420 the challenge of HSCs procurement for gene therapy of beta thalassemia: exploring plerixafor as mobilization agent

420 The Challenge of HSCs Procurement for Gene Therapy of beta Thalassemia Exploring Plerixafor as Mobilization Agent Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Socie[.]

Molecular Therapy Volume 21, Supplement 1, May 2013

Copyright © The American Society of Gene & Cell Therapy S161

HEMATOLOGIC AND IMMUNOLOGIC GENE & CELL THERAPY I

through both the TPO and TGF-beta pathways, with signifi cantly

increased levels of phosphorylation of MAPK and Smad proteins

respectively Biotinylation studies revealed increased levels of

cell surface receptors following mTOR inhibition Pharmacologic

disruption of endocytosis reversed the effects of rapamycin treatment,

with equivalent cytokine signaling in treated and untreated cells In

utero HSC transplantation studies revealed signifi cant enhancement

of HSC engraftment levels in rapamycin-treated cells compared to

fresh whole bone marrow controls (Figure 1)

Conclusions: These results support a role for mTOR in modulating

HSC signal transduction by regulation of cytokine receptor

endocytosis, resulting in optimized engraftment following

transplantation These studies will ultimately improve understanding

of how the manipulation of multiple signaling circuits may be

optimized to control self-renewal and quiescence of HSCs to best

support in vitro expansion and transplantation

Erythroid/Megakaryocyte Precursors from Cord

Blood CD34+ Cells with Hyperactive Variants of

Pharmacologically-Regulated Mpl

Eyayu Belay,1 Christophre P Miller,1 Amanda Kortum,1 Beverly

Torok-Storb,2 C Anthony Blau,1 David W Emery.1

1 Institute for Stem Cell & Regenerative Medicine, Univeristy

of Washington, Seattle, WA; 2 Clinical Research Division, Fred

Hutchinson Cancer Research Center, Seattle, WA.

The clinical utility of allogeneic umbilical cord blood (CB)

transplantation is limited by poor engraftment secondary to the

inadequate number of hematopoietic stem/progenitor cells per cord

and the delayed differentiation of these cells post-transplant We

have been investigating a means of expanding and activating these

hematopoietic stem/progenitor cells based on a pharmacologically

regulated Mpl cell growth switch This involves fusing the

signaling domain of the thrombopoietin receptor Mpl to an artifi cial

dimerization domain that is, in turn, specifi c for a small drug molecule

called a chemical inducer of dimerization (CID) We previously

reported that viral vector transduction of CB CD34+ cells with

a wild-type version of this Mpl cell growth switch allowed for a

20-fold, CID-mediated expansion of total cells in culture (Richard

 Blau, 2003) However, in contrast to similar studies with mouse

bone marrow which allowed for the virtually limitless expansion of

a primitive undifferentiated population in culture, these CB CD34+

cultures underwent terminal differentiation after a few weeks, with

a predominantly erythroid phenotype In order to determine whether

this performance could be improved by augmenting Mpl signaling,

we modifi ed the conventional hybrid Mpl cassette by deleting known

target sites involved in the degradation of Mpl These hyperactive

variants of Mpl in factor-dependent BaF/3 cells reduced the threshold

for CID responsiveness, with the strongest response achieved

by deleting an AP2 binding site involved in lysosomal targeting Studies with CB CD34+ cells demonstrated this delta-AP2 variant increased expansion of total cells nearly 3-fold, to an average of over

80 times the starting number by day 21 of culture (P=0.03) This included a 4.5-fold increase in the expansion of CD41a+/CD42b+ megakaryocytes by day 14 (P=0.01) and a 3.1-fold increase in the expansion of CD235a+/CD41a- erythroid cells by day 21 (P=0.03)

We also noticed a nearly 6-fold increase in an atypical CD235a+/ CD41a+ cell population (P=0.05), peaking at 14+/-3% of all cells

at day 14 of culture for the delta-AP2 vector Studies from others

in mice reported the emergence of a similar cell population under conditions of hematopoietic stress that exhibited an erythroid/ megakaryocyte bi-potential precursor phenotype In our studies, sorting and colony assays demonstrated a commensurate increase

in the number of clonogenic progenitor colonies, restricted largely

to the CD235a-/CD41a- population, although two of the 5 colonies grown from the CD235a+/CD41a+ population in CID exhibited a CFU-Mix phenotype with both erythroid and megakaryocytic cells

In conclusion, these hyperactive variants appear to improve the overall potency of the Mpl-based cell growth switch, with the greatest expansion seen with an apparently novel erythroid/megakaryocyte bi-potential cell population

Gene Therapy of beta-Thalassemia: Exploring Plerixafor as Mobilization Agent

Maria Rosa Lidonnici,1,5 Annamaria Aprile,1 Marta Frittoli,1,5 Giacomo Mandelli,1 Bernhard Gentner,1,2,5 Laura Bellio,3 Elena Cassinerio,4 Laura Zanaboni,4 Silvano Rossini,3 Maria Domenica Cappellini,4 Fabio Ciceri,2 Sarah Marktel,2 Giuliana Ferrari.1,5

1 H San Raffaele-Telethon Institute for Gene Therapy (HSR-TIGET), Milan, Italy; 2 Hematology and Bone Marrow Transplantation Unit, San Raffaele Scientifi c Institute, Milan, Italy; 3 Immunohematology and Transfusion Medicine Unit, San Raffaele Scientifi c Institute, Milan, Italy; 4 Università di Milano, Ca Granda Foundation IRCCS, Milan, Italy; 5 Università Vita-Salute San Raffaele, Milan, Italy.

Successful gene therapy of inherited blood diseases relies on transplantation and engraftment of a signifi cant dose of autologous genetically engineered hematopoietic stem cells (HSCs) Gene therapy trials in young pediatric patients are performed by transplanting CD34+ cells from steady state BM, while in adults G-CSF mobilized peripheral blood cells are the preferred targets In the context of gene therapy for thalassemia, the choice of HSC source

is crucial since intrinsic characteristics of patients (splenomegaly and thrombophilia) dictate caution in the use of G-CSF and prompt investigation of new agents A phase II clinical protocol exploring the use of Plerixafor as a single mobilizing agent in adult patients affected

by transfusion dependent beta-thalassemia was approved (EudraCT 2011-000973-30) and started in 2012 Plerixafor selectively and reversibly antagonizes the binding of SDF-1 to its receptor CXCR4 with subsequent egress of HSCs to the peripheral blood Aims of our trial were to explore the ability of Plerixafor in inducing safe and effective stem cells mobilization, to characterize mobilized stem/ progenitor cells in the BM and peripheral blood and to achieve gene transfer effi ciency in mobilized CD34+ cells at a level comparable to that obtained using steady state BM cells Four subjects were enrolled and treated by subcutaneously administration of Plerixafor at the single dose of 0.24 mg/kg followed by leukoapheresis Mobilization

of CD34+ cells occurred very rapidly with a peak between 7 to 9 hrs Only one patient received a second dose (0.40 mg/kg) at 24 hrs after the fi rst one and underwent a second leukoapheretic procedure Three out of four patients achieved the minimal target cell dose (2 x 106 cells/kg) and no severe adverse events occurred Purifi ed CD34+ cells

Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy

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HEMATOLOGIC AND IMMUNOLOGIC GENE & CELL THERAPY I

from leukoaphereses were analyzed for their biological and functional

properties, subpopulations composition and expression profi le In

vivo reconstitution potential and lymphomyeloid differentiation were

tested following transplantation in NSG mice We also compared

Plerixafor-mobilized peripheral blood cells with CD34+ cells derived

from BM pre- and post-Plerixafor treatment Cells were transduced

with GLOBE lentiviral vector, carrying the beta-globin gene, to

assess gene transfer effi ciency and transgene expression The results

indicate that cells mobilized by Plerixafor have a primitive phenotype

with a high in vivo hematopoietic reconstitution potential and are

effi ciently transduced, thus representing a suitable source of target

cells for gene therapy

Transplant and In Vivo Chemoselection of Mutant

MGMTP140K Gene-Modifi ed Hematopoietic Cells

Christopher R Burtner,1 Devikha Chandrasekaran,1 Brian C

Beard,1 Hans-Peter Kiem,1 Jennifer E Adair.1

1 Clinical Research Division, Fred Hutchinson Cancer Research

Center, Seattle, WA.

Engraftment of a suffi cient number of gene-modifi ed hematopoietic

stem cells (HSCs) to elicit a therapeutic effect remains a signifi cant

hurdle to the success of gene therapy for certain diseases where

gene-modifi ed cells do not have an intrinsic in vivo selective advantage over

non-modifi ed cells While drug resistance gene therapy in HSCs may

provide a strategy to overcome this limitation, the biological effects of

repeated chemoselective pressure on gene-modifi ed cells is unknown

It has been established that leukocyte telomere attrition correlates with

increasing age in patients, and HSC transplant has been demonstrated

to accelerate human telomere attrition in both the autologous and

allogeneic transplant setting Thus, we applied a quantitative PCR

method of leukocyte telomere length (LTL) measurement to determine

whether repetitive chemoselection after autologous transplant of

O6-benzylguanine (O6BG)-resistant methylguanine methyltransferase

(MGMTP140K) gene-modifi ed CD34+ cells caused accelerated

telomere shortening in both the pigtailed macaque (Macaca

nemestrina) and in three brain tumor patients enrolled in a clinical

trial (NCT #00669669) We did not observe telomere attrition with

age in the pigtailed macaque, consistent with telomere dynamics in

the rhesus macaque and likely due to variation among animals in

baseline telomere length Interestingly, in an analysis of 28 pigtailed

macaques receiving high-dose total body irradiation followed by

autologous MGMTP140K gene-modifi ed HSCs, we did not see a

decrease in LTL at time points immediately following and up to one

year post-transplantation In 16 animals that received subsequent

rounds of non-myeloablative O6BG and the alkylating agent

bis-chloroethylnitrosourea BCNU for chemoselection, a trend in LTL

shortening was observed that did not reach statistical signifi cance

In 11 animals for which longitudinal data is available, individual

telomere trends varied, but interestingly, 5 animals exhibited a

positive correlation between gene marking levels and telomere length

as a result of chemoselection These data suggest that expression of

the MGMTP140K transgene in modifi ed cells may exert a role in

telomere maintenance to a degree that corresponds with the level

of gene marking Analyses of LTL in gene modifi ed cells compared

to non-gene-modifi ed cells of different lineages are ongoing In

2 of 3 patients who received MGMTP140K-modifi ed mobilized

CD34+ cells after non-myeloablative conditioning with single-agent

BCNU, the administration of 3-4 rounds of O6BG and temozolomide

chemotherapy had no signifi cant effect on telomere length, although

LTL for all three patients at the beginning of study was lower than that

expected due to age alone However, in the third patient, a signifi cant

decline in gene marking was observed after 7 chemotherapy cycles

that corresponded with a >40% decline in LTL This is consistent

with the hypothesis that expression of MGMTP140K, measured as

the number of gene-modifi ed cells, positively regulates telomere maintenance in a way that counters the loss expected from the replicative demand of transplant and chemotherapy

Hematopoiesis in an NSG Transplantation Model

after In Vitro Expansion of Transduced Cord Blood

CD34 Cells

Reinhard Haemmerle,1 Ruhi Phaltane,2 Michael Rothe,1 Thomas Moritz,2 Ute Modlich.1

1 Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany; 2 REBIRTH Research Group Reprogramming and Gene Therapy, Hannover Medical School, Hannover, Germany.

Transplantation of genetically modifi ed hematopoietic stem cells (HSC) can cause severe adverse reactions in patients due to insertional deregulation of endogenous genes Recently, great efforts were made

to achieve expansion of transplantable HSCs by the usage of novel

cytokines and small molecules during extended culture periods As in

vitro culture required for cell modifi cation may actually contribute to

adverse reactions, prolongation of ex vivo culture time and the choice

of cytokines may further affect the clonal repertoire of retrovirally transduced HSCs following transplantation To address this question

we transduced and expanded human cord-blood CD34+ (CB-CD34+) cells under four different cytokine conditions: SCF, TPO, and FLT3L alone (1), plus GCSF (2)1, or plus StemReginin-1 (3)2; and SCF, TPO, FGF-1, IGFBP-2, Angptl5 (4)3 After 10 days of expansion, the progenies of 5x104 CB-CD34+ cells were transplanted into NOD SCID.IL2Rgc-/- (NSG) mice (4 groups, n=6 each) Transductions were performed on days 2 and 3 (2xMOI20) with the mutagenic gammaretroviral vector RSF91.eGFP.pre with effi ciencies of 90%-99% irrespective of the cytokine conditions Cells expanded in medium containing StemReginin-1 formed 1.2-3.8-fold more colonies

in methylcellulose assay than cells cultured in the other cytokine conditions Moreover, clearly more cells retained expression of the CD34 marker (34% vs 9.4%-19.9%) FACS analysis of blood and bone-marrow 24 weeks after transplantation showed no statistically signifi cant differences between the conditions in terms of engraftment (39.4% (1), 17.6% (2), 25.1% (3), 27.0% (4)) or mean GFP expression (24.7% (1), 63.5% (2), 52.7% (3), 25.3% (4)) due to high variations within all groups Blood lineage contributions of transduced cells did not indicate vector-induced clonal outgrowth or lineage skewing and histopathology remained normal Insertion site analysis was performed

by LAM-PCR and deep sequencing on 24 mice and four pre-transplant samples A total of 3198 unique integration sites clustering around transcription start sites were retrieved From each mouse on average

72 ± 21 integrations were recovered, a clear reduction compared

to the pre-transplant samples (305 ± 118) In six mice integrations with high read counts (>50% of all reads) potentially representing dominant clones were detected Furthermore, preliminary analysis by nrLAM-PCR in three mice revealed additional unique integrations that were not found by LAM-PCR One of these integrations with a high read count (84% of total reads) was located in the fourth intron

of ANGPT1 Although comparison between the four groups was diffi cult due to high variations within the groups, deep sequencing in individual humanized NSG mice revealed integrations near relevant cancer-related genes and apparently dominant integrations Next, the infl uence of the vector insertion on the outgrowth of potential dominant clones will be further addressed 1Neff et al., Blood, 2005;

2Boitano et al., Science, 2010; 3Zhang et al., Blood 2008