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Prepublished online as a Blood First Edition Paper on October 31, 2002; DOI 10.1182/blood-2002-07-2211.
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Blood, 15 March 2003, Vol. 101, No. 6, pp. 2175-2183
GENE THERAPY
The degree of phenotypic correction of murine  -thalassemia
intermedia following lentiviral-mediated transfer of a human  -globin
gene is influenced by chromosomal position effects and vector copy
number
Derek A. Persons,
Phillip W. Hargrove,
Esther R. Allay,
Hideki Hanawa, and
Arthur W. Nienhuis
From the Division of Experimental Hematology,
Department of Hematology and Oncology, St Jude Children's Research
Hospital, Memphis, TN.
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Abstract |
Increased fetal hemoglobin (HbF) levels diminish the
clinical severity of  -thalassemia and sickle cell anemia. A
treatment strategy using autologous stem cell-targeted gene transfer
of a  -globin gene may therefore have therapeutic potential. We
evaluated oncoretroviral- and lentiviral-based -globin vectors for
expression in transduced erythroid cell lines. Compared with
-globin, oncoretroviral vectors containing either a -spectrin or
-globin promoter and the  -globin HS40 element, a -globin
lentiviral vector utilizing the -globin promoter and elements from
the -globin locus control region demonstrated a higher
probability of expression. This lentiviral vector design was evaluated
in lethally irradiated mice that received transplants of
transduced bone marrow cells. Long-term, stable erythroid
expression of human -globin was observed with levels of
vector-encoded -globin mRNA ranging from 9% to 19% of total murine
-globin mRNA. The therapeutic efficacy of the vector was subsequently evaluated in a murine model of -thalassemia intermedia. The majority of mice that underwent transplantation expressed significant levels of chimeric m2h2
molecules (termed HbF), the amount of which correlated with the degree
of phenotypic improvement. A group of animals with a mean HbF level of
21% displayed a 2.5 g/dL (25 g/L) improvement in Hb concentration and
normalization of erythrocyte morphology relative to control animals.
-Globin expression and phenotypic improvement was variably lower in
other animals due to differences in vector copy number and chromosomal position effects. These data establish the potential of using a
-globin lentiviral vector for gene therapy of -thalassemia.
(Blood. 2003;101:2175-2183)
© 2003 by The American Society of Hematology.
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Introduction |
The hemoglobin disorders are highly prevalent,
recessive genetic diseases in which coinheritance of 2 defective globin
alleles results in severe hematologic disease. In patients with sickle cell anemia, the beta chain of hemoglobin S contains a substitution of
valine for glutamic acid at position 6.1 This substitution results in a change in surface charge that predisposes deoxygenated HbS
to polymerize, causing red cells to assume rigid sickled shapes leading
to vaso-occlusion, painful crisis, and organ damage. Defective synthesis of -globin in patients with severe -thalassemia due to
a variety of mutational mechanisms leads to the accumulation of
aggregates of unpaired, insoluble -chains that cause ineffective erythropoiesis, accelerated red cell destruction, and severe
anemia.2 Although palliative therapies improve the quality
and duration of life for many individuals, overall treatment for these
disorders remains unsatisfactory. A few patients with sickle cell
disease and a somewhat larger number with -thalassemia have been
cured with bone marrow (BM) transplantation from HLA-matched siblings, but such treatment is available for only a small minority of
patients.3,4 These considerations have made the
development of gene therapy for hemoglobin disorders a highly
desired goal.
Effective gene therapy for hemoglobin disorders will require (1)
relatively efficient gene transfer into repopulating, hematopoietic stem cells; (2) a method for achieving a substantial proportion (20%
or greater) of genetically modified, autologous stem cells in patients
without myeloablation; and (3) a globin vector configuration that
predictably results in durable, high-level expression in differentiating erythroid cells after the gene therapy procedure. During the past few years, significant progress has been made in all 3 areas.
Initial success in genetic modification of repopulating stem cells from
mice with murine oncoretroviral vectors was achieved in the early 1980s
but extension of that success to large animal models and humans in
early stage clinical trials has been highly problematic.5-8 More than a decade of focused effort to
improve oncoretroviral gene transfer has resulted in levels of
genetically modified cells of up to 5% to 20% in some but not all
studied nonhuman primates and up to 5% to 10% in a few patients in
recent human gene marking trials.9,10 Lentiviral vectors
have inherent biologic advantages over murine oncoretroviral vector
particles, and it is hoped that this will translate into improved stem
cell gene transfer efficiency.11,12 Extensive experience
using primitive human hematopoietic cells from cord blood and more
limited experience with cytokine-mobilized cells from adult volunteers
suggest that this system may provide improved gene transfer efficiency
of repopulating stem cells.13-16
Several approaches have been explored for amplifying genetically
modified stem cell populations by in vivo selection in order to obtain
therapeutically relevant levels of corrected cells.17 A
system based on a variant methylguanine, methyltransferase drug resistance gene in which temozolomide and O6-benzylguanine
have been used for stem cell selection appears promising.18 The ability to dramatically amplify a
minority population of genetically modified cells without limiting
myelosuppression has allowed this system to be used to ameliorate the
-thalassemia phenotype with genetically modified and selected,
normal stem cells in the absence of myeloablation (D.A.P. and B. Sorrentino, manuscript submitted).
In many respects, the most difficult aspect of developing gene therapy
for the hemoglobin disorders has been to identify a vector genome
configuration that sustains high-level, erythroid-specific gene
expression in developing erythroblasts. In early studies, vectors that
contained the -globin gene driven by its own promoter were shown to
transduce murine hematopoietic stem cells but globin gene expression
was either absent or very low.19,20 Discovery of the locus
control region (LCR) upstream of the human -like globin gene locus
initiated new efforts to make useful globin gene retroviral
vectors.21,22 The functional elements from the LCR,
detected as hypersensitive (HS) sites in nuclear chromatin, conferred high-level, relatively position-independent expression of
globin genes in transgenic mice.23 However, incorporation of HS elements rendered oncoretroviral vectors unstable during passage
in vitro or attempted transduction of primitive hematopoietic cells.
Systematic elimination of cryptic splice and polyadenylation sites or
empiric evaluation of the many potential orientations and order of
individual HS sites yielded vectors that transferred a -globin gene
into primitive murine hematopoietic cells.24,25 These
vectors failed to express therapeutic levels of globin and in some
instances exhibited significant silencing over time.26,27
The goal of our studies was to derive a vector capable of high-level
expression of the human -globin gene. Patients with either
-thalassemia or sickle cell genotypes display significant disease
amelioration in the setting of increased fetal hemoglobin (HbF)
levels.2 HbF (22) is a
potent, natural antisickling hemoglobin in part because of formation of
mixed tetramers, 2S, that do not
participate in polymer formation.28 Furthermore, enhanced,
vector-mediated expression of the -globin gene could improve
0-thalassemia without the risk of an immune response to
the -globin protein that might occur if it is expressed endogenously
for the first time in the developing erythroblasts of these
individuals. At the onset of this work, 3 potential approaches to
direct vector-mediated -globin expression were emerging: (1) the use
of alternative erythroid-specific promoters such as the -spectrin or
ankyrin promoters that do not depend on LCR elements29,30;
(2) the use of the HS40 element from the -globin locus, rather than
LCR elements, to augment expression of alternative erythroid promoters or the -globin promoter31,32; or (3) the use of
optimized -globin LCR elements to enhance expression of a promoter-driven -globin gene. Recently, HIV-based lentiviral
vectors have been shown to be capable of transferring complex genomes
containing extended LCR elements linked to a -globin
gene.33,34 Phenotypic correction has been achieved in both
-thalassemia intermedia and sickle cell anemia murine models. Here
we report the development of a novel self-inactivating, -globin
lentiviral vector containing -globin LCR elements that can direct
therapeutic expression in a murine model of -thalassemia intermedia.
Our -globin lentiviral vector will provide an alternative
therapeutic approach to the hemoglobin disorders based on increasing
levels of HbF.
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Materials and methods |
Plasmid construction
Plasmids containing the various vector genomes were assembled
using standard recombinant DNA technology. The details of all plasmid
constructions are available upon request. The specific HS fragments
used in the HS432-A vector have been previously
described.35 A plasmid, AC553, containing the -spectrin
promoter was a gift from Dr D. Bodine (National Institutes of Health
[NIH], Bethesda, MD).29 The 255-bp -globin
HS40 fragment was a gift from Dr G. Atweh (Mount Sinai Medical Center,
New York, NY).31
Oncoretroviral and lentiviral vector preparation
VSV-G pseudotyped oncoretroviral vector particles were prepared
using a 3-plasmid system (gene transfer vector plasmid, Gag/Pol plasmid, and VSV-G envelope plasmid) by transient transfection of 293T
cells as previously described.36 Lentiviral vector
particles were prepared as described previously, with some
modifications.16 In brief, 293T cells were transfected
with a mixture of plasmid DNA consisting of 6 µg pCAGkGP1R
(Gag/Pol), 2 µg pCAG4-RTR2 (Rev/Tat), 2 µg pCAG-VSVG (VSV-G
envelope), and 10 µg of gene transfer vector plasmid per 10-cm dish
using the calcium phosphate precipitation technique. At 24 hours after
transfection, the residual calcium-phosphate-DNA precipitate was
removed by washing with 10 mL of phosphate-buffered saline (PBS).
Serum-free X-VIVO 10 medium without phenol red (BioWhittaker, Walkersville, MD) was then added to each plate of cells. Then, 18 hours later, medium conditioned by vector-producing cells was harvested, cleared by low-speed centrifugation, and filtered through a
0.2-µm pore-sized cellulose acetate filter. In some cases, the conditioned medium was concentrated 100- to 200-fold by
ultracentrifugation for 90 minutes at 25 000 rpm at 4°C using a
Beckman Sw28 rotor (Beckman Instruments, Palo Alto, CA).
Aliquots of vector preparations were quick frozen and kept at
 80°C.
Titering of unconcentrated vector preparations was performed using NIH
3T3 cells and enumerating, by fluorescence-activated cell-sorter
(FACS) analysis, the percentage of green fluorescent protein-positive (GFP+) cells derived by
transduction with serial dilutions of conditioned media. For vectors
not containing the GFP reporter, titers were determined by comparing
the signal intensity of vector genome transfer into NIH 3T3 cells, as
assessed by Southern blot analysis, relative to the signal obtained
using CL10.1 murine stem cell virus (MSCV) GFP of known GFP
titer.16
Culture and transduction of mouse erythroleukemia (MEL)
cells
MEL cells were maintained in Dulbecco modified Eagle medium
(DMEM) supplemented with 10% fetal bovine serum (FBS), 50 units/mL penicillin G, and 50 µg/mL streptomycin. In all experiments,
transduction was performed by exposing 50 000 cells to a mixture of 1 mL viral supernatant (titer range of 1-5 × 105
transducing units [TU]/mL) with 1 mL growth medium in the presence of
polybrene at a final concentration of 6 µg/mL. Then, 24 hours later, vector-containing medium was replaced with fresh medium and cells were expanded. The GFP+ fraction was subsequently
purified by flow cytometry and returned to culture. Erythroid induction
was accomplished by adding 3 µM N,N'-hexamethylenebisacetamine and 10 µM
hemin. Cells were harvested after 6 days and evaluated for -globin
and GFP expression using the FACSCalibur (Becton Dickinson
Immunocytochemistry Systems, San Jose, CA) as described in "FACS
analysis of red cells for expression of human -globin"
below. The data were analyzed using the Cell Quest System
(Becton Dickinson Immunocytochemistry Systems).
Transduction and transplantation of murine BM cells
Mice heterozygous for the knocked-out -major and -minor
globin gene locus and previously characterized as having a severe -thalassemia phenotype that most closely represents human
-thalassemia intermedia were bred onto the HW80 background
(histocompatible with C57Bl/6J mice) as previously
described.37,38 BM cells from -thalassemic or normal
HW80 mice (B6.C-TyrcH1b Hbbd/By,
Jackson Laboratory; Bar Harbor, MA) were harvested from the femurs and
tibias 48 hours after treatment with 150 mg/kg 5-fluorouracil (Pharmacia; Kalamazoo, MI). Cells were placed into DMEM culture medium containing 20% FBS (Hyclone; Logan, UT) and 20 ng/mL
murine interleukin-3 (IL-3), 50 ng/mL murine IL-6, 50 ng/mL murine stem cell factor (all obtained from R&D Systems, Minneapolis, MN). After 48 hours, cells were collected, washed with PBS, and
8 × 106 cells were pelleted and resuspended in 1.5 to
2.0 mL of concentrated vector (2-3 × 108 TU/mL)
containing the above-stated concentration of serum/cytokines and
polybrene at 6 µg/mL. This mixture was placed into a RetroNectin (TAKARA Shuzo, Otsu, Shiga, Japan)-coated (20 µg/cm2)
6-well plate and incubated at 37°C in a humidified incubator with 5%
CO2. After 6 hours, additional growth medium (supplemented with cytokines and serum as above) was added to the culture to a final
volume of 4 mL and the cells were further incubated overnight. The
following day, cells were collected, washed with PBS, and resuspended
in PBS containing 2% FCS. Lethally irradiated (1050 cGy) C57Bl/6J mice
received transplants of 1 to 2 × 106 cells by
tail-vein injection.
Hematologic analysis
Blood samples were obtained by retro-orbital puncture of
anesthetized mice. Complete blood counts were obtained using an
automated blood cell analyzer as previously described.38
Peripheral blood (PB) films were prepared by standard methods. Hb
cellulose acetate gel electrophoresis and quantitation of Hb bands was
performed as previously described.38 An AlphaImager 2200 visualization system (Alpha Innotech, San Leandro, CA) was used to
estimate the relative proportions of the Hb bands. "HbF" bands
could not be accurately quantified below the 4% level and all animals
in this category are referred to as less than 4% HbF mice.
Reticulocyte counts were estimated as described by using flow
cytometric analysis to determine the proportion of red cells staining
with the RNA binding dye thiazole orange (Aldrich, Milwaukee,
WI).39
FACS analysis of red cells for expression of human
-globin
We used 5 to 10 µL of blood to fix, permeabilize, and stain
red cells with a biotinylated monoclonal antibody against the human
-globin chain (Perkin-Elmer Wallac, Norton, OH) as previously described.38 In brief, 5 µL of the monoclonal antibody
was incubated for 30 minutes on ice with permeabilized cells. After
washing, cells were then incubated in a 1:200
streptavidin-phycoerythrin (PE) secondary reagent (Southern
Biotechnology, Birmingham, AL) in order to identify cells stained with
the primary antibody. Red cells were gated on by light scatter
characteristics and analyzed for PE fluorescence using a FACSCalibur.
Ribonuclease protection assay
Preparation of RNA from PB samples was done using Rnazol B
(Tel-Test; Friendswood, TX) according to the manufacturer's
specifications. Determination of human -globin and murine -globin
mRNA levels as assessed by the relative amounts of their respective
exon 2 protected fragments was performed using an RPAII RNase
protection assay kit as described previously according to the
manufacturer's specifications.38 In the current studies,
a riboprobe containing both antisense human A-globin and
murine -globin sequences was used (gift from Dr D. Bodine).40 This riboprobe is characterized by having equal specific activities of the human A-globin and murine
-globin sequences, thereby allowing a direct comparison of
A-globin and -globin mRNA levels. The relative level
of lentiviral vector-encoded human A-globin mRNA
compared with endogenous murine -globin mRNA was determined by
dividing the signal of the human A-globin protected
fragment by the value of the signal of the murine -globin protected
fragment. To normalize expression per vector copy relative to an
endogenous -globin gene, this value was further divided by the
estimated vector copy number and multiplied by a factor of 4 to correct
for the number of -globin genes. A Molecular Dynamics (Sunnyvale,
CA) Storm Phosphoimager and its accompanying software were
used to visualize and quantitate the protected fragments.
DNA analysis
MEL cell, PB leukocyte (PBL), BM, and spleen colony DNA samples
were prepared, digested with the restriction enzymes, and subjected to
Southern blot analysis as previously described.38 XmaI was used to liberate full-length oncoretroviral
proviral fragments. The enzyme BglII, which cuts at the ends
of the provirus and liberates a near unit length provirus, and enzyme
KpnI, which cuts once within the provirus, were used to
verify the presence of unrearranged lentiviral vector and determine the
number of vector integrations, respectively. A radiolabeled GFP or
viral rev-responsive element (RRE) DNA probe was hybridized with the blot and the resulting hybridizing bands were visualized and
quantitated using the Molecular Dynamics Storm Phosphoimager and its
accompanying software.
Determination of average vector copy number by semiquantitative
polymerase chain reaction (PCR)
Semiquantitative PCR was performed on the PBL genomic DNA of
-thalassemic mice that received transplants of BM transduced with
the -globin vectors. The standard samples were prepared by making
dilutions of DNA from a MEL cell line containing a single, integrated
copy of the d432-A MSCV GFP provirus into mouse
spleen DNA. This MEL cell clone also contains only one copy of the
mouse -major gene due to monosomy 7 (data not shown). Standards
included a range of copy number from 0.05 to 1.0. The signal from mouse
-major was used as a loading control. Duplex PCR (25 cycles) was performed using an MJ Research PTC-200 peltier thermocycler
(Watertown, MA). Primers were as follows: 5' mouse -major primer
5'-cctatcctctgcctctgcta-3' and 3' primer 5'-cttctggaaggcagcctgtg-3'; 5'
-globin primer 5'-agcaacctcaaacagacacc-3' and 3' primer
5'-ggccactccagtcaccatctt-3'. DNA template (250 ng) was used
and the reaction mixture contained 32P-labeled
deoxycytidine 5'-triphosphate (dCTP) (ICN, Irvine, CA) to
label the amplified products. A Molecular Dynamics Storm Phosphorimager was used to quantitate the signals of the amplified products and the
vector copy number was calculated by comparing the / ratio of the
unknown sample to the / ratio of the standards of known copy
number using standard linear regression analysis.
Spleen colony-forming unit (CFU-S) assay
BM cells (0.5-1.0 × 105) from
mice receiving primary transplantations were transplanted into normal
C57Bl/6J mice pretreated with 900 cGy irradiation. At 13 days
following transplantation, mice were killed and
well-separated, discrete splenic colonies were carefully dissected and
a single cell suspension was prepared. A portion of the cells was used
to prepare genomic DNA for determination of vector copy number, as
described in "DNA analysis" above, while the rest of the
sample was subjected to fixation, permeabilization, and staining with
the TER119-PE antibody, which recognizes erythroid cells, and a
fluorescein isothiocyanate (FITC)-labeled antibody against
human -globin. The cells were analyzed using a FACSCalibur.
Statistical analysis
The probability of a statistically significant difference
between the mean values of 2 data sets was determined by a 2-tailed Student t test using InStat 2.03 software from Apple
Computers (Cupertino, CA).
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Results |
Evaluation of -globin oncoretroviral and lentiviral
vectors
Initially, we sought to develop an oncoretroviral vector utilizing
an erythroid promoter coupled with the HS40 enhancer from the
-globin locus.41 The -spectrin promoter, which
directs high-level expression of the -spectrin cytoskeletal protein
in developing erythroblasts,29,42 and a 130-bp -globin
promoter were chosen for testing. -Globin expression cassettes
utilizing these promoters coupled with the upstream HS40 enhancer were
placed in an MSCV-based oncoretroviral vector. These
constructs were also designed to express the GFP marker under the
transcriptional control of the viral long terminal repeat (LTR) (Figure
1A; MSCV-GFP HS40 spectrin
A and MSCV-GFP HS40globin
A).43,44 Vector particles were derived
for both constructs (4-5 × 105 IU/mL) and used to
transduce MEL cells. A pool of GFP+ cells for each
vector was then isolated for study. Southern blot analysis demonstrated
unrearranged transfer of both vector genomes (Figure 1B). -Globin
expression in each pool was then assessed by FACS analysis following
induction to terminal erythroid differentiation. Despite having a
2-fold lower average vector copy number (Figure 1B), the cell pool
transduced with the MSCV-GFP HS40 globin A vector
consistently demonstrated a much higher proportion of -globin-expressing cells than the MSCV-GFP HS40
spectrin A-transduced cell pool (Figure
1C, top panel). However, the majority of transduced cells failed to
express the globin cassette, suggesting that expression of both globin
vectors suffered, in varying degrees, from significant position
effects. This interpretation was verified by the observation that
individual GFP+ clones from the MSCV-GFP
HS40globin A-transduced cell population
also variably expressed -globin (data not shown). To ascertain
whether a potentially stronger enhancer might alleviate the position
effects we observed with these vectors, the HS40 element was replaced
with DNA fragments from the -globin LCR consisting of HS sites 4 (756 bp), 3 (898 bp), and 2 (374 bp) (MSCV-GFP HS432 globin
A; Figure 1A). However, as we and others have observed in the past, the LCR-containing vector was produced at extremely low titer
(< 102 TU/mL), precluding evaluation of this
design.
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| Figure 1.
Improved -globin vector expression using -globin
LCR elements in a lentiviral vector backbone.
(A) Schematic representations of oncoretroviral- and lentiviral-based
-globin vectors. MSCV-based oncoretroviral vectors express GFP from
the viral LTR and contain the different A-globin
cassettes in reverse orientation. The -spectrin (576) and
-globin promoters (130), along with their respective 5'
untranslated sequences, were fused to the A-globin
coding sequence at position 3 and +1 relative to the endogenous
translational start site. A 255-bp fragment containing the -globin
HS40 enhancer element was placed adjacent to the promoters. The same
-globin promoter, -globin coding sequences, and 3' untranslated
and downstream sequences (466 bp) were also placed downstream of
-globin LCR elements containing sites HS4 (756 bp), HS3 (898 bp),
and HS2 (374 bp) in both oncoretroviral and HIV-based lentiviral vector
backbones. The -globin gene contained a 720-bp deletion in the
second intron in all of the vectors. The lentiviral vector HS432
-A MSCV-GFP also contained an MSCV LTR-driven GFP
cassette. The GFP cassette was removed to yield
HS432-A. d432-A contained a 311-bp
deletion in HS4 outside of the "core" element. The -globin
enhancer ( 3' Enh) or the -globin 3' regulatory element
( 3' RE) was placed downstream of the -globin coding sequences to
derive 2 additional lentiviral vectors. (B) Southern blot analysis,
using a radiolabeled GFP probe, of DNA from MEL cells transduced with
the oncoretroviral vectors MSCV-GFP HS40spectrinA and
MSCV-GFP HS40globinA and the lentiviral vector
HS432-A MSCV-GFP. DNA (10 µg) from each
GFP+ MEL cell pool was digested to release a full- or near
full-length proviral fragment. The respective plasmid DNAs for each
construct were concurrently digested and run as controls for correct
molecular size. (C) FACS analysis for -globin expression (PE
fluorescence) following erythroid induction of MEL cells transduced
with vectors containing the -globin expression cassette driven by
the 5' regulatory elements as indicated above each dot plot. The top
row of dot plots shows representative results from 2 independent
experiments comparing the 2 HS40-based oncoretroviral vectors. The
bottom 2 dot plots are representative results from 3 independent
experiments comparing the oncoretroviral-based HS40/-globin promoter
vector and lentiviral-based HS432/-globin promoter vector. The
percentage of -globin-positive cells relative to all transduced,
GFP+ cells is indicated for each dot plot. MEL cells
transduced with a vector encoding only GFP failed to show staining for
-globin (data not shown).
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Over the course of these studies, we developed an HIV-based lentiviral
vector system that includes a self-inactivating gene transfer vector
backbone and distribution of the packaging functions (Gag/Pol, Rev/Tat,
and envelope) among 3 separate plasmids.16 The
HS432globinA assembly contained in the
above-described oncoretroviral vector was placed into the lentiviral
backbone that also contained an MSCV-driven GFP reporter
(HS432-A MSCV-GFP; Figure 1A). In contrast to the
poor titer observed with the corresponding oncoretroviral vector
MSCV-GFP HS432globinA, VSV-G-pseudotyped
preparations of the -globin lentiviral vector had titers of about
0.5-1.0 × 105 TU/mL. The vector genome was transferred
unrearranged into MEL cells (Figure 1B). We consistently observed a
substantially greater fraction of GFP+ MEL cells expressing
the -globin lentiviral vector compared with cells with a similar
vector copy number transduced with the HS40 -promoter -globin
oncoretroviral vector (Figure 1C, lower panel). Correspondingly,
-globin mRNA levels were higher in the cells transduced with the
lentiviral vector (9% of endogenous murine -globin vs 5%).
Human -globin gene expression in murine erythrocytes following
lentiviral-mediated stem cell gene transfer
To test the -globin lentiviral vector in vivo, a vector lacking
the MSCV-GFP cassette was generated (HS432-A; Figure
1A). The titer of the resulting, single gene -globin vector was 5- to 10-fold higher (0.5 to 1.0 × 106 TU/mL) than the
vector containing the GFP reporter, as assessed by Southern blot
analysis of genome transfer to NIH 3T3 cells (data not shown). However,
Northern blot analysis of RNA from 293T cells producing the single gene
vector indicated the presence of a significant amount of a truncated
viral genomic transcript corresponding to a potential premature
polyadenylation occurring within the HS4 element (data not shown). A 3'
rapid analysis of cDNA ends (RACE) analysis confirmed premature
polyadenylation occurring within the HS4 element (H.H., unpublished
observations, March 2001). This site was deleted from the HS4
element (reducing its size from 756 bp to 445 bp) in the vector plasmid
(yielding d432-A) and resulted in a further
improvement in titer to approximately 3 to 5 × 106
TU/mL. Addition of either the 3' -enhancer ( 3' Enh)
element45 or a 3' regulatory element of the
A-globin gene ( 3' RE),46 both located
downstream from the respective endogenous genes, was made separately to
this vector to potentially improve -globin expression (Figure 1A).
All 3 vectors were of similar titer and were transmitted without
rearrangement to NIH 3T3 target cells (Figure
2 and data not shown).
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| Figure 2.
Unrearranged d432-A 3'RE vector is present in
the genomic DNA from PB leukocytes and spleen cells of animals 32 weeks
following transplantation with vector-transduced BM cells.
Southern blot analysis, using a 32-P labeled RRE probe, of
the indicated genomic DNA digested with BglII. This
enzyme cuts once in the 5' LTR and immediately inside the 3' LTR of the
integrated d432-A 3'RE vector (Figure 1A), thereby
liberating a near unit length proviral form. DNA samples are
as follows: (plasmid) d432-A 3'RE vector plasmid DNA
mixed with genomic NIH3T3 DNA; (NIH3T3) NIH3T3 cells transduced with
the d432-A 3'RE vector; (Neg) naive NIH 3T3 cells; (1 copy) MEL cells harboring a single copy of d432-A
MSCV GFP (BglII digestion liberates a subgenomic
vector fragment); PB, and (Spl) spleen of the indicated mice. Genomic
DNA (15 µg) was used in all cases except for the PB lanes, which
contained 5 µg. The arrow indicates a 7.2-kb hybridizing fragment,
which is of the correct predicted size of an integrated, unrearranged
d432-A 3'RE provirus.
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Normal murine BM cells were transduced with the
d432-A 3'RE vector and transplanted into lethally
irradiated normal C57Bl/6J mice. Flow cytometric analysis 17 weeks
after transplantation demonstrated pancellular expression of -globin
in PB red cells (Figure 3A). The pattern
and level of -globin expression was stable out to 32 weeks after
transplantation (data not shown). Human -globin mRNA expression was
documented by a ribonuclease protection assay (Figure 3B) at levels
ranging from 9% to 19% (mean, 11.8% ± 2.4%) of the level of
total mouse -globin mRNA. This translated to a level of 12% to 25%
(mean, 15.7% ± 3.2%) per vector copy per copy of -globin.
Southern blot analysis of DNA obtained from PB and spleen cells from 2 representative animals demonstrated the presence of a hybridizing band
of the correct molecular size of the unrearranged provirus (Figure 2).
Compared with the band intensity of a cell line containing a single
copy of an integrated lentivirus, the average vector copy number, when adjusted for the amount of DNA loaded, in the PB and spleen of these
mice was approximately 3. These data established the functionality of
this vector in vivo and led us to evaluate its potential for correcting
the phenotype of animals with -thalassemia intermedia.
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| Figure 3.
Human A-globin expression in normal
murine red cells.
(A) FACS analysis for human -globin expression (HbF-PE fluorescence)
in the red cells of normal mice 17 weeks following transplantation with
normal BM cells transduced with the d432-A 3'RE
lentiviral vector. Shown at top is a histogram for -globin
expression (solid lines) in the red cells of a -thalassemic mouse
harboring 2 copies of a -spectrin promoter-driven -globin
transgene (THAL 2). Below are histograms for -globin expression
in the red cells of mice that underwent transplantation (mouse number
indicated at right). The dotted line indicates the staining profile of
red cells from a control mouse that did not undergo transplantation.
(B) RNase protection analysis for the levels of vector-encoded
A-globin and endogenous murine -globin mRNAs.
RNA (0.25 µg) from reticulocytes of the mice indicated above
each lane was hybridized to a 32P-labeled antisense
riboprobe that protects both exon 2 of the -globin gene and exon 2 of the murine -globin gene. The relative / ratio, indicated
below each lane, was obtained by dividing the -globin signal (h)
by the -globin signal (m). Neg indicates negative control mouse;
Tg, sample from the above-described
-thalassemic/-globin transgenic mouse.
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Phenotypic correction of murine -thalassemia intermedia
following lentiviral-mediated transfer of a human -globin
gene
These experiments were initially designed to compare the
expression and therapeutic efficacy of the d432-A
vector and 2 modified versions containing either the regulatory element
downstream from the A-globin gene ( 3' RE) or the
enhancer from downstream of the human -globin gene ( 3' Enh;
Figure 1A). Because -thalassemic mice are somewhat limited in
availability and we wished to obtain a significant number of recipient
mice for study of the different vectors, we transplanted
-thalassemic BM cells transduced with the various vectors into
lethally irradiated, normal C57Bl/6J mice. We have previously shown
that normal recipients of -thalassemic BM acquire the
-thalassemic phenotype following hematologic
reconstitution.38 In addition to these 3 vector cohorts, 2 control cohorts of mice received transplants of either mock-transduced
-thalassemic BM cells or normal BM cells transduced with a
lentiviral vector encoding only GFP.
At 15 weeks following transplantation, PB was obtained from the mice
that underwent transplantation, and the hematologic parameters and red
cell -globin expression were evaluated. Complete hematologic reconstitution with the donor graft occurred in all recipient mice as
verified by the absence on Hb electrophoresis analysis of the
endogenous "single" Hb phenotype (data not shown). In an initial
analysis, the average level of -globin production, as reflected by
the mean fluorescence intensity (MFI) of -globin-positive red cells, did not significantly vary among the 3 groups of animals receiving cells transduced with 1 of the 3 -globin vectors (data not
shown). Accordingly, subsequent data analysis encompassed all of the
mice that received transplants of -globin-transduced -thalassemic cells in order to better evaluate the impact of the
level of -globin gene expression on the degree of correction of the
thalassemic phenotype. All 21 mice that received -globin vector-transduced cells displayed -globin-positive red cells (range, 7%-90%), as judged by FACS analysis. The majority of the animals displayed expression in more than 40% of their red cells with
a concomitant improvement in hematologic parameters (Figure 4A and Table
1). Significant amounts of chimeric
m2h2 hemoglobin molecules (termed
HbF) were observed in the red cell lysates from these
animals that underwent transplantation as assessed by cellulose acetate
electrophoresis (Figure 4B). Densitometric scanning of the cellulose
acetate gels allowed estimation of the percent HbF relative to that of
the endogenous mouse hemoglobin molecules. The PB content of HbF
constituted more than 10% of the total hemoglobin in 14 of 21 animals.
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| Figure 4.
Lentiviral vector-mediated human A-globin
expression in erythrocytes derived from transplanted, -thalassemic
repopulating cells transduced with the d432--A
lentiviral vectors.
(A) FACS analysis for human A-globin expression (HbF-PE
fluorescence) in -thalassemic red cells 15 weeks following
transplantation with -globin vector-transduced -thalassemic BM
cells. At top is the histogram from the -thalassemic/-globin
transgenic control animal. Below are histograms of -globin
expression from representative mice (mouse numbers indicated) that
received transplants of genetically modified cells (solid lines). The
percentage of red cells with unequivocal -globin expression is shown
at the right of each histogram along with the average PBL vector copy
(VC) number. The dotted line indicates the staining profile of red
cells from a mouse that received transplants of mock-transduced
-thalassemic BM cells. (B) Cellulose acetate Hb
electrophoresis gels were used to separate the different Hb species of
red cell lysates as indicated by the arrow to the right. This
-thalassemic mouse strain has the "diffuse" Hb pattern
characterized by an uppermost
m2m species and a faster
migrating m2 species. Chimeric
m2h2 molecules migrate faster than the
endogenous murine Hb species as demonstrated in the
-thalassemic/-globin transgenic mouse lane (Tg). No endogenous
murine "single" Hb molecules, which migrate between the
"diffuse" and chimeric species, were observed, indicating full
donor engraftment. M indicates mouse that received transplants of
mock-transduced -thalassemic BM cells; numbered lanes represent
samples from representative mice that received transplants of
-globin vector-transduced -thalassemic BM cells. % F indicates
the quantity of the chimeric m2h2
species, estimated by densitometry, as a proportion of all Hb species.
% F cells indicates the proportion of red cells staining for human
-globin by FACS analysis. Vector copy number is the average copy
number in PBLs as estimated by DNA PCR. For reference, the uppermost
m2m band makes up approximately
20% of total mouse Hb.
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The degree of correction of the -thalassemic phenotype correlated
closely with the total level of HbF (Figure
5). The hematologic characteristics of
all of the animals, after grouping according to the observed level of
HbF, are shown in Table 1. As we have previously observed, animals that
received transplants of mock-transduced -thalassemic BM cells
accurately reproduced the -thalassemic phenotype (Figures 5B,
6, and Table 1). Animals with HbF levels ranging from 10% to 15% (mean, 12.6%) exhibited a 1 g/dL (10 g/L) improvement in Hb concentration, whereas animals having
total HbF levels of greater than 15% (mean, 20.8%) displayed nearly complete hematologic correction with a 2.5 g/dL (25 g/L) increase in Hb
level. Animals that received transplants of -globin
vector-transduced BM cells and having less than 4% HbF had minimal
improvement in Hb concentration compared with control animals that
received transplants of mock-transduced -thalassemic BM cells. The
degree of correction of red cell indices, reticulocyte count, and red
cell morphologic abnormalities also correlated with the level of HbF
(Table 1 and Figure 6).
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| Figure 5.
The degree of amelioration of the anemia of
-thalassemia correlates with the level of HbF resulting from
lentiviral vector-encoded human -globin.
(A) Hb concentration versus HbF in animals that received transplants of
-thalassemic BM cells transduced with -globin lentiviral vectors.
Animals with less than 4% HbF were excluded from the analysis because
the levels of HbF could not be accurately determined below this level.
Linear regression analysis was used to generate the trend line, which
had a correlation coefficient
(r2) of 0.80. (B) Hb
concentration is shown as a function of the mean level of HbF in the
designated groups of animals that underwent transplantation. Mock
indicates animals transplanted with mock-transduced -thalassemic BM
cells; normal control, animals that received transplants of normal BM
cells transduced with a lentiviral vector, CL10.1
MSCV-GFP, that encodes only GFP. Error bars represent the
standard error of the mean.
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Abstract
Introduction