Fetal gene therapy of ?-thalassemia in a mouse model
Xiao-Dong Han*†, Chin Lin*, Judy Chang*, Michel Sadelain‡, and Y. W. Kan*§
*Cardiovascular Research Institute, Institute of Human Genetics and Department of Medicine, University of California, San Francisco, CA 94143;
and‡Memorial Sloan–Kettering Cancer Center, New York, NY 10021
Contributed by Y. W. Kan, April 9, 2007 (sent for review February 15, 2007)
Fetuses with homozygous ?-thalassemia usually die at the third
potentially be a target for fetal gene therapy. We have previously
established a mouse model of ?-thalassemia. These mice mimic the
human ?-thalassemic conditions and can be used as preclinical
models for fetal gene therapy. We tested a lentiviral vector
element, and the ?-globin gene promoter directing either the EGFP
or the human ?-globin gene. We showed that the GFP expression
was erythroid-specific and detected in BFU-E colonies and the
erythroid progenies of CFU-GEMM. For in utero gene delivery, we
did yolk sac vessel injection at midgestation of mouse embryos.
The recipient mice were analyzed after birth for human ?-globin
gene expression. In the newborn, human ?-globin gene expression
was detected in the liver, spleen, and peripheral blood. The human
?-globin gene expression was at the peak at 3–4 months, when it
reached 20% in some recipients. However, the expression declined
at 7 months. Colony-forming assays in these mice showed low
abundance of the transduced human ?-globin gene in their BFU-E
and CFU-GEMM and the lack of its transcript. Thus, lentiviral
vectors can be an effective vehicle for delivering the human
?-globin gene into erythroid cells in utero, but, in the mouse
model, delivery at late midgestation could not transduce hemato-
poietic stem cells adequately to sustain gene expression.
in utero gene transfer ? lentiviral vector ? yolk sac vessel injection
?-Thalassemia is a hereditary disorder caused by deficient or
absent production of ?-globin. ?-Globin gene mutation fre-
quency is high among many populations, and the severe form has
the highest prevalence in Southeast Asia. Hydrops fetalis asso-
ciated with hemoglobin Bart syndrome is caused by complete
absence of ?-globin and is usually not compatible with postnatal
life. Hemoglobin H disease, caused by a deletion and/or muta-
tion affecting three of the ?-globin genes, results in hemolytic
anemia of variable severity. The prenatal genetic diagnosis for
?-thalassemia has been clinically available for many years (1). A
few patients with homozygous ?-thalassemia have survived by
early and regular blood transfusions (2–6), and hemoglobin H
disease is usually treated symptomatically.
Recombinant lentiviral vectors have been shown to be effec-
tive in transducing nondividing hematopoietic stem cells (7–10).
By using lentiviral vectors carrying the ?-globin transcription
units and ex vivo transduction, therapeutic ?-hemoglobin syn-
thesis has been demonstrated in ?-thalassemic mice (11)) as well
as the antisickling capability of the ?-globin variant in a trans-
genic mouse model of sickle cell disease (12).
Direct in vivo delivery of the therapeutic viral vector has been
shown in animal models to be an effective alternative to the ex vivo
approach (13, 14). Particularly, direct in utero viral vector transfer
has been demonstrated to result in widespread transduction and
long-term correction of transduced genes in animal models of
human genetic diseases such as lysosomal storage disease, Crigler–
Najjar disease, and Duchenne muscular dystrophy (15–17).
In this study, we investigated the efficacy of direct in utero
delivery of a lentiviral-based human a-globin gene to a mouse
model of ?-thalassemia. We demonstrated erythroid specific
he human ?-globin genes are duplicated, and four copies
of ?-globin genes are present in the diploid genome.
expression of the transduced human ?-globin gene and relatively
high levels of expression of the human ?-globin gene in mice
receiving the lentiviral vector by yolk sac vessel injection at
midgestation. However, the expression decreased to low levels
on long-term follow up.
Lentiviral Vector Construction. The lentiviral vector used in this
study was derived from a TNS9 vector (11), which contains an
extended ?-promoter, ?-proximal enhancers, and genomic frag-
This vector has been shown in ex vivo transduction experiments
to be effective in transferring the therapeutic human ?-globin
gene into murine hematopoietic stem cells (11, 18–20). All
elements in the original vector remained except for the replace-
ment of the ?-globin gene with either the human ?-globin gene
or the cDNA-encoding GFP (Fig. 1A). Southern blot analysis of
genomic DNA from infected mouse erythroleukemia (MEL)
cells showed a single band corresponding to the intact proviral
vector (Fig. 1B).
Erythroid-Specific Expression of GFP. To evaluate whether gene
and MEL cells were infected with the viral supernatant of dANS9-
cppt-egfp. GFP expression was detected when these cells were
induced toward erythroid differentiation (Fig. 2A), whereas no
expression was detected in infected 293 cells (data not shown).
When this vector was used to infect primary murine bone marrow
cells, GFP expression was detected in BFU-E colonies or erythroid
lineage of CFU-GEMM colonies, but not in cells of other lineages
derived from the same progenitors (Fig. 2B).
Direct Delivery of the Lentiviral Vectors into Fetuses by Yolk Sac
Vessel Injection. Direct administration of the therapeutic viral
vectors into the fetus can eliminate multiple manipulating steps
associated with ex vivo gene transfer. Therefore, a modified yolk
sac vessel injection protocol was used in this study to deliver the
viral vector systematically into the fetuses (21, 22). By injecting
Trypan blue dye as tracer, we could see blue color appearing
within seconds in the fetal circulation. Fig. 3 shows the site of
injection (Left), followed immediately by the appearance of the
dye in the yolk sac vessels (Center). They can be distinguished
from the uterine wall vessels, which were not stained blue. In
addition, the dye was concentrated in the liver as shown by the
high dye intensity in its location (Right). This is important for
targeting hematopoietic progenitors because the fetal liver is a
major site for hematopoietic stem cell homing and development.
Author contributions: X.-D.H. and Y.W.K. designed research; X.-D.H. and C.L. performed
research; J.C. and M.S. contributed new reagents/analytic tools; X.-D.H., M.S., and Y.W.K.
analyzed data; and X.-D.H. and Y.W.K. wrote the paper.
The authors declare no conflict of interest.
Abbreviations: cppt, central polypurine tract; MEL, mouse erythroleukemia.
†Present address: ViroMed Laboratories, 6101 Blue Circle Drive, Minnetonka, MN 55343.
§To whom correspondence should be addressed at: Institute of Human Genetics, 513
Parnassus Avenue, HSW 901B, University of California, San Francisco, CA 94143-0793.
© 2007 by The National Academy of Sciences of the USA
May 22, 2007 ?
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In the mouse, fetal liver erythropoiesis starts from day 11,
progressing to around day 14, when the erythroblastic islands are
established (23). Although it would be preferable to target the
fetal liver during the early part of this period when it is relatively
richer in hematopoietic stem cells (24), we injected on day 14.5
with earlier injections.
Efficacy of Gene Transfer. To investigate the effectiveness of gene
transfer of the dANS9-cppt-egfp and dANS9-cppt-ha vectors via
yolk sac vessel injection, we analyzed the transgene expression in
newborn recipient mice. GFP expression was detected in spleen
for dANS9-cppt-egfp (Fig. 4A) and human a-globin specific
transcripts in liver for dANS9-cppt-h? (Fig. 4B).
Long-Term Expression of Erythroid-Specific Lentiviral Vectors.Primer
extension analysis was performed to measure the transgene
expression relative to the endogenous mouse ?-globin gene
expression in peripheral blood at different time points after birth
(Fig. 5A). Expression of the transduced human ?-globin gene
was detectable at ?70 days and reached peak levels at 130 days
after birth, indicating the expansion of the targeted erythroid
progenitors. The human ?-globin transcript was expressed at a
level equal to 20% of the mouse ?-globin transcript in one
mouse. However, in all of the mice, expression declined to the
low level of 5% or less in recipient’s peripheral blood 7 months
after birth (Fig. 5B).
Possible Reason for the Decline in Transgene Expression. We har-
vested bone marrows from two of the mice that had shown a
decline in ?-globin gene expression and cultured BFU-E and
CFU-Emix from them. Fifty-five individual colonies were ana-
lyzed for the human ?-globin DNA by PCR and for the presence
of human and mouse ?-globin mRNA by RT-PCR. Twenty-four
of these colonies were also cultured in the presence of
6 of these 55 colonies, and ?-globin transcripts were found in
none, with or without 5-azacytidine. In contrast, mouse ?-globin
mRNA was detected in 49 of these colonies, showing that the
RNA prepared from them were of good quality. Hence, the
decline in gene expression could be due to the loss of cells that
contained the human ?-globin transgene and to the silencing or
level too low to be detected in the few that retained the
In this study, we investigated the approach of intrauterine
therapy for ?-thalassemia in an ?-globin gene knockout mouse
model that we have constructed (25). Homozygous ?-thalasse-
mia appears to be a good candidate for intrauterine gene therapy
because prenatal diagnosis can be made by DNA analysis early
in pregnancy, and the homozygously affected fetuses often
survive up to the third trimester of pregnancy or to birth.
Although several newborns with this disease survived when they
respectively and GFP coding region by the hatched box. The splice donor (SD) and acceptor (SA), the packaging sequence (?), and rev-response element (RRE),
indicates the 3? LTR deletion and cppt central polypurine tract. (B) Southern blot analysis of transduced MEL cells. Genomic DNA of MEL cells infected with
dANS9-cppt-h? vector was digested with restriction enzyme SacI and hybridized with the human ?-globin probe. The upper band represents the expected size
for the proviral vector, and the lower band was due to cross hybridization with the endogenous mouse ?-globin gene. The ?/HindIII DNA molecular markers are
indicated in kilobases.
Structures and integration of the lentiviral vectors. (A) Exons and introns of the human ?-globin gene are represented by filled and open boxes
K562 and MEL were infected with the lentiviral vector dANS9-cppt-egfp
cells. (B) Mouse bone marrow cells were infected with the lentiviral vector
dANS9-cppt-egfp and cultured in complete methylcellulose medium supple-
was detected under fluorescent microscope. GFP-positive cells were observed
in CFU-Emix (Left) and BFU-E (Right). Images with UV light (Upper) and visible
and UV light (Lower) are shown. The arrows indicate the nonerythroid colony
that was negative for GFP.
www.pnas.org?cgi?doi?10.1073?pnas.0702457104 Han et al.
received transfusion prenatally or immediately after birth, the
anemia is invariably severe, and they invariable require regular
transfusion beginning from birth. Thus, the disease is more
severe than ?-thalassemia, where the clinical manifestation is
much more variable, with some patients having the less severe
intermediate form. Even those who require transfusion may not
need it until a few months or a few years of age. Successful
intrauterine gene therapy in ?-thalassemia would therefore
obviate subsequent transfusion.
Using a dye as a marker, we showed that injection into the yolk
sac vein resulted in concentration of the dye in the liver. Van der
Wegen et al. (26) reported the successful treatment of UDP-
glucuronosyltransferase deficiency in a rat model of Crigler–
the liver is a distinct advantage for treating globin disorders
because the fetal liver is a hematopoietic organ. However, some
vectors will also be expected to reach the systemic circulation.
Hence, the use of erythroid-specific promoters and enhancers is
desirable. Lentiviral vectors were chosen to deliver the GFP and
the ?-globin genes because they could transduce hematopoietic
cells efficiently and were successful in treating mouse models of
?-thalassemia and sickle cell anemia by ex vivo transduction of
hematopoietic cells. Indeed, we also found that our vector
specifically expressed GFP in erythroid cell lines and in ery-
throid colonies cultured from mouse bone marrow cells.
The ?-globin gene we used was controlled by the ?-LCR
because such a construct has been shown to express the ?-globin
efficiently in transgenic mice (27). Control by the ?-globin
elements would also be expected to maintain gene expression
into postnatal life. Intrauterine injection resulted in expression
of the ?-globin gene at birth and reached a peak of up to 20%
uwv, uterine wall vessel. (Right) Concentration of the dye in fetal liver after injection.
In utero delivery of lentiviral vector by yolk vessel injection. (Left) Needle insertion site. (Center) Dye in yolk sac vessel after injection. ysv, yolk sac vessel;
injection. (A) GFP expression in frozen section of the spleen after injection of
the 350-bp human ?-globin RT-PCR product with Pml 1 yields 180- and 170-bp
bands and with HindIII 230/120-bp bands. The left side shows injected new-
born mouse liver, and the right side shows human fetal liver control. U,
undigested; H, HindIII; P, Pml I.
Gene expression in the newborn mice after in utero lentiviral
a), 90 days (lane b), 130 days (lane c), and 210 days (lane d) after birth. c1 and
c2 are positive (human fetal liver) and negative (uninjected mouse) controls
human and mouse ?-globin mRNA determined by PhosphorImager.
Han et al.
May 22, 2007 ?
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no. 21 ?
in some mice at ?3–4 months. However, the level of expression
declined to ?5% at approximately the seventh month. The
decline could be due to several factors. Very few erythroid
colonies cultured from the bone marrow of the mice after their
?-globin expression has declined contained the ?-globin trans-
gene. This suggests that the decline may be due to the loss of the
transgene as a result of the inadequate transduction of hema-
topoietic stem cells, and most of the transduced cells were later
progenitors. Because of technical difficulty and the high mor-
tality rate, delivery of the lentiviral vectors close to day 14.5 of
gestation and not to day 11 may account for the transduction of
the later progenitor cells. Unlike effective fetal treatment of
metabolic diseases, where the liver cell could be targeted during
most of the duration of pregnancy, treatment of hematopoietic
disorders may have a much narrower window for effective
hematopoietic stem cell targeting. Our inability to detect human
?-globin mRNA in the few human ?-globin gene-containing
colonies may be due to the low level of the transcripts or to gene
silencing that cannot be reactivated by demethylating agents.
In summary, we demonstrated the erythroid-specific expres-
sion of the human ?-globin gene after the lentiviral-based vector
delivery at midgestation by yolk sac vessel injection. However,
the expression declined at 7 months. The decline may be due to
the inability to transduce early hematopoietic stem cells by
delivery at 14.5 days of gestation or to gene silencing. Larger-
animal models in which the fetal circulation can be accessed
earlier may well be used to test the efficacy of in vivo lentiviral
transduction of hematopoietic stem cells.
Materials and Methods
Construction of the Lentiviral Vectors. The human ?-globin gene
fragment spanning NcoI and PstI sites was used to replace the
?-globin gene in the lentiviral vector TNS9 (11). The central
polypurine tract (cppt) fragment was amplified from the pol region
HapI sites downstream of the 3? ?-enhancer. The primers used for
the cppt fragment amplification were as follow: cppt-5-HpaI,
TCGCGTTAACTTTTAAAAGAAAAGGGGGG and cppt-3-
TTTG. The orientation of cppt was checked by sequencing. Sub-
sequently, the human ?-globin gene in the resulting vectors,
dANS9-cppt-h?, was replaced with the EGFP gene fragment from
Production and Purification of Lentiviral Vector.Thevector,dANS9-
cppt-h? or dANS9-cppt-egfp, was cotransfected with pCMV
?R8.9 (14) and pMD.G (28) into human embryonic kidney cells
293FT as described (8). Briefly, 6 ? 108of 293FT cells in 750 ml
of DMEM containing 10% FBS (D10F) were plated into a
polylysine-precoated cell factory unit (Nalge Nunc Interna-
tional, Rochester, NY) and transfected the next day with 580 ?g
of dANS9-cppt-h? or dANS9-cppt-egfp, 430 ?g of pCMV?R8.9,
and 145 ?g of pMD.G in a 5% CO2incubator at 37°C for 24 h.
After the transfection medium was removed, the cells were
washed once with DMEM, and the transfected cells were
incubated for another 24 h in fresh D10F supplemented with 10
mM sodium butyrate (Sigma, St. Louis, MO) and 20 mM Hepes.
ml of DMEM containing 5% FBS and 20 mM Hepes for virus
production. The viral supernatants were collected on three
consecutive days, spun at 3,500 rpm [RC-3B; Sorvall (Newtown,
CT) at 4°C for 10 min and filtered through a 0.45-?m low-
protein-binding filter (Millipore, Bedford, MA)]. The viral su-
pernatant was concentrated by two rounds of ultracentrifugation
at 25,000 rpm, 15°C for 100 min in a SW32Ti rotor in Optima
L-90K Ultracentrifuge (Beckman, Fullerton, CA). The final viral
pellet was resuspended in 1 ml of saline containing 4 ?g/ml
Polybrene and stored at ?80°C until use.
Virus titer was determined by a p24 antigen ELISA, following
the manufacturer’s instructions (ZeptoMetrix, Buffalo, NY).
The number of erythroid-specific infectious viral particles was
determined by infecting MEL cells with dANS9-cppt-egfp len-
tiviral vector, followed by induction for 4–5 days with 5 mM
N,N?-hexamethylene bisacetamid (HMBA; Sigma).
Cell Lines and CFU Assay. MEL and K562 cells were carried in
RPMI medium 1640 containing 10% of FCS. For infection, cells
were incubated with the viral solution containing 10 ?g/ml of
Polybrene for 4–6 h, followed by induction for 5 days with
HMBA for MEL cells and hemin for K562 cells, respectively.
Bone marrow cells were prepared from the ?-globin gene
knockout mice and infected overnight with the viral solution in
the presence of 10 ?g/ml Polybrene and plated into complete
methylcellulose medium containing IL-3, IL-6, SCF, and eryth-
ropoietin (M3434; StemCell Technologies, Vancouver, BC, Can-
ada). The colonies were scored after 10 days of incubation.
CFUs were similarly prepared from the bone marrow of two
of the injected mice that had a decline of ?-globin expression
of 5 ?M 5-azacytidine (Sigma). After 12 days, cells from an
individual BFU-E or CFU-Emix colony were transferred to a
2-ml tube containing ULTRASPEC RNA solution, and total
RNA and DNA were isolated according to the manufacturer’s
instruction and DNA and RNA determined as described.
Southern Blot Analysis. Genomic DNA was isolated from infected
MEL cells by proteinase K digestion and sodium chloride precip-
itation. Ten micrograms of DNA were digested with ScaI, electro-
phoresed on a 1% agarose gel, transferred to a Hybond-n ?
membrane (Amersham Biosciences, Piscataway, NJ), and hybrid-
sodium phosphate buffer and 6% SDS. After washing at 65°C, the
hybridized products were detected by chemiluminescence by using
an antidigoxigenin alkaline phosphatase conjugate and the CSPD
substrate (Roche, Indianapolis, IN).
Animal Model and Yolk Sac Vessel Injection. The ?-globin knockout
mice were produced as described (25). The heterozygous (??/
??) females were bred with the homozygous (??/??) males.
Pregnant mice at day 14.5 of gestation were anesthetized by i.p.
injection of 0.1 ml of 2.4% tribromoethanol (Sigma–Aldrich) per
10 g of body weight. A midline laparotomy (1–1.5 cm) was
performed to expose horns of the gravid uteri. The yolk sac
vessels of individual embryos were visualized under a dissecting
microscope. The injection was done by inserting a glass needle
volume was controlled through a PB600–1 repeating dispenser
(Hamilton, Reno, NV). Ten to 15 ?l of lentiviral vector equiv-
alent to 5 ? 105to 1 ? 106infectious viral particles was injected
into each embryo. After injection, the uteri were returned to the
abdominal cavity and the abdomen was closed with 4–0 silk
suture. The mice were allowed to recover in a warm cage. All
animal experiments were carried out according to the institu-
tional guidelines for animal use.
Table 1. Number of CFUs assayed for human ?-globin transgene
and mRNA and mouse mRNA
h DNAh RNAm RNA
www.pnas.org?cgi?doi?10.1073?pnas.0702457104Han et al.
RT-PCR Analysis. Peripheral blood samples or tissues were lysed in
ULTRASPEC RNA solution (Bioteck Laboratories, Houston,
TX), and total RNA was isolated, following the manufacturer’s
instructions. For RT-PCR, cDNA was synthesized from 1 ?g of
total RNA in 10 ?l of RT buffer containing 5 mM dNTP, 10 ?M
oligo d(T), 10 units of RNase inhibitor, and 2 units of M-MLV
reverse transcriptase at 37°C for 50 min, followed by 70°C for 15
min. PCR was carried out by using the following primers:
H?1266-: AAGCCAGGAACTTGTCCAGG. The reactions
were first denatured at 94°C for 5 min, followed by 40 cycles of
94°C for 30 seconds, 62°C for 30 seconds, and 72°C for 30
seconds. The final extension was carried out at 72°C for 5 min.
The PCR products were digested with restriction enzyme Pml I
or HindIII and analyzed by electrophoresis on 2% agarose gel.
Primer Extension Analysis. A primer extension assay was done by
using the AMV Reverse Transcription Primer Extension Sys-
tem (Promega, Madison, WI) with ?-[32P]ATP-labeled prim-
ers as the following: m?-98 5?-AGCAGCCTTCTCAGCAT-
CAG, resulting in a 98-nt band for mouse ?-globin; m?-53
5?-TGATGTCTGTTTCTGGGGTTGTG; h?-82 5?-CGTTG-
GTCTTGTCGGCAGGAAAC. The labeled primers were an-
nealed to 1 ?g of total RNA, and the reaction was carried
our according the manufacturer’s instruction. The intensity of
radioactive bands was determined with PhosphorImager
We thank Hao He (Memorial Sloan–Kettering Cancer Center) for the
TNS9 human ?-globin vector. This work was partially supported by
National Institutes of Health Grants DK016666 and HL053762.
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