4 Institute of Emerging Diseases and Innovative Therapies (iMETI), CEA, Fontenay aux Roses, France.
The long term safety of an improved LentiGlobin vector was evaluated by transplanting transduced Lin-depleted bone marrow cells isolated from-thalassemia (Hbbth1/th1) mice into syngeneic
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HEMATOLOGIC AND IMMUNOLOGIC GENE & CELL THERAPY II
myeloablated recipients The therapeutic effi cacies and potential
toxicity (histopathology, hematology) and insertion site analyses
were analyzed in 54 primary transplants A signifi cant improvement
of hemoglobin concentration, hematocrit level, red blood cell counts
and reticulocyte percentage were observed in mice transplanted with
bone marrow cells transduced with the LentiGlobin HPV569 lentiviral
vector or the LentiGlobin BB305 lentiviral vector compared to the
group transplanted with untransduced cells in the primary recipients
The improvement of -thalassemic phenotype was similar between
mice transplanted with cells transduced with LentiGlobin HPV569
lentiviral vector and mice transplanted with cells transduced with
LentiGlobin BB305 lentiviral vector No toxicological changes
were observed in the treated groups A comparative large scale
integration site analysis has been performed on mouse bone marrow
samples transduced with the LentiGlobin HPV569 vector and with
the LentiGlobin BB305 lentiviral vector and the analyses of the pre
transplant samples showed a highly polyclonal vector integration
profi le in both LentiGlobin vectors (BB305, HPV569) IS analyses in
samples derived from the transplanted - thalassemic (Hbb th1/th1)
mice revealed an oligo-/polyclonal hematopoietic repopulation for
both LentiGlobin (BB305: 3508 unique IS; HPV569: 4257 unique IS)
vectors Secondary transplants were also conducted (total of 108 mice)
to evaluate the toxicity of LentiGlobin vectors BB305 and HPV569 in
CD45.1 C57BL/6 mice following a single intravenous administration
of “secondary” bone marrow obtained from -thalassemic Hbb th1/
th1mice originally transplanted four months earlier with syngeneic
Lin depleted mouse bone marrow cells transduced with LentiGlobin
BB305 and Lentiglobin HPV 569 lentiviral vectors The secondary
transplant mice are sacrificed and necropsied six months after
transplantation and an extensive histopathology evaluation are
performed to assess hematopoietic homeostatis, clinical chemistry,
hematology and bone marrow cytology In addition, comprehensive IS
analyzes is being performed on DNA samples isolated from the bone
marrow of the secondary recipients.A comprehensive data set will
be presented on both the primary and secondary transplant animals
Insulated LV Driven by FANCA-Promoter towards
GT in Fanconi’s Anemia
Emilie Bayart,1 Caroline Duros,1 Alexandre Artus,1 Stephanie
Lemaire,1 Odile Y D Cohen-Haguenauer.1,2
1 CNRS UMR 8113, Ecole Normale Superieure de Cachan, Cachan,
France; 2 Oncogenetics-Medical Oncology, Hopital St-Louis
Sorbonne Paris-Cité, Faculté de Médecine Paris-Diderot, Paris,
France.
Fanconi anemia is a rare inherited genomic instability syndrome,
with severe bone-Marrow (BM) defi ciency developing around age 7
The 15 known genes are involved in DNA repair, redox metabolism
and differentiation FA patients’ cells exhibit hypersensitivity to DNA
crosslinking agents such as Mitomycin C (MMC) In a previous study,
we have: (i) demonstrated that BM dysfunction can be ameliorated
in protecting cells from oxidative stress in growing cells with both
hypoxia and N-Actyl-Cystein in the culture medium and (ii) achieved
in vitro and in vivo long-term reconstitution with retrovirus transduced
cells from unfractionated patients’ BM (Cohen-Haguenauer et al,
2006) With view to reducing the risk for GT induced malignancies
in this cancer-prone disease, we have developed a new generation of
genetically stable insulated vectors which have been used to shuttle the
FANCA cDNA Transduction of HSC72 FA cells resulted in functional
correction with overcoming the G2M block on high doses MMC
and restoring FancD2 mono-ubiquitination In genetically corrected
primary mesenchymal cells from FANCA-/- patients the transgene
is stably expressed and the insulator sequences preserved after 18
months of sustained growth Three different internal promoters –
with distinct potentials- have been tested: the strong viral derived
Fr-MuLV-U3, the housekeeping hPGK or the FANCA homologous promotor (FANCendo) that we have engineered The latter mediates low basal expression levels with EGFP1 mean fl uorescence reduced
by one and two ranges compared to hPGK or viral promoters, respectively In primary MSCs cells grown under Mitomycin C while retrieving protection from oxidative stress, the earliest and most signifi cant recovery is monitored in cells transduced with FANCendo Data will be shown in CD34+ cells effi ciently transduced with the FANCendoInsLV and grown over 8 weeks in LTCIC without toxicity and patients’ samples This new insulated lentivector driven by FANCA-homologous promoter shows improved safety and effi cacy profi le towards gene therapy in Fanconi’s anemia