Blood First Edition Paper, prepublished online February 26, 2010; DOI 10.1182/blood-2009-08-240556
A zinc-finger transcriptional activator designed to interact with the γ-globin gene promoters
enhances fetal hemoglobin production in primary human adult erythroblasts
Running Title: Zinc finger-mediated induction of fetal hemoglobin
GENE THERAPY
Andrew Wilber,1 Ulrich Tschulena2*, Phillip W. Hargrove,3 Yoon-Sang Kim,3
Derek A. Persons,3 Carlos F. Barbas III,2and Arthur W. Nienhuis3
Department of Surgery and Simmons Cooper Cancer Institute, Southern Illinois
University School of Medicine, Springfield, IL; 2Skaggs Institute for Chemical Biology and
the Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA;
3
Department of Hematology, St Jude Children’s Research Hospital, Memphis, TN.
1
Correspondence:
Arthur W. Nienhuis, M.D.
Department of Hematology
St. Jude Children’s Research Hospital
262 Danny Thomas Place, MS 341
Memphis, TN 38105
arthur.nienhuis@stjude.org
Phone: 901-595-2752
Fax: 901-595-2176
*Current Address:
Ulrich Tschulena, Ph.D.
Division of Molecular Genome Analysis
German Cancer Research Center
Im Neuenheimer Feld 580
69120 Heidelberg, Germany
Copyright © 2010 American Society of Hematology
ABSTRACT
Fetal hemoglobin is a potent genetic modifier of the severity of β-thalassemia and sickle cell
anemia. We utilized an
in vitro
culture model of human erythropoiesis in which late stage
erythroblasts are derived directly from human CD34 + hematopoietic cells to evaluate HbF
production. This system recapitulates expression of globin genes according to the
developmental stage of the originating cell source. When cytokine-mobilized peripheral blood
CD34+ cells from adults were cultured, background levels of HbF were 2% or less. Cultured cells
were readily transduced with lentiviral vectors when exposed to vector particles between 48
and 72 hours. Among the genetic elements which may enhance fetal hemoglobin production is
an artificial zinc-finger transcription factor, GG1-VP64, designed to interact with the proximal γglobin gene promoters. Our data show that lentiviral-mediated, enforced expression of GG1VP64 under the control of relatively weak erythroid-specific promoters induced significant
amounts of HbF (up to 20%) in erythroblasts derived from adult CD34 + cells without altering
their capacity for erythroid maturation and only modestly reducing the total numbers of cells
that accumulate in culture following transduction. These observations demonstrate the
potential for sequence specific enhancement of HbF in patients with β-thalassemia or sickle cell
anemia.
INTRODUCTION
All species that use hemoglobin for oxygen transport switch the composition of hemoglobin
during development. 1-3 In humans, embryonic hemoglobins are produced early during
hematopoiesis when erythropoiesis is predominantly in the yolk sac. During the fetal period,
comprising the last two trimesters of development, fetal hemoglobin is produced in erythroid
cells populating the liver. Beginning with the perinatal period, which initiates several weeks
just before the end of gestation and continues during the first year of life, fetal hemoglobin
(HbF) is progressively replaced by adult hemoglobin (HbA). All hemoglobin molecules are
composed of tetramers of two different types of globin chains. Alpha-globin encoded on
chromosome 16 in humans is common to both fetal and adult hemoglobin; the switch from HbF
(α2γ2) to HbA (α2β2) reflects replacement of γ-chains in hemoglobin tetramers with β-chains
encoded by linked genes on chromosome 11. Fetal hemoglobin production continues in adult
humans at low levels with individual variation subject to genetic control. 4
The level of HbF production is inherited as a quantitative trait and is of significant clinical
relevance given its role in ameliorating the severity of the principle hemoglobin disorders,
sickle cell anemia and β-thalassemia.1,4 Individuals homozygous for these mutations in their βglobin genes and who also have genetic characteristics leading to enhanced fetal hemoglobin
production present with a less severe clinical syndrome than those in which fetal hemoglobin
production is more limited. Over five decades of clinical studies supporting these facts has
triggered an intense interest in the mechanisms which control developmental switching. 1,2
These mechanistic studies have led to the identification of a number of agents which enhance
fetal hemoglobin production
in vivo
.5 The most widely used drug, hydroxyurea,6 has been
approved for the treatment of adult patients with sickle cell disease following a randomized
clinical trial which demonstrated its benefit. 7
Transgenic mouse models have been utilized to study the molecular mechanisms of human
hemoglobin switching.8,9 Much has been learned from such models regarding the distribution
of regulatory elements within the β-globin locus and potential influence of various
transcriptional factors on the relative synthesis of γ- and β-globin.1-3 The embryonic (ε),
duplicated γ-genes (Gγ and Aγ), the poorly expressed δ-globin gene and the functional β-globin
gene are encoded on chromosome 11 in order of their developmental expression. Upstream
from this set of genes is the locus control region (LCR) that is comprised of five hypersensitive
sites which have both insulating and enhancer activity. 1-3
13
Many transcriptional activators
and repressors, most of which are neither tissue or developmental stage specific, are known to
interact with specific sequences throughout the globin loci in modulating globin gene
expression.1-3
Studies in mouse models suggest that sequential expression of the individual globin genes
throughout development occurs as a combination of competition between promoters
regulating transcription of ε-,γ-, and β-globin genes for the LCR as well as autologous silencing
of the ε gene at the end of early embryogenesis and of the γ-genes during the perinatal switch
leaving the adult β-globin gene to interact with the LCR throughout adult life. 1-3, 10-12 However,
mouse models are limited by the fact that a mouse has no fetal globin equivalent. Indeed
embryonic hemoglobins are produced during mouse embryogenesis and adult hemoglobin
production begins relatively early during fetal development and is the predominant hemoglobin
made in fetal liver. Recent studies suggest that human γ-globin production in transgenic mice is
limited to the embryonic erythroid compartment and that mouse fetal liver cells lack human γglobin derived from transgenic loci 13.
These considerations have prompted us to focus on the use of human primitive hematopoietic
cells from different developmental stages to derive erythroid cultures of maturing erythroblasts
that can be used to evaluate the molecular mechanisms of switching. We have employed a two
stage liquid culture system14 to generate pure populations of mature erythroblasts from
primitive hematopoietic cells from different developmental stages. This culture system results
in very low levels of fetal hemoglobin production in cells derived from adult bone marrow or
from cells mobilized into the peripheral blood of adults with a cytokine. Primitive
hematopoietic cells from earlier developmental stages are committed to generating
erythroblasts making fetal hemoglobin under identical culture conditions. Furthermore, we
have shown that the primitive erythroid cells that expand early in culture are transduced with
high efficiency by lentiviral vectors and therefore, potentially useful for evaluation of globin
gene vectors and those encoding genetic elements designed to enhance fetal hemoglobin
production.
Among the genetic elements which may enhance fetal hemoglobin production is an artificial
transcription factor designed to interact with the proximal γ-globin gene promoters.15
Advances in the engineering of polydactyl zinc-finger transcription factors has made it possible
to produce zinc-finger proteins capable of recognizing any 18-bp stretch of DNA. 16 Zinc finger
domains linked to a transcriptional activator domain were constructed to bind to 18-bp
segments of the proximal γ-globin gene promoters.15 Of the three independent artificial
transcriptional factors which were assembled, one binding to the 18-bp segment of the γpromoter which includes position -117 proved to be the most active at augmenting γ-globin
production in K562 human erythroleukemia cells. The -117 position of the Aγ-promoter is the
site of a naturally occurring mutation resulting in hereditary persistence of fetal hemoglobin 17,18
and thus this region of the γ-globin promoter is known to be relevant to modulation of γ-globin
gene expression. This artificial transcriptional activator, termed GG1-VP64, was subsequently
shown to augment γ-globin production in continually proliferating bone marrow cells from
transgenic mice harboring the entire human β-globin locus.19 These results prompted us to
undertake studies designed to evaluate the impact of expression of GG1-VP64 on γ-globin
expression in maturing adult erythroblasts.
METHODS
Purification of human CD34+ cells
Peripheral blood cells from normal volunteers were collected with a Cobe Spectra continuous
flow blood cell separator following mobilization with recombinant human granulocyte-colony
stimulating factor (G-CSF) given for four days according to a clinical protocol approved by the
Institutional Review Board of St. Jude Children’s Research Hospital. CD34 + cells were purified
using anti-CD34+ antibodies linked to magnetic microbeads.20 Purified CD34+ cells from normal
human bone marrow, cord blood and fetal liver were purchased commercially (Lonza,
Walkersville, MD). The cells were initially cultured for expansion in Iscove’s Modified
Delbecco’s Medium (IMDM) containing 20% fetal bovine serum (FBS; Hyclone, Thermo
Scientific, Waltham, MA), hSCF (10ng/mL), hIL-3 (1 ng/mL), erythropoietin (2 units/mL),
dexamethasone and β-estradiol (10-6 M each). At the end of the expansion phase, the cells
were pelleted and transferred into IMDM containing 20% FBS, erythropoietin (2 units/mL) and
insulin (10 ng/mL) for differentiation. Cells were incubated at 37 oC in a humidified atmosphere
of 5% CO2 and maintained at a density of 1x105-1x106 cells/mL by supplementing cultures every
other day with fresh media. Cell numbers and viability were determined by trypan blue
exclusion. Cell morphology was assessed by Wright-Giemsa staining of cytocentrifuge
preparations and images acquired with an Olympus Bx41 Upright microscrope equipped with a
DP70 digital camera and DP manager software (Olympus).
Plasmid constructions
Self-inactivating (SIN) lentiviral vectors beginning with the plasmid, pCL20cMp-GFP, to which
had been added the modified woodchuck post-transcriptional RNA processing element (WPRE),
were derived using components of our HIV-based lentiviral vector system previously
described.21 The internal Mp promoter is a modified Murine Stem Cell Virus (MSCV) long
terminal repeat (LTR) from which non-essential U5 sequences have been eliminated.
(i) GFP Vectors: Erythroid specific vectors encoding for expression of GFP were created by
replacing the Mp promoter in pCL20cMp-GFP with either a minimal ankyrin-1 promoter 22,23 or a
human β-spectrin gene promoter24 thereby generating pCL20cAnk-GFP or pCL20cSp-GFP,
respectively. For construction of pCL20cAnk-GFP, a PCR product was amplified using the
primers Ankyrin-F (5’-ACG CGT TTC GAA GGG GCA ACG AGG-3’) and Ankyrin-R (5’-ACC GGT GGG
AAT TGC CGC CGA AGG-3’) using DNA of a plasmid containing the minimal ankyrin promoter as
a template.22 The resulting 281-bp product containing a
Mlu
I site 5’ and
Age
I site 3’ was cloned
into pCR2.1-Topo (Invitrogen, Carlsbad, CA) and confirmed by sequence analysis. An
Mlu
I-
Age
I
fragment containing the ankyrin-1 promoter was recovered from the pCR2.1-Topo clone and
ligated into the same sites in pCL20cMp-GFP replacing the Mp promoter. Construction of
pCL20cSp-GFP was similarly achieved by PCR amplification of the β-spectrin promoter on
plasmid DNA23 using the primers Spectrin-F (5’-ACG CGT TAA TTC GAA GGG AGG-3’) and
Spectrin-R (5’-ACC GGT GCA ATT GAC AGC GG-3’). The resulting 454-bp product flanked by
introduced
Age
Mlu
I and
Age
I sites was cloned into pCR2.1-Topo and subsequently excised as
I fragment to replace the Mp promoter in pCL20cMp-GFP.
Mlu
I-
(ii) GG1-VP64 Vectors: A rhesus variant of GG1-VP64 was designed in anticipation of
performing in vivo experiments in non-human primates (Supplemental Figure 1) and shown to
be equally active as human GG1-VP64 on the human γ-promoter in the transcient luciferase
assay in HeLa cell (Supplemental Figure 2). The rhesus variant was used in all subsequent
experiments. The β-spectrin promoter was excised as a 568-bp
pCR2.1-Topo clone described above and cloned between
Mfe
Eco
I and
RI-
RI fragment from
Eco
Eco
RI sites of an
intermediate plasmid 5’ to the internal ribosome entry site (IRES) GFP sequences to create pSpiG. This vector was linearized with
coding sequences recovered as a
Eco
Bam
RI (blunt) to allow for insertion of the 803-bp GG1-VP64
HI-
Eco
RI fragment from a retroviral expression vector
analogous to pMx-gg1-VP64-HA15 and rendered blunt with Klenow DNA polymerase generating
pSp-GG1-VP64-iG. The Sp-iG or Sp-GG1-VP64-iG intermediates were excised as
fragments and cloned into pCL20cMp-GFP digested with
Mlu
I (blunt)-
Eco
Stu
I-
Sna
BI
RI (blunt) replacing the
Mp-GFP cassette to create pCL20cSp-iG or pCL20cSp-GG1-VP64-iG, respectively. To construct
pCL20cAnk-GG1-VP64-iG, a 1407-bp
Nco
I-
Nco
I fragment including GG1-VP64 and IRES
sequences was ligated into pCL20cAnk-GFP linearized by partial digest with
Nco
I.
Lentiviral vector production and gene transfer
Lentiviral vector particles pseudotyped with vesicular stomatitis virus G (VSV-G) protein were
prepared using a four-plasmid system by transient transfection of human embryonic kidney
293T-cells using the calcium phosphate precipitation technique as previously described. 21
Eighteen hours after transfection, cells were washed twice with phosphate buffered saline
(PBS) and fresh medium was added to each plate of cells. Twenty-four hours later, the medium
containing vector particles was harvested, cleared by low-speed centrifugation, and filtered
through a cellulose acetate filter of 0.22 μm pore size.
stored at −80°C until use.
Viral supernatants were aliquoted and
Vector preparations were thawed and titered on K562 human
erythroleukemia cells based on GFP expression as determined by flow cytometry. Transduction
of primitive hematopoietic cells was accomplished by transferring them to retronectin coated
plates (50 μg/cm2; Fisher Scientific, Pittsburg, PA) approximately 48 hours after the cultures
were initiated. All samples were supplemented with protamine sulfate (10 μg/mL) and vector
particles were added to the culture medium to achieve various multiplicities of infection (MOI).
Following overnight exposure to virus, the cells were harvested with enzyme-free cell
dissociation solution (Millipore, Billerica, MA), washed in IMDM and returned to the expansion
medium.
Flow cytometry
Cells at various stages of differentiation were rendered into single-cell suspensions for flow
cytometric analysis. Live cells were identified and gated by exclusion of 7-amino-actinomycin D
(7-AAD; Beckton-Dickinson, Franklin Lakes, NJ) and tested for expression of GFP or cell surface
receptors with antibodies specific for CD34, CD45, CD71, and CD235 conjugated to either
phycoerythrin (PE) or allophycocyanin (APC) on a FACSCalibur System (Beckton-Dickinson) using
CellQuest analysis software (BD Biosciences, Heidelberg, Germany).
DNA analysis for lentivirus vector copy number
The average vector copy number in transduced CD34 + cell populations was determined by
Southern blot analysis or quatitative polymerase chain reaction (qPCR) performed on DNA from
cells harvested 1-2 days after initiation of erythroid differentiation culture conditions. Genomic
DNA was isolated using the Gentra Puregene DNA Extraction kit (Qiagen, Valencia, CA).
(i) Southern hybridization. Southern blotting was performed as previously described. 25 DNA
samples were digested with
Bgl
II which cuts within each provirus to release a near full length
,
unit, electrophoresed through 0.8% agarose gel, and then blotted onto nylon membrane.
Equivalent loading of lanes was confirmed by ethidium bromide staining of gels prior to DNA
transfer to the nylon filters. A radiolabeled 761-bp fragment encoding for HIV-1 RRE element
was hybridized with the blot and the signal intensity of the hybridizing band for each DNA
sample was compared to that of the DNA from a K562 clone harboring a single vector copy
using a Molecular Dynamic Storm 860 Phosphorimager (Sunnyvale, CA) and its accompanying
software.
(ii) qPCR analysis. The conditions used for detecting integrated HIV vector sequences,
establishing the strandard curve, normalizating reactions and calculating final vector copy
number (VCN) were conducted according to conditions previously described 25 using the
StepOne Plus™ Real-time PCR System (Applied Biosystems, Foster City, CA) and the following
modifications. PCR amplification of integrated HIV vector sequences was achieved using the
primers FPLV-(5’-ACC TGA AAG CGA AAG GGA AAC-3’) and RTLV-(5’-CAC CCA TCT CTC TCC TTC
TAG CC-3’) and amplicon specific probe (5’-6-FAM-AGC TCT CTC GAC GCA GGA CTC GGC-3’)
(Applied Biosystems, Foster City, CA). The average VCN was calculated by establishing a
standard curve of K562 DNA containing a single copy of the HIV vector genome serially diluted
with native K562 DNA to yield mixtures containing 1, 0.5, 0.25, 0.1, and 0.01 vector copies
where DNA was normalized using primers and a probe specific for human N-RAS. 26 The final
VCN of each sample was adjusted by dividing the copy number by 1.5 based on the triploid
nature of the K562 cell line genome.
Hemoglobin analysis
Cells (10-15 million) were harvested at various times during the differentiation phase of
erythroid culture, lysed in 40 microliters of hemolysate reagent (Helena Laboratories,
Beaumont, TX), and refrigerated overnight before centrifuging at 14,000 rpm for 10 minutes at
4oC to remove cellular debris. The cleared supernatant was used for characterization of
hemoglobin production by cellulose acetate hemoglobin (Hb) electrophoresis or high
performance liquid chromatography (HPLC) using methodologies previously established in our
laboratory.25
RESULTS
Differentiation of erythroid progenitors from various developmental stages
We initially tested our two-stage
in vitro
model of human erythropoiesis for the ability to
recapitulate the developmental pattern of hemoglobin expression associated with the
developmental stage of the originating primary cell source. For this purpose, approximately
1x105 purified CD34+ cells in the range of 94-99% purity, as determined by flow cytometry (data
not shown), from adult peripheral blood (PB) following G-CSF administration, adult bone
marrow (BM), cord blood (CB) or fetal liver (FL) were established in liquid culture under
conditions designed to foster expansion and differentiation (Figure 1A). After 7 days in the
proliferative phase, the cells were transferred into medium designed to mediate terminal
erythroid maturation. Cell division continued for 12 days and resulted in a 1000-fold expansion
in the total number of cells (Figure 1B). Erythroid maturation was monitored by flow
cytometry. The vast majority of maturing erythroid cells from PB, BM and CB were late stage
erythroblasts reflected by nearly complete enrichment for expression of transferrin receptor
(CD71; 99%) and glycophorin A (CD235; >96%) which peaked by culture day 14 and was
maintained until cultures were terminated 6 days later (Figure 1C). While the majority of cells
in cultures of FL were transferrin positive (98%), there was a residual fraction of cells that
remained glycophorin A negative (<25%) at the end of the culture period. Morphological
evaluation following cytocentrifuge preparations and Giemsa staining indicated that the
majority of cells in all of the cultures were terminally maturing erythroblasts (Figure 1D).
Cellulose acetate hemoglobin electrophoresis demonstrated that virtually all of the hemoglobin
in erythroblasts derived from PB and BM CD34+ cells was adult hemoglobin (>92%) (Figure 1E-F
and Supplemental Figure 3). Alternatively, erythroblasts derived from CB CD34 + cells had
roughly equal proportions of HbF and HbA and the majority of hemoglobin in erythroblasts
derived from FL CD34+ cells was HbF (>94%) (Figure 1E-F).
Lentiviral vector-mediated transduction of adult erythroid progenitors
To evaluate the effect of lentivirus-mediated gene transfer on erythroid differentiation, dividing
cells derived from mobilized PB hematopoietic progenitors were collected 48 hours after
initiation of culture and 3-4x105 cells transferred to 24-well suspension tissue culture plates
coated with retronectin before overnight exposure to lentiviral vector particles encoding for
GFP added to the culture medium to achieve a MOI of 5 or 10 (Figure 2A). The following day,
the cells were lifted from plates, returned to expansion medium and cultured until day 7 at
which point they were transferred into the differentiation medium. Greater than 1000-fold
expansion of the mock transduced cells as well as cells transduced with the GFP vector at MOIs
of 5 or 10 was documented (Figure 2B). Transduction was highly efficient with the majority of
cells demonstrating expression of the GFP marker as shown by flow cytometry analysis on days
8, 11 and 14 of culture (Figure 2C). Loss of the CD34 + antigen, diminution in expression of CD45
and acquisition of expression of transferrin (CD71) and glycophorin A (CD235) was documented
for both mock and transduced cell populations (Figure 2D; Surface Marker Expression). Where
the seed cell populations were relatively large and heterogeneous in size, the average cell size
diminished as the cultures progressed down the erythroid maturation pathway until the
majority of cells were found in a single peak (Figure 2D; Size) consistent with maturing
erythroblasts as documented by morphological evaluation (Figure 2D; Cytospin). Furthermore,
manipulation of mobilized PB CD34+ cells following vector transduction did not alter the adult
pattern of hemoglobin production (HbA >96%; HbF <2%) as shown in Figures 2E and 2F.
Interested in achieving erythroid specific expression of transactivators
in vivo
and conscious of
the potential need to have promoters of various strengths available for our planned studies of
genetic elements that enhance fetal hemoglobin production, we also tested the minimal
ankyrin-122-23 and β-spectrin24 promoters in our erythroid culture system (Figure 3A). Cells
from mobilized PB were transduced as described above and monitored for GFP expression by
flow cytometry (Figure 3B). As we had found previously (Figure 2C), the MSCV LTR mediated
very high expression of the GFP marker, as reflected by the 10-fold higher mean fluorescent
intensity (MFI) relative to that observed with the erythroid specific promoters at the earliest
time point (Day 5) following transduction with a progressive decrease in MFI as the cells
diminished in size during erythroid maturation. In contrast, the β-spectrin promoter was much
weaker initially, but exhibited a small progressive increase in MFI over time as erythroblasts
matured (Figure 3B and Supplemental Figure 4). The minimal ankyrin-1 promoter gave a
somewhat higher level of expression than the β-spectrin promoter at early stages of culture,
and also exhibited a modest (approximately 2-fold) increase in expression as erythroid
maturation progressed (Figure 3B-C). Despite the lower levels of expression from the ankyrin
promoter, the copy number on Southern blot was higher than with the MSCV promoter. We
infer that the copy number in cells transduced with the spectrin vector was nearly equivalent
based on the fact that the proportion of GFP cells were equivalent but insufficient DNA was
available for direct analysis. In all cases, gene transfer demonstrated no appreciable effect on
erythroid cell development when GFP- and GFP+ fractions of bulk cell populations were
monitored for co-expression of the erythroid markers CD71 and Glycophorin A (Supplemental
Figure 4).
Enhanced fetal hemoglobin production in adult erythroid cells by the zinc finger-based
transcriptional activator GG1-VP64
In preliminary experiments, we found that the LTR driven GG1-VP64 transactivator consistently
retarded division of erythroid cells during the expansion phase of our culture system and which
was coincident with an appreciable reduction in the GFP + cell fraction over time (Supplemental
Figure 5). Accordingly, GG1-VP64 vectors were constructed in which the erythroid-specific and
comparably weaker ankyrin-1 or β-spectrin promoters were used to regulate transcription of
the transgene (Figure 4A). Transduction of cells according to our standard experimental format
between 2 and 3 days after initiation of culture with the vectors encoding GG1-VP64 under the
control of the erythroid specific promoters β-spectrin resulted in only minimal retardation of
subsequent cell proliferation compared to the controls (Figure 4B). Shown are the results from
two donors; in one, the total number of cells at the end of culture expressing the GG1-VP64
was 87% the number of cells expressing the control vector and in the second donor, the
percent of cells expressing GG1-VP64 was 69% of control. In these experiments, approximately
50-60% of the cells were successfully transduced and terminal erythroid maturation was
documented at the end of culture by expression of glycophorin A (Figure 4C). Induction of HbF
ranged from approximately 12-21% (Figure 4D-E) compared to 1-2% for cells transduced with
the control vector.
+
Normalization of HbF to the transduced cell population (GFP
fraction; Donor 1, Sp-
GG1 (40%) and Donor 2, Sp-GG1 (51%) calculates to an elevation of HbF to 30% or 41% in transduced
cells, respectively.
An experiment performed with mobilized peripheral blood CD34 + cells from a
third adult donor gave similar results (data not shown). To demonstrate that production of HbF
was present only in the fraction of cells transduced with GG1-VP64, the cells derived from
mobilized peripheral blood CD34+ cells from donor 1 were sorted into GFP- and GFP+ fractions
(Figure 5A, inset boxes). The morphology of the two sorted cell populations was similar (Figure
5B-C). Significant amounts of HbF were present only in the GFP + fraction (Figure 5C-D) as
predicted.
DISCUSSION
Human primitive hematopoietic cells from different developmental stages are already
committed to production of different hemoglobin types in developing erythroblasts when
cultured under identical conditions. Erythroblasts derived from adult peripheral blood and
bone marrow cells make HbA, erythroblasts derived from cord blood CD34 + cells make a
mixture of fetal and adult hemoglobin whereas erythroblasts derived from CD34 + cells from
fetal liver make HbF. We have demonstrated that early erythroid progenitors derived from
cytokine-mobilized adult peripheral blood CD34+ cells can be transduced with lentiviral vectors
without altering their subsequent capacity for proliferation and differentiation and pattern of
hemoglobin production. Lentiviral vector-mediated delivery of a synthetic zinc-finger
transcriptional factor, GG1-VP64, under the control of relatively weak erythroid-specific
promoters with varying expression kinetics induced significant amounts of HbF in erythroblasts
derived from transduced, mobilized peripheral blood CD34 + cells from adults without altering
their capacity for erythroid maturation and only modestly reducing the total numbers of cells
that accumulate in culture following transduction.
Our results with respect to the commitment of primitive hematopoietic cells that initiate
erythropoiesis in culture and hemoglobin phenotype are consistent with much earlier results
obtained with clonal hematopoietic cultures in which erythroid colonies form in semisolid
media.27 In these studies, erythroid colonies developed from the primitive progenitors, Burst
Forming Unit-erythroid (BFU-E), present in fetal liver contained HbF whereas those derived
from adult hematopoietic tissue contained predominantly HbA. BFU-E from newborns
generated colonies containing a mixture of HbF and HbA. 27 The liquid culture system provides
large numbers of differentiating erythroblasts from the different developmental stages that will
be useful for comparative molecular analysis of the globin loci with respect to chromatin
structure, epigenetic modifications and transcriptional factor binding as has recently been
reported by others in erythroblasts developed from adult progenitors. 28,29 While such studies
should yield important information regarding how the developmental pattern of gene
expression is maintained during erythropoiesis, the initiating events which result in
commitment to hemoglobin phenotype in early progenitors may be more challenging to
discern. Available evidence suggests that genes that are ultimately expressed in a lineage
restricted manner may be activated in very early progenitor cells and that lineage-specific
transcriptional activators expressed at basal levels in progenitor cells may participate in gene
potentiation.30,31 The order of expression of specific transcriptional factors has been shown to
direct hierarchical specification of hematopoietic lineages. 32 In a recent study, RUNX1 was
shown to be responsible for early chromatin unfolding without assembling into a stable
transcription factor complex on the PU.1 promoter. 33 If such mechanisms are involved in the
developmental commitment with respect to hemoglobin phenotype in early hematopoietic
cells, their discovery and elucidation may prove challenging.
Relevant to studies intending to characterize genes or microRNAs with potential for enhancing
HbF expression, our culture system is characterized by a very low basal level of HbF in
erythroblasts derived from adult CD34+ cells at all analyzed time points during the maturation
process. Other culture conditions result in higher levels of fetal hemoglobin in cells developed
from adult BFU-E. Early studies using immunofluorescence demonstrated that burst colonies
were segmented with respect to HbF production suggesting that commitment to produce
limited amounts of HbF by these adult cells were occurring only after one or a few divisions in
contrast to developmental control of HbF in which BFU-E are more fully committed with
respect to hemoglobin phenotype.34 A similar pattern of commitment during early
erythropoiesis is thought to result in the heterocellular distribution of HbF in normal adults in
whom up to 8% of red cells contain small and variable amounts of HbF.1,4 The increased
production of HbF in adult erythroblasts under stress has been attributed to acceleration of
erythroid maturation allowing for synthesis of γ-globin, which normally occurs to a limited
extent in the earliest erythroblasts, 35 to persist during erythroid maturation. A cell stress
signaling model of fetal hemoglobin induction has been proposed as recently reviewed. 36 Early
studies demonstrated that Stem Cell Factor (SCF) induces fetal hemoglobin production in
cultures of purified BFU-E37 and recent studies have shown that the combination of SCF and
Transforming Growth Factor-β (TGF-β) consistently cause a significant increase in HbF in
cultures of adult cells to levels up to 20% with a pancellular distribution. 38 We have used a
relatively low concentration of SCF of 10ng/mL in our cultures to achieve a low baseline level of
HbF production whereas a concentration of 50ng/mL is typically used when HbF synthesis is
maximized.38
The zinc-finger transcriptional factor that we have shown induces HbF in maturing erythroblasts
derived from adult CD34+ cells was designed to function as a transcriptional activator (Figure
6A) and indeed it has been shown to enhance expression from a minimal γ-promoter in a
reporter assay (Reference 15 and Supplemental Figure 2B). However, the region in the γ-globin
gene promoter to which it binds includes a sequence called the direct repeat element because
it is tandemly repeated in the ε promoter (Figure 6B). This element has been implicated in
adult stage, γ-globin gene silencing.39 The direct repeat element interacts with nuclear receptor
chicken ovalbumin upstream promoter-transcriptional factor II (COUP-TFII) 40 and the direct
repeat erythroid definitive binding proteins, (DRED) TR2/TR4. 41 Both COUP-TFII and DRED have
been implicated in repression of γ-globin expression. Recent results indicate that SCF induces γglobin gene expression by decreasing COUP-TFII expression. 42 Displacement of these proteins
that are involved in silencing the γ-globin gene during the adult stage of erythropoiesis by the
zinc-finger transcription factor may also be involved in enhancing HbF production in maturing
adult erythroblasts. Recent work in the Orkin lab has shown that BCL11A acts as a stagespecific repressor in silencing HbF expression in adult human erythroid cells, 13,43 but it has been
shown to bind to the intergenic region and thus may not be directly affected by expression of
the zinc finger transcription factor. The mechanism for silencing the γ genes in human adult
erythroid cells appears to be complex and redundant. 44-46 Other proteins that interact with the
region or nearby where GG1-VP64 is designed to bind (Figure 6B) are transcriptional activators
such as KLFII47 or stage selector protein48 or may act as an activator or repressor in the case of
NF-Y, depending on exactly where it binds and the complexes it forms with other proteins. 49
In addition to local effects (i.e. direct activation of the γ-globin promoters or perturbed
repressor-binding at this target sequence), it is possible that GG1-VP64 makes possible longrange interactions between the LCR or transcription factor complexes and γ-globin promoter by
attenuating chromatin condensation (Figure 6A). The GG1-VP64 target sequence includes the
guanine residue at nucleotide position -117 which when mutated to adenine results in
increased production of γ-globin in cases of Greek non-deletion HPFH17,18. Generation of
transgenic mice with this mutation in the γ-globin gene resulted in persistent expression of fetal
hemoglobin.50 While Berry and colleagues indicated that persistence of γ-globin was correlated
with loss of Gata-1 binding at the γ- promoter, the precise molecular mechanism has not been
fully resolved. A study analyzing DNA-protein interactions at the γ-globin promoter in K562
cells, which express γ but not β-globin, revealed that an unknown protein interacts with the
promoter in the CCAAT-box located next to position -117 rendering the TTG motif sensitive to
dimethyl sulfate.51 Thus, it is possible that binding of GG1-VP64 to sequences which include the
-117 position results in retention of an open chromatin structure that allows interaction with
the LCR during the course of erythroid cell maturation.
Enforced expression of the zinc-finger transcriptional activator inhibits cell proliferation in our
culture system, which we have largely avoided by regulating transcription with relatively weak,
erythroid-specific promoters. Our plans are to test the capacity of the zinc-finger
transcriptional activator in vivo both alone and concurrently with γ-globin gene addition to
augment HbF levels. For this, we will use a non-human primate model, the pigtailed macaque
(Macaca nemestrina), the stem cells of which have been shown to be amenable to lentiviral
vector mediated gene transfer with HIV-based vector systems. 52 We are also exploring the use
of tamoxifen modulated nuclear accumulation of the transcriptional activator in a further level
effort to control its activity in order to achieve a beneficial effect with respect to HbF
production while further reducing the unwanted inhibition of cell proliferation. Studies in the
non-human primate should also provide information regarding the potential safety of this
approach as a strategy for enhancing fetal hemoglobin in patients with thalassemia or sickle cell
disease.
Acknowledgments: We thank Dr. David Bodine (Hematopoiesis Section, Genetics and
Molecular Biology Branch, NHGRI, National Institutes of Health, Bethesda, MA) and Dr. Patrick
Gallagher (Department of Pediatrics, Yale University School of Medicine, New Haven, CN) for
the Ankyrin-1 and β-Spectrin promoters. We thank Qi Wang, Ph.D., for having cloned the rhesus
γ-globin gene promoter fragment. We thank the flow cytometry laboratory of Anna
Travelstead for expertise in flow cytometry studies and FACS analysis. We thank Pat Streich for
help in preparing the manuscript. The work of Andrew Wilber, Phillip W. Hargrove, Yoon-Sang
Kim, Derek A. Persons and Arthur W. Nienhuis was supported by NHLBI PO1HL053749, the
Assisi Foundation of Memphis and the American Lebanese Syrian Associated Charities. The
work of Ulrich Tschulena and Carlos F. Barbas III, was supported by the Skaggs Institute for
Chemical Biology.
Authorships:
Contribution: A.W. designed and performed research, analyzed data and contributed to the
writing of the manuscript; UT, PWH and YSK contributed to performing the research; D.A.P.,
C.F.B. and A.W.N. participated in designing the research and writing the paper and A.W.N. was
responsible for the overall organization of the research effort.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Arthur W. Nienhuis, M.D., Department of Hematology, St. Jude Children’s
Research Hospital, 262 Danny Thomas Place, MS#341, Memphis, TN 38105; e-mail:
arthur.nienhuis@stjude.org
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Figure 1:
stages.
Expansion and differentiation of erythroid cells from different developmental
(A) Schematic representation of the two-phase erythroid culture model showing the
experimental time frame and additions to the culture medium during expansion and
differentiation. (B) Total cell numbers derived from 1 x10 5 CD34+ cells from cytokine mobilized
peripheral blood (PB), adult bone marrow (BM), cord blood (CB) or fetal liver (FL) over the initial
12 days of culture. (C) Flow cytometry analysis for expression of CD71 (transferrin receptor)
and CD235 (glycophorin A) where the percentages indicate the proportion of cells considered
positive and (D) morphology of Wright-Giemsa stained cytocentrifuge preparations (original
magnification 60x) after 20 days of culture. (E) Cellulose acetate Hb electrophoresis of
erythroblast lysates from cultured PB, BM, CB and FL CD34 + cells and whole blood (WB) from an
adult after 12 or (F) 16 days of culture.
Figure 2: Lentiviral vector-mediated transduction of erythroid progenitors derived from
cytokine mobilized peripheral blood CD34
+
cells.
(A) Schematic representation of the lentiviral
vector encoding for expression of GFP from a modified MSCV LTR sequence (Mp). (B)
Expansion of cultured cells following transduction. (C) GFP expression is indicated as a function
of time after transduction of cells exposed to vector particles at a multiplicity of infection (MOI)
of 10. (D) Phenotypic comparison of cells on the day of transduction (left panels) and 14 days
later at the time of termination of the cultures (right panels) by flow cytometry for expression
of the CD34, CD45, CD71, and CD235 surface markers where percentages indicate the
proportion of cells considered positive, cell volume , and cell morphology (Wright-Giemsa
Staining, original magnification 60x). (E) The results of cellulose acetate Hb electrophoresis of
lysates from terminal stage erythroblasts from two separate experiments ( vertical lines have
been inserted to indicate a repositioned gel lane) and (F) HPLC analysis of hemoglobins present in
erythroblasts at the end of culture which were derived from cells exposed to vector particles at
a MOI of 10. The percentages indicate the proportion of hemoglobin species.
Figure 3: Marker gene expression from constitutive or erythroid specific promoters in
maturing erythroblasts.
(A) Schematic representation of the lentiviral vectors encoding for
expression of GFP from the MSCV, Ankyrin-1, or β-Spectrin promoters. The PRE element is not
present in the MSCV vector. (B) GFP expression by erythroblasts derived from CD34 + cells
transduced with vectors having the MSCV, spectrin or ankyrin promoter after various times
following transduction where the percentages and mean fluorescence intensity (MFI) are
indicated for the proportion of cells considered positive.The percentage of glycophorin A
positive cells at the same time points are shown above the GFP profiles. (C) Southern blot
analysis of genomic DNA extracted from transduced cells, digested with
BglII, an enzyme which
cuts twice within the vector genome and probed with a RRE fragment common to all vectors
from the 5’ end of the genome. DNA size marker is shown in the leftmost lane (vertical lines
have were inserted to indicate where lanes have been repositioned). Numbers below each lane
on the image represent the vector copy number as determined by desitometry analysis, relative
to controls 1.0 or 0.5, that consist of DNA from a K562 clone that contains a single copy of an
integrated lentiviral vector either used directly (1.0) or diluted 1:1 with native K562 DNA to
establish a sample with a copy number of 0.5.
Figure 4: Induction of HbF by GG1-VP64 in erythroblasts derived from adult mobilized
peripheral blood CD34
+
cells.
(A) Schematic diagram of the empty vector control (top) and
GG1-VP64-encoding vectors (bottom) where transcription is regulated by the erythoid-specific
Ankyrin-1 (Ank) or β -Spectrin (Sp) promoters, respectively. The results obtained with two
separate donors are shown in B-E. Abbreviated designation of the vectors are Spectrin-ires-GFP
control vector (Sp-iG); Ankyrin-GG1-VP64-ires-GFP (Ank-GG1); and Spectrin-GG1-VP64-ires-GFP
(Sp-GG1). (B) Cell numbers as a function of time in culture following transduction. (C) Flow
cytometry analysis for expression of GFP and CD235 (Glyocophorin A) in erythroblasts at the
end of culture where the percentages indicate the proportion of cells considered positive. (D)
Hemoglobin electrophoresis of lysates from erythroblasts at the end of culture ( vertical lines
have been inserted to indicate a repositioned gel lane).
Numbers below each lane on the images
represent the vector copy number as determined by Southern Blot analysis and desitometry
analysis or quantitative PCR, for the various cell populations. (E) HPLC analysis of lysates from
erythroblasts at the end of culture where the percentage of HbF is reported for each condition.
Figure 5: HbF is present only in the transduced fraction of cultured erythroblasts.
(A) FACS
sorting of GFP- and GFP+ fraction of erythroblasts derived from adult mobilized peripheral blood
CD34+ cells transduced with SpGG1-VP64 vector where the morphology of sorted cell
populations are presented as Wright-stained cytocentrifuge preparations (original
magnification 60x) imbedded within histograms for GFP negative and GFP positive cell
populations, respectively. (B) Hemoglobin electrophoresis of lysates from various cell
populations (vertical lines have been inserted to indicate a repositioned gel lane). (C) HPLC analysis
of lysates from the GFP- and GFP+ cell populations.
Figure 6: Chromatin Structure and organization of the
γ-globin gene promoter. (A) Potential
changes in adult stage chromatin structure from condensed (left) to flexible (right) induced by
binding of GG1-VP64 (tennis ball structure) to the γ-globin gene promoters. Depicted are
interactions that exist between the the locus control region (LCR) and delta ( δ)- and beta-(β )
globin genes during normal adult erythoid development compared to potential new
interactions between the LCR and the γ-globin genes caused by binding of GG1-VP64 (adapted
from Bank2 with permission). (B) Location of binding sites for selected endogenous
transcriptional activators (shaded light grey) and repressors (shaded dark grey) within the
proximal γ-globin gene promoter with respect to the HPFH-117 target sequence. Abbreviations
used are: KLF11, fetal Kruppel-like factor; HPFH, hereditary persistence of fetal hemoglobin;
COUP-TFII, nuclear receptor chicken ovalbumin upstream promoter-transcription factor II;
DRED, direct repeat erythroid definitive binding protein TR2/TR4; NF-Y, nuclear transcription
factor-Y; CP2; transcription factor CP2; NF-E4, nuclear transcription factor-erythroid 4.