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Blood, Vol. 93 No. 10 (May 15), 1999:
pp. 3276-3285
Transcriptional Targeting of Retroviral Vectors to the Erythroblastic
Progeny of Transduced Hematopoietic Stem Cells
By
Alexis Grande,
Bianca Piovani,
Alessandro Aiuti,
Sergio Ottolenghi,
Fulvio Mavilio, and
Giuliana Ferrari
From TIGET, Istituto Scientifico H.S. Raffaele, Milano, Italy; and
the Department of Genetics, University of Milano, Milano, Italy.
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ABSTRACT |
Targeted expression to specific tissues or cell lineages is a
necessary feature of a gene therapy vector for many clinical applications, such as correction of hemoglobinopathies or thalassemias by transplantation of genetically modified hematopoietic stem cells. We
developed retroviral vectors in which the constitutive viral enhancer
in the U3 region of the 3' LTR is replaced by an autoregulatory
enhancer of the erythroid-specific GATA-1 transcription factor gene.
The replaced enhancer is propagated to the 5' LTR upon
integration into the target cell genome. The modified vectors were used
to transduce human hematopoietic cell lines, cord blood-derived CD34+ stem/progenitor cells, and murine bone marrow
repopulating stem cells. The expression of appropriate reporter genes
( LNGFR, EGFP) was analyzed in the differentiated progeny of
transduced stem cells in vitro, in liquid culture as well as in
clonogenic assay, and in vivo, after bone marrow transplantation in
lethally irradiated mice. The GATA-1 autoregulatory enhancer
effectively restricts the expression of the LTR-driven proviral
transcription unit to the erythroblastic progeny of both human
progenitors and mouse-repopulating stem cells. Packaging of viral
particles, integration into the target genome, and stability of the
integrated provirus are not affected by the LTR modification. Enhancer
replacement is therefore an effective strategy to target expression of
a retroviral transgene to a specific progeny of transduced
hematopoietic stem cells.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
RETROVIRAL VECTORS are widely used to
integrate and express exogenous genes into a variety of animal cells
and provide a safe and relatively efficient gene transfer tool for
human gene therapy applications.1-3 In some of these
applications, controlling the expression of a transferred, therapeutic
gene at quantitative and qualitative (tissue- or cell-specific) level
is a critical, and often limiting, factor. The most frequently used
backbone for the construction of retroviral vectors is the Moloney
murine leukemia virus (MoMLV). Transcription from the MoMLV long
terminal repeat (LTR) is dependent on the viral promoter/enhancer
elements located in the U3 region of the 5' LTR, which allows
constitutive expression of a transferred gene in most cell types.
Conversely, tissue-specific or inducible expression is difficult to
obtain. Although an independently controlled transcriptional unit can be transferred and expressed in the framework of a retroviral vector,
the strong activity of the MoMLV enhancer/promoter usually interferes
with, or prevents, regulation and function of most eukaryotic
cis-acting elements, even in the proper cell
context.4-6 Alternative designs have been developed in an
attempt to overcome this problem, from self-inactivating vectors, in
which deletion of viral elements allows expression from an internal
promoter,7-9 to vectors in which a transgene is transcribed
in opposite orientation with respect to the viral transcription
unit,10 to double-copy vectors in which a complete minigene
is inserted into the LTR upstream from the U3 region.11
These strategies frequently raise other problems, such as generation of
antisense transcripts, proviral instability, or decreased viral titer.
Attempts to redirect the promiscuous MoMLV LTR transcriptional activity
by adding to or replacing the viral enhancer with heterologous control
elements from other viruses or cellular genes have been partially
successful, allowing investigators to change the vector
tropism12 or increase transgene expression in specific
tissues13 or cell contexts.14
For correction of genetic disorders such as hemoglobinopathies or
thalassemias, in which the ultimate goal is to restrict transgene
expression to a specific progeny the erythropoietic compartmentof
the hematopoietic stem cell, transcriptional targeting of the vector is
a mandatory requirement. We report the development of targeted
retroviral vectors obtained by replacing the LTR extended viral
enhancer with an autoregulatory enhancer (ARE) of the
erythroid-specific, zinc finger transcription factor
GATA-1.15,16 The GATA-1 ARE is a 200-bp, conserved element
upstream of the GATA-1 erythroid-specific promoter ( 856 to
655) and contains two palyndromic GATA-1 binding sites. This
element was previously shown to restrict transcription of a
heterologous promoter to human or murine erythroblastic cell lines in
transient transfection.17,18 The LTR modification was
introduced in retroviral vectors encoding either a truncated form of
the human p75, low-affinity nerve growth factor receptor ( LNGFR), or
a humanized form of the green fluorescent protein (EGFP) to allow
detection of transgene expression with single-cell resolution by flow
cytometry19 or in situ in a colony-forming assay. Human
cord blood-derived hematopoietic stem/progenitor cells, including the
CD34+/CD38low fraction, and mouse repopulating
bone marrow (BM) stem cells were transduced by the modified vectors and
analyzed for transgene expression in the differentiated progenies in
vitro or after BM transplantation ex vivo. We show that enhancer
replacement does not affect transduction and stability of the
retroviral vectors and effectively restricts LTR-driven expression to
the erythropoietic progeny of transduced stem cells.
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MATERIALS AND METHODS |
Retroviral vectors and producer cell lines.
The SFCM retroviral vector, encoding the LNGFR cDNA under the
control of the viral LTR, was previously described.20 The SFCM vector was derived by replacing the enhancer and CAAT box in
the U3 region of the 3' LTR, from the Nhe I site to
position 60 from the transcription start site, with a 23-bp
polylinker. The GATA-SFCM vector was obtained by cloning the 0.2-kb
BamHI fragment of the pSV0GATA plasmid18 containing
the GATA-1 ARE in the SnaBI site of the SFCM LTR polylinker.
The LGSN vector was constructed by cloning the
Eco47III/Sca I fragment of the pEGFP-C3 plasmid
(Clontech, Palo Alto, CA) containing the EGFP gene in
the Hpa I site of the LXSN vector.21 The GATA-LGSN
vector was obtained by replacing the LNGFR insert of GATA-SFCM with the EGFP insert of LGSN.
Packaging lines for LGSN and SFCM were generated by transinfection in
the amphotropic GP+envAm1222 and ecotropic
GP+E-8623 cells, as previously described,20
whereas those for SFCM, GATA-SFCM, and GATA-LGSN were obtained by
plasmid transfection and selection in 0.8 mg/mL G418 (Boehringer
Mannheim, Mannheim, Germany). Viral titers were assayed
by transfer of G418 resistance to NIH-3T3 fibroblasts.
Transduction of hematopoietic cells.
Mouse erythroleukemia (MEL) cells were transduced as
described.24 Human hematopoietic K562, Kasumi-1, U937, and
MOLT-3 cell lines (ATCC, Rockville, MD) were grown in
RPMI 1640 (GIBCO, Grand Island, NY) supplemented with
10% fetal calf serum (FCS), transduced by 48 hours of cocultivation
with irradiated (100 Gy) packaging cells in the presence of polybrene
(8 µg/mL), and selected as bulk cultures in 1.0 to 1.5 mg/mL of G418.
Human CD34+ cells were purified from the Ficoll
(Lymphoprep; Nycomed Pharma, Oslo, Norway) mononuclear
cell fraction of umbilical cord blood by a two-round separation
procedure using the CD34 magnetic cell isolation kit (MiniMacs;
Miltenyi, Auburn, CA). CD34+ cells (5 to 10 × 104) were cultured in Iscove's modified
Dulbecco's medium (IMDM; GIBCO) containing 20% FCS, 10 ng/mL human
interleukin-3 (huIL-3), huIL-6, and human stem cell factor (huSCF; all
from R&D Systems, Minneapolis, MN) for 48 hours and
transduced by multiple cycles of infection with retroviral supernatant
in the presence of polybrene (4 µg/mL). Transduction was performed in
the upper chamber of Transwell (Costar, Cambridge, MA)
coculture plates above the murine, clonal stromal cell line CBR-BM,
D#1.25 Cells were then cultured for an additional 48 hours
in medium supplemented with 4 U/mL human erythropoietin (huEpo;
Janssen-Cilag, Geel, Belgium).
Transduced CD34+ cells were grown in liquid culture in
X-VIVO 20 serum-free medium (Biowhittaker, Walkersville,
MD) supplemented with 10 ng/mL huSCF and 4 U/mL huEpo
to induce erythroid differentiation or were grown in IMDM supplemented
with 10% FCS, 10 ng/mL huSCF, huIL-6, huIL-3, and human
granulocyte-macrophage colony-stimulating factor (huGM-CSF) to induce
myeloid differentiation and were then analyzed by flow cytometry.
Colony-forming assay was performed by plating 5 to 10 × 102 transduced CD34+ cells in 1 mL of
methylcellulose culture medium (Stem Cell Technologies, Vancouver,
British Columbia, Canada) containing 50 ng/mL huSCF, 10 ng/mL huGM-CSF, 10 ng/mL huIL-3, 3 U/mL huEpo, and 1.2 to 1.8 mg/mL
G418. Burst-forming unit-erythroid (BFU-E) and colony-forming unit-granulocyte-macrophage (CFU-GM) colonies were
scored 10 to 14 days after plating, harvested, and analyzed for
LNGFR expression by immunocytochemistry.
Analysis of transduced cells.
Expression of LNGFR was monitored by flow cytometry (FACScan; Becton
Dickinson, Mountain View, CA) using the murine
antihuman p75-NGFR monoclonal antibody (MoAb) 20.4 (ATCC), conjugated
to fluorescein isothiocyanate (FITC). Human cell surface phenotype was
determined by flow cytometry using phycoerythrin (PE)-conjugated antihuman CD34 (Becton Dickinson), Glycophorin A (GpA) and CD13 (DAKO
A/S, Glostrup, Denmark), and tricolor (TC)-conjugated
antihuman CD38 (Becton Dickinson) MoAbs. Mouse cell phenotyping was
performed using PE-conjugated antimouse Sca-1, TER-119, and Gr-1
(PharMingen, San Diego, CA) antibodies. The
FITC-conjugated, antimouse CD45.1 MoAb (PharMingen) was used to
evaluate donor-host chimerism in BM-transplanted mice. Isotype-matched
nonspecific antibodies were used as controls.
Cyto-centrifuged cell preparations were acetone-fixed and analyzed for
LNGFR expression by immunocytochemistry with alkaline phosphatase
antialkaline phosphatase (APAAP) complex following standard methods.
Samples were incubated for 1 hour with 20.4 MoAb (1:50 dilution),
washed, incubated for 30 minutes with rabbit antimouse IgG (DAKO A/S;
1:25 dilution), and incubated with APAAP complex (DAKO A/S; 1:50
dilution) for an additional 30 minutes. The reaction was developed in
Tris-buffered saline, pH 8.2, containing Naphtol AS-MX, Fast Red TR
salt, and levamisole (all from Sigma, St Louis, MO).
Slides were counterstained with hematoxylin before analysis.
RNA analysis.
Total cellular RNA was extracted by guanidine-isothiocyanate,
poly(A)+ selected by oligo(dT)-cellulose chromatography,
size-fractionated on 1% agarose-formaldeyde gel, Northern blotted to
nylon membranes, and hybridized to 107 dpm of
[32P]-labeled probes for the neomycin phosphotransferase
(NeoR), GATA-1,26 and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) genes, as previously described.19
Transduction of murine BM and BM transplantation.
Murine BM cells were harvested from C57Bl/6-Ly-5.1 mice
(B/6.SJL-CD45a-Pep3b; Jackson Laboratories, Bar
Harbor, ME) by flushing femurs and tibiae;
prestimulated for 24 hours in IMDM supplemented with 20% FCS, 10 ng/mL
muSCF, muIL-3, and huIL-6 (all from R&D Systems); and infected by
coculture for 72 hours with irradiated (100 Gy) ecotropic packaging
cells in the presence of polybrene (4 µg/mL). Transduced cells were
analyzed for efficiency of gene transfer by flow cytometry and injected
(1.0 to 1.5 × 107 cells/mouse) into the tail vein of
recipient 8-to 12-week-old male C57BL/6 mice (Charles River, Calco,
Italy) irradiated with two split doses of 400 cGy 4 hours apart. Eight weeks after BM transplantation, animals were killed
and hematopoietic tissues were collected for FACS and
immunocytochemical analysis.
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RESULTS |
Targeting retroviral vectors by LTR enhancer replacement.
The GATA-1 ARE was inserted in the U3 region of the 3' LTR of the
previously described SFCM vector,20 in which the cDNA for
the marker gene LNGFR and a NeoR gene are under the control of the
viral LTR and of an internal SV40 promoter, respectively (Fig 1B). The modification is transferred
to the 5' LTR upon reverse transcription and integration of the
provirus, allowing the chimeric LTR to direct transcription of the
LNGFR transgene. We first replaced the LTR enhancer direct repeats
(from position 419 to 149 from the transcription start
site) with the GATA-1 ARE. The residual activity of this partially
deleted LTR was still constitutively high in all tested cells, although
reduced approximately fivefold as compared with a wild-type LTR (data
not shown). We therefore generated the SFCM vector by deleting the
viral enhancer and CAAT box (from position 419 to 60),
leaving intact only the TATA and initiator elements (Fig 1B). The
GATA-SFCM vector was constructed by cloning the GATA-1 ARE in the LTR
of SFCM (Fig 1B). Retroviral vectors containing the EGFP gene as an
alternative reporter (LGSN and GATA-LGSN) were built following the same
strategy. Amphotropic and ecotropic producer cell lines were
established by transfecting hybrid-LTR plasmid vectors and isolating
G418-resistant clones.
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| Fig 1.
(A) Northern blot analysis of transcripts from K562
(erythroblastic), 3T3 (fibroblastic), MOLT-3 (T-lymphocytic), and
Kasumi-1 (myeloblastic) cell lines transduced with SFCM (lanes ),
GATA-SFCM (lanes G), and SFCM (lanes S) retroviral vectors, after
hybridization to NeoR (upper panels), GATA-1 (middle panels), or GAPDH
(lower panels) probes. Transcripts from the viral LTR (LTR) or the
internal SV40 (SV) promoters are indicated on the left. (B) Schematic
representation of the SFCM retroviral provirus and 5' LTR and of
the modified 5' LTRs of the SFCM and GATA-SFCM vectors. LTR
and SV40 promoters are indicated by arrows. (A)n indicates
the polyadenylation site. The LTR U3 wild-type enhancer (MoMLV-E), the
GATA-1 enhancer (GATA1-E), the LTR R and U5 regions, and the CAAT and
TATA elements are indicated.
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Efficiency of gene transfer was assayed by infecting GATA-1-expressing
cell lines (human K562 for amphotropic and MEL cells for ecotropic
vectors) and counting LNGFR+ or EGFP+ cells
by FACS analysis 48 hours after infection. Positive cells ranged from
5% to 25% in cultures transduced by enhancer-replaced LTR vectors and
from 50% to 80% in cultures transduced by wild-type LTR vectors (data
not shown). The differences correlated with the viral titers assayed on
NIH/3T3 cells, which ranged from 104 to 105
cfu/mL for SFCM, GATA-SFCM, and GATA-LGSN vectors to 106
cfu/mL for SFCM and LGSN vectors.
The GATA-SFCM vector is expressed only in erythroblastic cell lines.
The transcriptional activity of the chimeric LTR was tested in
different cell lines of hemato-lymphopoietic origin. Human myeloblastic
(Kasumi-1), monoblastic (U937), erythroblastic (K562), and
T-lymphoblastic (MOLT-3) cell lines and murine erythroblastic (MEL) and
fibroblasts (NIH 3T3) cells were transduced with the SFCM, SFCM, and
GATA-SFCM retroviral vectors and selected as bulk cultures for
resistance to G418. Southern blot analysis of vector integration showed
that modifications in the 3' LTRs were correctly transferred to
the 5' LTRs, originating intact proviruses integrated at multiple
sites in all target cell populations (data not shown). Expression of
vector-specific transcripts was analyzed by Northern blotting of
poly(A)+ RNA and hybridization to a NeoR-specific probe.
LTR-driven transcripts were never detected in cells transduced with the
SFCM vector (Fig 1A, lanes ), indicating that deletion of viral
enhancer/CAAT sequence virtually abrogated transcription from the LTR
promoter. Genomic transcripts derived from the modified LTR of the
GATA-SFCM vector were detected only in K562 cells, and not in Kasumi-1, MOLT-3, NIH 3T3 (Fig 1A, lanes G), and U937 (not shown) cells. K562
(Fig 1A) and MEL (not shown) were the only cell lines to express GATA-1
transcripts at detectable levels. Subgenomic, SV40 promoter-driven
transcripts were present in all samples (Fig 1A). Transcripts from the
GATA-modified LTR were accumulated in K562 cells at levels of
approximately 50% of those derived from the wild-type LTR, as
quantitated by phosphorimaging after normalization for GAPDH mRNA
contents (Fig 1A, lanes G v S). The significant difference in
size between the LTR-driven transcripts of GATA-SFCM and SFCM vectors
is due to the use of a full-length (1.5 kb) rather than a truncated
(0.9 kb) version of the LNGFR cDNA in the SFCM vector used in this
particular experiment.
Cell surface expression of LNGFR was quantitatively analyzed by flow
cytometry in all G418-selected cell lines. Expression from the
GATA-SFCM vector was observed only in K562 and MEL cells (Fig 2, dotted lines), with mean
fluorescence values approximately sixfold and threefold lower,
respectively (169 v 1,001 and 50 v 135 arbitrary units
[AU]), than those of SFCM-transduced cells (Fig 2, bold lines). In
all the other cases, LNGFR expression profiles of
GATA-SFCM-transduced and SFCM-transduced cells were indistinguishable (NIH3T3 and Kasumi-1 are shown in Fig 2 as an example).
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| Fig 2.
FACS analysis of surface LNGFR expression in
erythroblastic (K562 and MEL) and nonerythroblastic (3T3 and Kasumi-1)
cell lines transduced by SFCM (solid lines), GATA-SFCM (dotted
lines), and SFCM (bold lines) vectors. Cells were stained with
FITC-conjugated anti-LNGFR antibody.
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Expression of the GATA-SFCM vector is restricted to the
erythroblastic progeny of transduced human
CD34+ cells.
Human CD34+ cells from cord blood were transduced by
multiple cycles of infection with viral supernatants in the presence of cytokines (huIL-3, huIL-6, and huSCF) and in coculture with the murine
stromal cell clone CBR-BM, D#1. This clone was previously described to
support survival and proliferation of human
CD34+/CD38low hematopoietic stem
cells,25 leading to higher yield and increased gene
transfer efficiency compared with standard protocols based on cytokines
only (Aiuti et al, manuscript in preparation).
SFCM-transduced and GATA-SFCM-transduced CD34+ cells were
analyzed for expression of LNGFR by multiparameter flow cytometry 48 hours after infection. The efficiency of transduction of primitive
hematopoietic progenitors, identified by a low side scatter profile and
the CD34+/CD38low phenotype
(Fig 3, upper panel), ranged from 55%
(SFCM-transduced cells) to 23% (GATA-SFCM-transduced cells; Fig 3,
lower panel), correlating with the difference in viral titer. Both
wild-type and the enhancer-replaced LTRs were expressed in
CD34+/CD38low cells, although at different
levels (mean fluorescence of 1,003 and 214 AU, respectively). These
data indicate efficient transduction of primitive hematopoietic
progenitors, which were induced to proliferate without losing the
primitive, CD34+/CD38low phenotype.
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| Fig 3.
Multiparametric FACS analysis of human cord blood
CD34+ cells 48 hours after transduction with SFCM and
GATA-SFCM vectors. The forward (FSC) and side (SSC) scatter plot is in
the upper-left panel. Expression of LNGFR (lower panels) was
analyzed in the low-SSC, CD34+, CD38low gated
cell population (boxed in the upper-right panel). The percentage of
LNGFR+ cells (boxed) is indicated in the lower panels.
Cells were stained with FITC-conjugated anti-LNGFR, TC-conjugated
anti-CD38 (X axis) and PE-conjugated anti-CD34 (Y axis) antibodies. C,
control, untransduced cells.
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To test the expression of the chimeric LTR in the differentiated
progeny of human hematopoietic progenitors, transduced
CD34+ cells were grown in liquid culture in conditions that
allow both myeloid and erythroid differentiation. LNGFR expression
was evaluated by FACS analysis of cells double-stained with antibodies
against either erythroid (GpA) or myeloid (CD13) lineage-specific
surface markers. A representative experiment is shown in
Fig 4. After 6 days of culture and 48 hours after
transduction (day 6, left panel in Fig 4), the proportion of
CD34+ cells expressing the LNGFR marker ranged from 32%
in cultures infected with SFCM to 13% in cultures infected with
GATA-SFCM, with mean fluorescence values of 1,613 and 555 AU,
respectively. After induction of differentiation, LNGFR was
expressed in a comparable fraction of GpA+ and
CD13+ cells derived from SFCM-transduced progenitors
(35.4% v 30%), whereas it was expressed preferentially in
GpA+ and only in a few CD13+ cells (14.7%
v 3.6%) derived from GATA-SFCM-transduced progenitors (day 8, middle and right panels in Fig 4). Quantitative expression of LNGFR
was comparable in GpA+ cells transduced with SFCM and
GATA-SFCM (246 v 218 AU), whereas in CD13+ cells
transduced with GATA-SFCM, it was fivefold lower than in those
transduced with SFCM (155 v 730 AU).
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| Fig 4.
Expression of LNGFR in liquid cultures of control (C),
SFCM-transduced, and GATA-SFCM-transduced CD34+ cells.
Cultures were analyzed 48 hours after transduction for expression of
LNGFR and CD34 (day 6) and 2 days after induction of differentiation
for expression of LNGFR and the lineage-specific markers GpA and
CD13 (day 8). CD34+, GpA+, and
CD13+ cells are shown in blue, red, and green colors,
respectively. Values indicate the percentage of LNGFR+
cells in the CD34+, GpA+, or
CD13+ fractions (boxed). Cells were stained with
FITC-conjugated anti-LNGFR (X axis) and PE-conjugated anti-CD34, GpA,
and CD13 (Y axis) antibodies.
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To provide an independent assay of the expression properties of the
enhancer-replaced LTR, transduced CD34+ cells were grown in
clonal culture in the presence of G418. Colonies were morphologically
scored as BFU-E, CFU-GM, or CFU-GEMM between 10 and 14 days from
plating. Transduction efficiency in clonogenic progenitors (percentage
of G418-resistant colonies) ranged from 19% to 26% for the SFCM
vector and from 7.5% to 12% for the GATA-SFCM vector and was overall
higher for CFU-GM (15% to 51%) than for BFU-E (3.5% to 12.5%)
progenitors, with no significant difference between the two vectors
(data not shown). BFU-E and CFU-GM colonies were individually picked,
pooled (60 to 80 colonies/pool), and analyzed for LNGFR expression
by immunocytochemistry. As shown in
Fig 5, both BFU-E and CFU-GM
colonies derived from SFCM-transduced clonogenic progenitors expressed
cytoplasmic and surface LNGFR (upper panels). Conversely, only BFU-E
colonies derived from GATA-SFCM-transduced progenitors were positive
for LNGFR staining (Fig 5, lower panels). Colony identification was
confirmed by staining for GpA and CD13 (not shown).
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| Fig 5.
Immunocytochemical staining for LNGFR in pools of
BFU-E (left) and CFU-GM (right) colonies from methylcellulose clonal
cultures of CD34+ hematopoietic progenitors transduced
with the SFCM (upper panels) and GATA-SFCM (lower panels) vectors.
LNGFR+ cells appear in red after APAAP staining. The
bar is 10 µm.
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To study gene expression at the level of single colonies,
CD34+ cells were transduced with the LGSN and GATA-LGSN
vectors carrying the EGFP marker gene. Clonogenic assay was performed
under the same conditions described for LNGFR vectors, and
G418-resistant, EGFP+ colonies were scored in situ under an
inverted fluorescence microscope. In a representative experiment, EGFP
was expressed in both CFU-GM (99 of 102) and BFU-E (48 of 50) colonies
differentiated from LGSN-transduced progenitors and in most of the
accessory cells in the plates (Fig 6A and
B). On the contrary, EGFP expression was detected only in BFU-E
colonies (13 of 14 v 1 of 30 CFU-GM colonies) in the progeny of
GATA-LGSN-transduced CD34+ cells (Fig 6C through F).
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| Fig 6.
Expression of EGFP in G418-resistant colonies grown in
methylcellulose from CD34+ hematopoietic progenitors
transduced with LGSN (A and B) and GATA-LGSN (C through F) vectors.
Green-fluorescence and bright-field views of the same fields are shown
in the left and right panels, respectively.
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Overall, these results show that the GATA-SFCM LTR is expressed in
CD34+ hematopoietic progenitors, strongly downregulated in
cells undergoing myelo-monocytic differentiation, and persistently
expressed, at levels comparable to those of a wild-type LTR, in cells
undergoing erythroid differentiation.
Lineage restriction of LNGFR expression in mice
engrafted with GATA-SFCM-transduced BM cells.
To test the expression of the enhancer-replaced LTR in the progeny of
hematopoietic stem cells in vivo, 1 to 1.5 × 107 BM
cells from C57Bl/6-Ly-5.1 (CD45.1+) donor mice were
transduced by coculture with ecotropic SFCM and GATA-SFCM packaging
cells and transplanted into lethally irradiated C57Bl/6
(CD45.2+) recipient mice. Transduction efficiency of murine
progenitors, expressed as a percentage of G418-resistant colonies in
methylcellulose assay, averaged 80% for SFCM and 23% for GATA-SFCM
vectors, respectively. Eight weeks posttransplant, BM cells from
recipient animals were analyzed for expression of LNGFR and
erythroid (TER-119) and myeloid (Gr-1) lineage-specific markers.
Engraftment of donor cells in the BM, indicated by the proportion of
CD45.1+/total CD45+ cells, averaged 66.5% ± 9.1%, with no significant difference between the two groups of
mice (data not shown). In mice transplanted with SFCM-transduced BM,
the proportion of LNGFR+ cells averaged 3% in the
Sca-1+ stem cells, 6% in the TER-119+ cells,
and 4% in the Gr-1+ cells (Fig
7, right panels). In recipients of GATA-SFCM-transduced BM,
LNGFR+ cells were detected at significant level (7%)
only in the erythroblastic, TER119+ cell population (Fig 7,
left panels). Quantitative expression of LNGFR was comparable in
TER119+ cells transduced with SFCM and GATA-SFCM (69 v 116 AU). These results indicate that expression of the
GATA-1-modified LTR is restricted to the erythroblastic lineage also
in the progeny of transduced mouse-repopulating stem cells.
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| Fig 7.
Expression of LNGFR in the BM of a mouse 2 months
after transplantation of SFCM-transduced (right) and
GATA-SFCM-transduced (left) BM cells. Cells were stained with
FITC-conjugated anti-LNGFR (X axis) and PE-conjugated anti-Sca1, TER
119, and Gr-1 (Y axis) antibodies. Positivity to Sca1 and TER 119 was
analyzed in the blast/lymphocyte-gated cells, and positivity to GR-1
was analyzed in the granulocyte-gated cells.
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To analyze the activity of the GATA-1-modified LTR at all stages of
erythroblast differentiation, expression of TER119 and LNGFR was
assayed in three different subpopulations of BM cells, gated according
to their forward-scatter (FSC) profile. These populations contain
erythroid cells at different, morphologically recognizable stages of
maturation.27 As shown in Fig
8, immature large erythroblasts (fraction A), more mature small
erythroblasts (fraction B), and mature erythrocytes (fraction C)
contain a comparable percentage of cells expressing LNGFR (between
7% and 8%). However, quantitative levels of LNGFR appeared to
decrease with the maturation stage (mean fluorescence decreasing from
86 AU in fraction A to 22 AU in fraction C; see Fig 8). These results
indicate that the GATA-1-responsive LTR is active at all stages of
murine erythroid differentiation.
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| Fig 8.
Expression of LNGFR in erythoid cells
(TER119+) at different stages of differentiation,
obtained from the BM of a mouse 2 months after transplantation of
GATA-SFCM-transduced BM cells. Cells were stained with FITC-conjugated
anti-LNGFR (X axis on the right panels) and PE-conjugated anti-TER
119 (Y axis on the right panels) antibodies. Positivity to both markers
was analyzed in three separate BM fractions, gated according to their
forward scatter (SSC, Y axis in the left panels) profile and
containing, respectively, large erythroblasts (A), mature erythroblasts
(B), and erythrocytes (C).
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DISCUSSION |
Transcriptional control of an integrated retroviral vector is mastered
by the 5' LTR of the provirus, which exerts a strong influence on
the activity of any additional enhancer/promoter element placed within
the viral transcription unit. Traditional ways to overcome this
transcriptional interference include disabling the LTR by enhancer
deletion or cloning alternative transcription units upstream of the
5' LTR U3 region or in the opposite transcriptional orientation.
Each design has advantages and limitations, and none can be considered
of general applicability.6 We have tried to redirect,
rather than disable, the activity of the 5' LTR promoter by
replacing the constitutive U3 extended enhancer (direct repeats + CAAT
box) with the 200-bp ARE from the murine GATA-1 gene. Retroviral vectors in which the enhancer-replaced LTR drive the expression of
appropriate reporter genes (LNGFR or EGFP) were used to transduce human primitive hematopoietic progenitors
(CD34+/CD38low) and mouse-repopulating stem
cells. Analysis of transgene expression in vitro as well as in vivo
indicated that the GATA-1 ARE effectively restricts transcription from
the LTR promoter to the erythroid progeny of the transduced stem cells.
The activity of the modified LTR was in fact quantitatively comparable
to that of the wild-type LTR in erythroblasts and erythropoietic cell
lines, at the level of both RNA accumulation and protein synthesis,
whereas it was significantly lower in human
CD34+/CD38low and murine Sca1+
progenitors and virtually absent in other cell types. Transcription of
the modified LTR appears to be activated in cycling progenitors, downregulated in myelo-monocytic cells, and maintained at sustained levels in all stages of erythroid differentiation, strictly paralleling the expression pattern of the GATA-1 gene during differentiation of
human primary hematopoietic cells.28,29 The LTR
modification had no effect on integration and stability of the provirus
in the target cell genome. Enhancer replacement is therefore an
effective strategy to target transcription of a retroviral genome to a
specific progeny of stem cells transduced ex vivo and reintroduced into an active hematopoietic system.
These data provide new insight into the role of the ARE in the
physiological regulation of the GATA-1 gene. In adult mice, expression
of GATA-1 is restricted to hematopoietic cells and Sertoli cells of the
testis by the activity of two alternative promoters, designated IE and
IT, located approximately 8 kb apart on the X chromosome.30
The proximal, erythroid-specific IE promoter drives transcription of
GATA-1 at low levels in multipotential hematopoietic progenitors and at
high levels in differentiating erythroblasts, megacaryocytes, and mast
cells.31 Initiation and maintenance of GATA-1 transcription
in development and differentiation of both primitive and definitive
erythropoietic cells was shown to be under the control of multiple,
DNase hypersensitive elements located upstream (from 3,900 to
2,700) and downstream (first intron) of the IE
promoter.32-35 The ARE (856 to 655 from the IE promoter), which contains two GATA-1 binding sites, was originally identified by in vitro footprinting17 and shown to restrict expression of a heterologous promoter to erythroblastic cell lines in a
transient transfection assay.17,18 Although its role has not been addressed by direct mutagenesis in vivo and remains therefore uncertain, this element might cooperate in erythroid-specific maintenance of GATA-1 gene expression by activating a GATA-1-driven auto-regulatory loop.17,18 Our data provide the first in
vivo evidence for such a role by showing that transcription of a
transgene under the control of the GATA-1 ARE is activated in early
hematopoietic progenitors (Sca1+) and maintained at
significant levels throughout erythropoietic differentiation with a
kinetics virtually overlapping that of the endogenous GATA-1 gene. The
auto-regulatory circuit established by the ARE is therefore sufficient
to maintain GATA-1-dependent transcription in differentiating
erythroblasts. This circuit might be essential to sustain GATA-1
transcription at appropriate levels in erythroid cells, the maturation
of which is dose-dependent with respect to GATA-1 levels.33
As a strategy for transcriptional targeting, manipulating the LTR
control elements offers a number of advantages. First, it provides a
more efficient vector design, which allows us to use the major viral
transcription unit to express the gene of interest under the form of a
spliceable RNA. Second, it allows the use of a second, independently
regulated internal promoter to control an additional function, such as
a positive (NeoR or LNGFR) or negative (HSV-TK) selectable marker
for selecting packaging cells or eliminating transduced cells in vivo.
Third, the presence of a genomic control element into the LTR could
reduce the chances of chromatin-mediated inactivation of the integrated
provirus. This is known to affect the long-term maintenance of MoMLV
LTR-driven gene expression particularly in stem cells36-38
and has been circumvented in the past by replacing the MoMLV enhancer
with that of viruses with different tropism.39-41 The use
of genomic instead of viral sequences could have the additional
advantage of promoting transcriptionally active chromatin
configurations only in the cells in which the transgene is
transcriptionally targeted.
We had previously shown that the simple insertion of a muscle-specific
enhancer into an intact MoMLV LTR is sufficient to restrict the
transcriptional activity of the viral promoter to differentiated muscle
fibers.13 However, the same design was unsuccessful in the
case of the GATA-1 ARE, which has no effect in modulating the activity
of the LTR if inserted upstream of the viral enhancer (G.F.,
unpublished results). Replacing, rather than adding
sequences to, the viral enhancer therefore appears to be a more
efficient strategy to restrict transcriptional activity of retroviral
vectors, although its general applicability needs to be tested in
additional contexts. In fact, the LTR architecture imposes a number of
constrains upon the elements that it can accommodate, such as the
distance between the enhancer and the promoter and the sequence of the
enhancer itself, and it is reasonable to assume that some tissue-or
cell-specific elements could be unable to drive the TATA-containing LTR
promoter in an appropriate fashion. Unpredictable behavior of chimeric
LTRs has already been observed in the past.42 Maintaining
the LTR CAAT element downstream from the replaced enhancer increases
the transcriptional efficiency of the chimeric LTR by increasing the
activity of the basal promoter, although this necessarily limits the
stringency of the tissue- or cell lineage-specificity14
(see Results). The overall size of the inserted fragment can also be a
problem: an LTR much bigger than its normal size reduces both viral
titer and stability of proviral integration, most likely because of
decreased efficiency of reverse transcription.14,43,44
Although titer problems could be overcome in a number of ways, a high
frequency of rearrangement would limit the practical use of certain
enhancer-replaced vectors.
The vector targeting strategy described in this paper could be
potentially relevant for gene therapy of hemoglobinopathies. Current
attempts to build retroviral vectors for expression of  -globin in
erythropoietic cells are based on the use of a full -globin gene and
part of the -globin locus control region (LCR), with the rationale
of reproducing, on a smaller size scale, the high-level and
position-independent expression provided by the interaction between the
LCR and the -globin promoter.45,46 However, genetic
instability severely limits the possibility of using full-length LCR
sequences in the context of a retroviral vector,47,48
whereas small LCR fragments are apparently unable to reduce the
integration-dependent variability of transgene expression observed with
MoMLV-based vectors.24,49 Indeed, the very concept that the
LCR has a role in maintaining an open chromatin structure to the globin
locus has been challenged by more recent evidence.50,51 Binding of erythroid-specific transcription factors, such as GATA-1, to
enhancers of erythroid-specific genes early in development or
differentiation could instead be a key factor in initiation and
maintenance of open chromatin configurations.52,53 Although our experiments do not directly address this issue, LTRs engineered with GATA-1-responsive elements could therefore provide a certain degree of position-independence to the expression of integrated proviruses in erythroid cells. A potential limitation of such a vector
design could be an insufficient level of -globin mRNA accumulation,
which even in the best conditions (use of a -globin promoter and
noncoding trailer sequences), is likely to remain lower than that
obtainable with an LCR. However, even a mild correction of the
-globin imbalance in maturing erythroblasts might be sufficient to
reduce ineffective erythropoiesis in severe -thalassemia
patients54 and significantly affect both morbidity and
clinical management of the disease. On the short-term, a
limited-correction gene therapy strategy based on autologous
transplantation of BM transduced with a transcriptionally targeted
retroviral vector could therefore be a safe and viable alternative to a
still elusive full functional replacement of the -globin locus.
| |
ACKNOWLEDGMENT |
The authors thank C. Moroni for the initial help in constructing and
testing hybrid LTRs and G. Torriani and R. Parma for the excellent
technical assistance.
| |
FOOTNOTES |
Submitted September 22, 1998; accepted January 13, 1999.
Supported by a core grant from the Italian Telethon Foundation and a
grant from the Istituto Superiore di Sanità.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Giuliana Ferrari, PhD, TIGET-H.S.
Raffaele, Via Olgettina, 58, 20132 Milano, Italy; e-mail:
ferrari{at}tigem.it.
| |
REFERENCES |
1.
Mulligan RC:
The basic science of gene therapy.
Science
260:926, 1993[Abstract/Free Full Text]
2.
Crystal RG:
Transfer of genes to humans: Early lessons and obstacles to success.
Science
270:404, 1995[Abstract/Free Full Text]
3.
Verma IM, Somia N:
Gene therapy-Promises, problems and prospects.
Nature
389:239, 1997[Medline]
[Order article via Infotrieve]
4.
Emerman M, Temin HM:
Genes with promoters in retrovirus vectors can be independently suppressed by an epigenetic mechanism.
Cell
39:449, 1984[Medline]
[Order article via Infotrieve]
5.
Emerman M, Temin HM:
Quantitative analysis of gene suppression in integrated retrovirus vectors.
Mol Cell Biol
6:792, 1986[Abstract/Free Full Text]
6.
Miller N, Whelan J:
Progress in transcriptionally targeted and regulatable vectors for genetic therapy.
Hum Gene Ther
8:803, 1997[Medline]
[Order article via Infotrieve]
7.
Yu S, von Ruden T, Kantoff PW, Garber C, Sciberg M, Rather U, Anderson WF, Wagner EF, Gilboa E:
Self-inactivating retroviral vectors designed for transfer of whole genes into mammalian cells.
Proc Natl Acad Sci USA
83:3194, 1986[Abstract/Free Full Text]
8.
Yee JK, Moores JC, Jolly DJ, Wolff JA, Respess JG, Friedmann T:
Gene expression from transcriptionally disabled retroviral vectors.
Proc Natl Acad Sci USA
84:5197, 1987[Abstract/Free Full Text]
9.
Naffakh N, Pinset C, Montarras D, Li Z, Paulin D, Danos O, Heard JM:
Long-term secretion of therapeutic proteins from genetically modified skeletal muscles.
Hum Gene Ther
7:11, 1996[Medline]
[Order article via Infotrieve]
10.
Karlsson S, Papayannopoulou T, Schweiger SG, Stamatoyannopoulos G, Nienhuis AW:
Retroviral-mediated transfer of genomic globin genes leads to regulated production of RNA and protein.
Proc Natl Acad Sci USA
84:2411, 1987[Abstract/Free Full Text]
11.
Hantzopoulos PA, Sullenger BA, Ungers G, Gilboa E:
Improved gene expression upon transfer of the adenosine deaminase minigene outside the transcriptional unit of a retroviral vector.
Proc Natl Acad Sci USA
86:3519, 1989[Abstract/Free Full Text]
12.
Davis B, Linney E, Fan H:
Suppression of leukaemia virus pathogenicity by polyoma virus enhancers.
Nature
314:550, 1985[Medline]
[Order article via Infotrieve]
13.
Ferrari G, Salvatori G, Rossi C, Cossu G, Mavilio F:
A retroviral vector containing a muscle-specific enhancer drives gene expression only in differentiated muscle fibers.
Hum Gene Ther
6:733, 1995[Medline]
[Order article via Infotrieve]
14.
Diaz RM, Eisen T, Hart IR, Vile RG:
Exchange of viral promoter/enhancer elements with heterologous regulatory sequences generates targeted hybrid long terminal repeat vectors for gene therapy of melanoma.
J Virol
72:789, 1998[Abstract/Free Full Text]
15.
Tsai SF, Martin DI, Zon LI, D'Andrea AD, Wong GG, Orkin SH:
Cloning of cDNA for the major DNA-binding protein of the erythroid lineage through expression in mammalian cells.
Nature
339:446, 1989[Medline]
[Order article via Infotrieve]
16.
Evans T, Felsenfeld G:
The erythroid-specific transcription factor Eryf1: A new finger protein.
Cell
58:877, 1989[Medline]
[Order article via Infotrieve]
17.
Tsai SF, Strauss E, Orkin SH:
Functional analysis and in vivo footprinting implicate the erythroid transcription factor GATA-1 as a positive regulator of its own promoter.
Genes Dev
5:919, 1991[Abstract/Free Full Text]
18.
Nicolis S, Bertini C, Ronchi A, Crotta S, Lanfranco L, Moroni E, Giglioni B, Ottolenghi S:
An erythroid specific enhancer upstream to the gene encoding the cell-type specific transcription factor GATA-1.
Nucleic Acids Res
19:5285, 1991[Abstract/Free Full Text]
19.
Mavilio F, Ferrari G, Rossini S, Nobili N, Bonini C, Casorati G, Traversari C, Bordignon C:
Peripheral blood lymphocytes as target cells of retroviral vector-mediated gene transfer.
Blood
83:1988, 1994[Abstract/Free Full Text]
20.
Ruggieri L, Aiuti A, Salomoni M, Zappone E, Ferrari G, Bordignon C:
Cell-surface marking of CD(34+)-restricted phenotypes of human hematopoietic progenitor cells by retrovirus-mediated gene transfer.
Hum Gene Ther
8:1611, 1997[Medline]
[Order article via Infotrieve]
21.
Miller AD, Rosman GJ:
Improved retroviral vectors for gene transfer and expression.
Biotechniques
7:980, 1989[Medline]
[Order article via Infotrieve]
22.
Markowitz D, Goff S, Bank A:
A safe packaging line for gene transfer: Separating viral genes on two different plasmids.
J Virol
62:1120, 1988[Abstract/Free Full Text]
23.
Markowitz D, Goff S, Bank A:
Construction and use of a safe and efficient amphotropic packaging cell line.
Virology
167:400, 1988[Medline]
[Order article via Infotrieve]
24.
Sadelain M, Wang CH, Antoniou M, Grosveld F, Mulligan RC:
Generation of a high-titer retroviral vector capable of expressing high levels of the human beta-globin gene.
Proc Natl Acad Sci USA
92:6728, 1995[Abstract/Free Full Text]
25.
Aiuti A, Friedrich C, Sieff CA, Gutierrez-Ramos JC:
Identification of distinct elements of the stromal microenvironment that control human hematopoietic stem/progenitor cell growth and differentiation.
Exp Hematol
26:143, 1998[Medline]
[Order article via Infotrieve]
26.
Caiulo A, Nicolis S, Bianchi P, Zuffardi O, Bardoni B, Maraschio P, Ottolenghi S, Camerino G, Giglioni B:
Mapping the gene encoding the human erythroid transcription factor NFE1-GF1 to Xp11.23.
Hum Genet
86:388, 1991[Medline]
[Order article via Infotrieve]
27.
Loken MR, Shah VO, Dattilio KL, Civin CI:
Flow cytometric analysis of human bone marrow: I. Normal erythroid development.
Blood
69:255, 1987[Abstract/Free Full Text]
28.
Sposi NM, Zon LI, Care A, Valtieri M, Testa U, Gabbianelli M, Mariani G, Bottero L, Mather C, Orkin SH, Peschle C:
Cell cycle-dependent initiation and lineage-dependent abrogation of GATA-1 expression in pure differentiating hematopoietic progenitors.
Proc Natl Acad Sci USA
89:6353, 1992[Abstract/Free Full Text]
29.
Cheng T, Shen H, Giokas D, Gere J, Tenen DG, Scadden DT:
Temporal mapping of gene expression levels during the differentiation of individual primary hematopoietic cells.
Proc Natl Acad Sci USA
93:13158, 1996[Abstract/Free Full Text]
30.
Ito E, Toki T, Ishihara H, Ohtani H, Gu L, Yokoyama M, Engel JD, Yamamoto M:
Erythroid transcription factor GATA-1 is abundantly transcribed in mouse testis.
Nature
362:466, 1993[Medline]
[Order article via Infotrieve]
31.
Orkin SH:
GATA-binding transcription factors in hematopoietic cells.
Blood
80:575, 1992[Free Full Text]
32.
Onodera K, Takahashi S, Nishimura S, Ohta J, Motohashi H, Yomogida K, Hayashi N, Engel JD, Yamamoto M:
GATA-1 transcription is controlled by distinct regulatory mechanisms during primitive and definitive erythropoiesis.
Proc Natl Acad Sci USA
94:4487, 1997[Abstract/Free Full Text]
33.
McDevitt MA, Shivdasani RA, Fujiwara Y, Yang H, Orkin SH:
A "knockdown" mutation created by cis-element gene targeting reveals the dependence of erythroid cell maturation on the level of transcription factor GATA-1.
Proc Natl Acad Sci USA
94:6781, 1997[Abstract/Free Full Text]
34.
McDevitt MA, Fujiwara Y, Shivdasani RA, Orkin SH:
An upstream, DNase I hypersensitive region of the hematopoietic-expressed transcription factor GATA-1 gene confers developmental specificity in transgenic mice.
Proc Natl Acad Sci USA
94:7976, 1997[Abstract/Free Full Text]
35.
Shivdasani RA, Fujiwara Y, McDevitt MA, Orkin SH:
A lineage-selective knock-out establishes the critical role of transcription factor GATA-1 in megacaryocyte growth and platelet development.
EMBO J
16:3965, 1997[Medline]
[Order article via Infotrieve]
36.
Palmer TD, Rosman GJ, Osborne WRA, Miller AD:
Genetically modified skin fibroblasts persist long after trasplantation but gradually inactivate introduced genes.
Proc Natl Acad Sci USA
88:1330, 1991[Abstract/Free Full Text]
37.
Gerrard AJ, Hudson DL, Brownlee GG, Watt FM:
Towards gene therapy for haemophilia B using primary human keratinocytes.
Nat Genet
3:180, 1993[Medline]
[Order article via Infotrieve]
38.
Challita PM, Kohn DB:
Lack of expression from a retroviral vector after transduction of murine hematopoietic stem cells is associated with methylation in vivo.
Proc Natl Acad Sci USA
91:2567, 1994[Abstract/Free Full Text]
39.
van Beusechem VW, Kukler A, Einerhand MPW, Bakx TA, van der Eb AJ, Van Bekkum DW, Valerio D:
Expression of human adenosine deaminase in mice transplanted with hemopoietic stem cells infected with amphotropic retroviruses.
J Exp Med
172:729, 1990[Abstract/Free Full Text]
40.
van Beusechem VW, Kukler A, Heidt PJ, Valerio D:
Long-term expression of human adenosine deaminase in rhesus monkeys transplanted with retrovirus-infected bone marrow cells.
Proc Natl Acad Sci USA
89:7640, 1992[Abstract/Free Full Text]
41.
Riviere I, Brose K, Mulligan RC:
Effects of retroviral vector design on expression of human adenosine deaminase in murine bone marrow transplant recipients engrafted with genetically modified cells.
Proc Natl Acad Sci USA
92:6733, 1995[Abstract/Free Full Text]
42.
Kaptein LC, Breuer M, Valerio D, van Beusechem VW:
Expression pattern of CD2 locus control region containing retroviral vectors in hemopoietic cells in vitro and in vivo.
Gene Ther
5:320, 1998[Medline]
[Order article via Infotrieve]
43.
Ferrari G, Rossini S, Nobili N, Maggioni D, Garofalo A, Giavazzi R, Mavilio F, Bordignon C:
Transfer of the ADA gene into human ADA-deficient T-lymphocytes reconstitutes specific immune function.
Blood
80:1120, 1992[Abstract/Free Full Text]
44.
Bordignon C, Notarangelo LD, Nobili N, Ferrari G, Casorati G, Panina P, Mazzolari E, Maggioni D, Rossi C, Servida P, Ugazio AG, Mavilio F:
Gene therapy in peripheral blood lymphocytes and bone marrow for ADA immunodeficient patients.
Science
270:470, 1995[Abstract/Free Full Text]
45.
Beuzard Y:
Towards gene therapy of hemoglobinopathies.
Semin Hematol
33:43, 1996[Medline]
[Order article via Infotrieve]
46.
Ioannou P:
Gene therapy for thalassaemia.
Gene Ther
3:746, 1996[Medline]
[Order article via Infotrieve]
47.
Novak U, Harris EA, Forrester W, Groudine M, Gelinas R:
High-level beta-globin expression after retroviral transfer of locus activation region-containing human beta-globin gene derivatives into murine erythroleukemia cells.
Proc Natl Acad Sci USA
87:3386, 1990[Abstract/Free Full Text]
48.
Leboulch P, Huang GM, Humphries RK, Oh YH, Eaves CJ, Tuan DY, London IM:
Mutagenesis of retroviral vectors transducing human beta-globin gene and beta-globin locus control region derivatives results in stable transmission of an active transcriptional structure.
EMBO J
13:3065, 1994[Medline]
[Order article via Infotrieve]
49.
Raftopoulos H, Ward M, Leboulch P, Bank A:
Long-term transfer and expression of the human -globin gene in a mouse transplant model.
Blood
90:3414, 1997[Abstract/Free Full Text]
50.
Reik A, Telling A, Zitnik G, Cimbora D, Epner E, Groudine M:
The locus control region is necessary for gene expression in the human -globin locus but not the maintainance of an open chromatin structure in erythroid cells.
Mol Cell Biol
18:5992, 1998[Abstract/Free Full Text]
51.
Epner E, Reik A, Cimbora D, Telling A, Bender MA, Fiering S, Enver T, Martin DIK, Kennedy M, Keller G, Groudine M:
The -globin LCR is not necessary for an open chromatin structure or developmentally regulated transcription of the native mouse -globin locus.
Mol Cell
2:447, 1998[Medline]
[Order article via Infotrieve]
52.
Orkin SH:
Regulation of globin gene expression in erythroid cells.
Eur J Biochem
231:271, 1995[Medline]
[Order article via Infotrieve]
53.
Martin DI, Fiering S, Groudine M:
Regulation of beta-globin gene expression: Straightening out the locus.
Curr Opin Gen Dev
6:488, 1996[Medline]
[Order article via Infotrieve]
54.
Weatherall DJ:
The thalassemias, in
Stamatoyannopoulos G,
Nienhuis AW,
Majerus PW,
Varmus H
(eds):
The Molecular Basis of Blood Diseases (ed 2). Philadelphia, PA, Saunders, 1994, p 157.
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TOP
ABSTRACT
INTRODUCTION