Correction of murine
-thalassemia after minimal lentiviral gene transfer and ho

Correction of murine
-thalassemia after minimal lentiviral gene transfer and
homeostatic in vivo erythroid expansion

*Olivier Negre,1-5 *Floriane Fusil,1-4 Charlotte Colomb,1-3 Shoshannah Roth,6 Beatrix Gillet-Legrand,1-5 Annie Henri,2,3
Yves Beuzard,1-5 Frederic Bushman,6 Philippe Leboulch,1-3,7 and Emmanuel Payen1-4
1Commissariat a` l’Energie Atomique (CEA), Institute of Emerging Diseases and Innovative Therapies (iMETI), Fontenay-aux-Roses, France; 2Inserm U962
CEA-iMETI, Fontenay-aux-Roses, France; 3Universite´ Paris XI, CEAiMETI, Fontenay-aux-Roses, France; 4Universite´ Denis Diderot-Paris VII, Institut
Universitaire d’He´matologie, Paris, France; 5Bluebird bio France, CEA-iMETI, Fontenay-aux-Roses, France; 6Department of Microbiology, University of
Pennsylvania School of Medicine, Philadelphia, PA; and 7Genetics Division, Brigham & Women’s Hospital and Harvard Medical School, Boston, MA
A challenge for gene therapy of genetic
diseases is to maintain corrected cell
populations in subjects undergoing transplantation
in cases in which the corrected
cells do not have intrinsic selective advantage
over nontransduced cells. For inherited
hematopoietic disorders, limitations
include inefficient transduction of stem
cell pools, the requirement for toxic myelosuppression,
and a lack of optimal
methods for cell selection after transduction.
Here, we have designed a lentiviral
vector that encodes human
-globin and
a truncated erythropoietin receptor, both
under erythroid-specific transcriptional
control. This truncated receptor confers
enhanced sensitivity to erythropoietin and
a benign course in human carriers. Transplantation
of marrow transduced with the
vector into syngenic thalassemic mice,
which have elevated plasma erythropoietin
levels, resulted in long-term correction
of the disease even at low ratios of
transduced/untransduced cells. Amplifi-
cation of the red over the white blood cell
lineages was self-controlled and averaged
 100-fold instead of  5-fold for

-globin expression alone. There was no
detectable amplification of white blood
cells or alteration of hematopoietic homeostasis.
Notwithstanding legitimate
safety concerns in the context of randomly
integrating vectors, this approach
may prove especially valuable in combination
with targeted integration or in situ
homologous recombination/repair and
may lower the required level of pretransplantation
myelosuppression. (Blood.
2011;117(20):5321-5331)
Introduction
Recent progress in the field of hematopoietic gene therapy has
raised the hope that patients afflicted with
-thalassemia and sickle
cell anemia will benefit from these novel therapeutic approaches.
Transplantation of hematopoietic cells (HCs) modified with lentiviral
vectors carrying the
-globin gene has resulted in long-term
correction of several mouse models of hemoglobin disorders1-4 and
very recently led to transfusion independency in a
-thalassemic
patient.5 Although the main advantages of infusing genetically
modified autologous cells are to avoid the risks of GVHD and
immunosuppressive pretransplant conditioning as well as to address
the lack of compatible donors, a drawback is the requirement
for toxic myeloablation.6 In addition, current gene transfer methods
are unable to transduce more than a fraction of hematopoietic stem
cells (HSCs),7 and the various in vivo selection strategies available
suffer from suboptimal efficacy and safety.8-10
Therapeutic and stable mixed chimerism has been observed in a
few
-thalassemic patients treated by allogeneic marrow transplantation
from HLA-identical relatives. A 20%-30% degree of hematopoiesis
of donor origin increased the hemoglobin (Hb) level high
enough to avoid RBC transfusions,11 consistent with the preferential
survival of normal erythroid cells as opposed to the high
apoptotic rate of erythroid precursors and RBC hemolysis in

-thalassemia.12 In murine models, a 10%-20% proportion of
normal donor cells resulted in significant improvement of anemia.13
As a consequence, reversion of the thalassemia phenotype despite
relatively low levels of corrected HCs is not out of reach by ex vivo
gene therapy with autologous transplantation.
Nevertheless, expansion of HCs appears unavoidable in a
minimally myeloablative setting. In mice, a very high dose of bone
marrow cells ( 20  106) had to be injected into
-thalassemic
recipients given 200 rads irradiation to achieve stable engraftment
and phenotypic improvement.14 Ex vivo HSC expansion is thus
appealing. However, cytokine-expanded marrow cells have a
defective long-term repopulating capability in irradiated15 as well
as nonmyeloablated mouse recipients,16 leading to low-level engraftment
of retroviral transduced cells in mice and patients in the
absence of a pretransplantation conditioning regimen.6,17 An alternative
approach is to confer a benign proliferative advantage to the
modified cells over the nontransduced cells in vivo.
Here, we sought to increase the proportion of corrected
erythroid cells in a murine model of
-thalassemia by coexpressing
a truncated form of the erythropoietin receptor (tEpoR) together
with a therapeutic
-globin chain by a lentiviral vector. In humans,
tEpoR causes primary familial and congenital polycythemia (PFCP),
a benign, autosomal-dominant erythrocytosis. PFCP is characterized
by an increased erythrocyte mass, which remains stable over
time, the absence of splenomegaly, normal white blood cell (WBC)
and platelet counts, generally low serum erythropoietin levels, the
Submitted January 8, 2010; accepted March 10, 2011. Prepublished online
as Blood First Edition paper, March 24, 2011; DOI 10.1182/blood-2010-01-
263582.
*O.N. and F.F. contributed equally to this work.
The online version of this article contains a data supplement.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
© 2011 by The American Society of Hematology
BLOOD, 19 MAY 2011
VOLUME 117, NUMBER 20 5321
From www.bloodjournal.org by guest on April 22, 2015. For personal use only.
absence of bleeding or thrombotic complications, hypersensitivity
of erythroid progenitors to erythropoietin in vitro, and no progression
to acute leukemias or myelodysplastic syndromes.18-21 In mice,
on ubiquitous expression of wild-type EpoR, the lineage commitment
of pluripotent hematopoietic progenitors is not biased,22,23
and pluripotent progenitor proliferation remains limited.24 The
tEpoR cDNA has been used in mouse transplant experiments to
induce the expansion of transduced mouse HSCs and SCIDrepopulating
cells in partially myeloablated recipients.25,26
We reasoned that the naturally elevated erythropoietin plasma
levels observed in
-thalassemia will provide a favorable environment
to trigger selective cell expansion resulting from tEpoR
expression. We show here that erythroid-specific coexpression of
human
-globin and tEpoR in thalassemic mice from lentivirally
transduced HSCs results in a major, yet self-controlled and
homeostatic, cell expansion restricted to the erythroid lineage.
Accordingly, disease correction was achieved in all thalassemic
mice that underwent transplantation even as a result of purposely
minimal transduction efficiency.
Methods
Cell culture, transduction, and BM cell transplantation
Vectors and production methods are described in supplemental Methods
(available on the Blood Web site; see the Supplemental Materials link at the
top of the online article). HSCs, hereafter called 5-fluorouracil (5-FU) cells,
were obtained from BM cells of male donors injected 4 days previously
with 150 mg/kg 5-FU (Sigma-Aldrich) and submitted to Lympholyte-M
density gradient purification (Cedarlane). Alternatively, stem cells were
purified from male BM by sorting CD105 Sca1 cells with the use of
magnetic beads (Miltenyi Biotec). Medullar lymphomyeloid and erythroid
cells were purified on the basis of the presence or absence of the CD45
antigen with magnetic beads (Miltenyi Biotec). Purity was checked by
cytometry with antibodies against CD45 and Ter119 antigens. All mouse
experiments were approved by the local committee of the Hematology
Institute of the Saint-Louis Hospital.
Before transduction, all cells were washed and suspended at a final
concentration of 1-2  106/mL in alpha-MEM medium (Invitrogen) containing
15% FCS, 100 ng/mL recombinant mouse stem cell factor, 6.25 ng/mL
interleukin-3, and 10 ng/mL interleukin-6 and grown at 37°C. All cytokines
were from Peprotech. rhEpo (3 U/mL; Roche Pharma) was added in
erythroid cell culture.
Transduction of 5-FU cells with gammaretroviral vectors (RV) started
40 hours later. Cells were exposed twice, 24 hours apart, to undiluted
retroviral supernatants on Retronectin (Takara)-coated Petri dishes in
alpha-MEM medium containing 8 g/mL protamine sulfate (SigmaAldrich),
decomplemented serum, and cytokines as described previously.
Two days after transduction, percentages of enhanced green fluorescent
protein-(eGFP)–positive cells (24%-32% as determined by flow cytometry
and unchanged 4 days later) were set to 10% with mock transduced cells.
Four million cells (including 4  105 eGFP-expressing cells) were injected
intravenously in lethally irradiated
-thalassemic female mice Hbbth-1/th-1.
27
In this experiment, MOI was 1 (twice).
-thalassemic recipients received
1100 rads (split dose of 550 rads over 3 hours) of total body irradiation.
Concerning lentiviral vectors, transduction started 16 hours after cell
isolation. 5-FU cells were exposed to vectors on Retronectin-coated Petri
dishes in StemPro-34 serum-free medium (Invitrogen) supplemented with
protamine sulfate and cytokines. Six hours later, cells were harvested by the
use of trypsin-EDTA solution (Cambrex BioScience) and a cell scraper. A
total of 500 000-750 000 transduced 5-FU cells were injected intravenously
into each
-thalassemic female recipient given total body irradiation. In
experiment 1, MOI was 20, and
-thalassemic recipient received 600 rads
(single dose). In experiment 2, 3 groups of mice received cells transduced at
MOI of 0.3, 2, or 10, respectively and 1100 rads (split dose of 550 rads over
3 hours).
-Thalassemic mice that undergo transplantation with cells
transduced with the LG and the LG/HA-Y1 mice are called LG- and
LG/HA-Y1 mice, respectively. In a third experiment, after a single
irradiation dose of 200 rads, 4
-thalassemic mice were injected with
25 000 CD105Sca1 cells each. Cells were transduced with LG/HA-Y1 at
a MOI of 20. For in vitro studies, bone marrow erythroid (CD45   ) and
lympho/myeloid (CD45) cells were transduced with LG/HA-Y1 at a MOI
of 50. RNA was extracted 2 days later.
Blood parameters
Blood samples were analyzed for hemoglobin and blood cell counts with
the use of an automated cell counter (Cell Dyn 3700; Abbot Diagnostic).
Hematocrit values were obtained by the manual centrifugation method. The
proportion of soluble hemoglobin versus total hemoglobin was determined
by the measurement of hemoglobin with the Drabkin reagent (SigmaAldrich)
in total hemolysate and in the supernatant of centrifuged (5 minutes
at 20 000g) hemolysate. Erythropoietin concentration was determined
by use of the Epo monoclonal enzyme immuno-assay kit (Medac Diagnostika)
with human Epo standards. Mouse and human hemoglobins were
separated by cation-exchange HPLC. Hemolysates were injected onto a
PolyCAT A column (PolyLC Inc)

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Andy,

If I understand the above article, this may be an improved gene transfer method that reduces the risks associated with myeloablation. 

Is that correct?

is it this new company working on gene Therapy?