GENE THERAPY

Correction of sickle cell disease by homologous recombination

in embryonic stem cells
Li-Chen Wu, Chiao-Wang Sun, Thomas M. Ryan, Kevin M. Pawlik, Jinxiang Ren, and Tim M. Townes

Previous studies have demonstrated that
sickle cell disease (SCD) can be cor-
rected in mouse models by transduction
of hematopoietic stem cells with lentiviral
vectors containing antisickling globin
genes followed by transplantation of these
cells into syngeneic recipients. Although
self-inactivating (SIN) lentiviral vectors
with or without insulator elements should
provide a safe and effective treatment in
humans, some concerns about inser-
tional mutagenesis persist. An ideal cor-
rection would involve replacement of the
sickle globin gene (␤S) with a normal
copy of the gene (␤A). We recently derived
embryonic stem (ES) cells from a novel
knock-in mouse model of SCD and tested
a protocol for correcting the sickle muta-
tion by homologous recombination. In
this paper, we demonstrate the replace-
ment of the human ␤S-globin gene with a
human ␤A-globin gene and the derivation
of mice from these cells. The animals
produce high levels of normal human
hemoglobin (HbA) and the pathology as-
sociated with SCD is corrected. Hemato-
logic values are restored to normal levels
and organ pathology is ameliorated.
These experiments provide a foundation
for similar studies in human ES cells
derived from sickle cell patients. Al-
though efficient methods for production
of human ES cells by somatic nuclear
transfer must be developed, the data in
this paper demonstrate that sickle cell
disease can be corrected without the risk
of insertional mutagenesis. (Blood. 2006;
108:1183-1188)
© 2006 by The American Society of Hematology

Introduction
Sickle cell disease (SCD) is an autosomal recessive disorder that
affects a significant proportion (approximately 1 in 500 individu-
als) of the African-American population. Hispanic, Arabic, Mediter-
ranean, and some Asian populations are also affected. More than
300 000 individuals worldwide and more than 70 000 in the United
States suffer from the disease. The molecular basis for sickle cell
disease is an A to T transversion in the sixth codon of the human
␤-globin gene.1,2 This simple transversion changes a polar glutamic
acid residue to a nonpolar valine in the ␤s-globin chain on the
surface of HbS (␣2␤S2) tetramers. The valine creates a hydrophobic
projection that fits into a natural hydrophobic pocket formed on Hb
tetramers after deoxygenation.3,4 The interaction of tetramers
results in the formation of HbS polymers/fibers that cause red blood
cells (RBCs) to become rigid and nondeformable and to occlude
small capillaries.5-10 These vasoocclusive events cause severe
tissue damage that can result in strokes, splenic infarction, kidney
failure, liver and lung disorders, painful crises, and other complica-
tions. Cycles of erythrocyte sickling also cause the cells to become
fragile, and lysis produces chronic anemia.
Sickle cell disease is normally a relatively benign disorder in the
first few months of life because human fetal hemoglobin (HbF) has
potent antisickling properties. HbF, which comprises 70% to 90%
of total hemoglobin at birth, is gradually replaced by HbS during
the first few months of life. Rising HbS levels result in the onset of
disease between 3 and 6 months of age. We recently produced a
knock-out/transgenic mouse model that mimics this switch from
HbF to HbS. The LCR ␥-␤S transgene in these animals was
designed to switch hemoglobins after birth11-13 rather than before
birth14-19 as observed in animals produced with cosmid, BAC, or
YAC transgenes. The LCR ␥-␤S transgenic animals are relatively
healthy at birth and then develop severe anemia when the switch to
HbS is completed at approximately 3 weeks of age. More recently,
we used the same ␥-␤S configuration to produce a knock-in mouse
model of sickle cell disease (T.M.R. and T.M.T., unpublished data,
November 2003). These animals complete the switch of human
hemoglobins (HbF to HbS) after birth and develop the same severe
anemia as the knock-out/transgenic mice during the first week of
life. We derived embryonic stem (ES) cells from these sickle
knock-in mice for the present studies.
Two groups have corrected SCD in mouse models by transduc-
tion of hematopoietic stem cells with lentiviral vectors containing
antisickling globin genes followed by transplantation of these cells
into syngeneic recipients.20,21 Although self-inactivating (SIN)
lentiviral vectors with or without insulator elements should provide
a safe and effective treatment for hemoglobinopathies,22 some
concerns about insertional mutagenesis persist.23 If viral integra-
tion inhibits a tumor-suppressor gene or activates an oncogene,
leukemia can result. Recent studies of children who received a
transplant of retrovirally transduced hematopoietic cells in France
reveal that this outcome can occur.24 An approach that bypasses this
problem is replacement of the sickle globin gene (␤S) with a normal
copy of the gene (␤A). In this paper, we demonstrate the replace-
ment of the human ␤S-globin gene with a human ␤A-globin gene by
homologous recombination, and the derivation of mice from these

From the Department of Biochemistry and Molecular Genetics, University of
Alabama at Birmingham, Schools of Medicine and Dentistry.
Submitted February 21, 2006; accepted April 4, 2006. Prepublished online as
Blood First Edition Paper, April 25, 2006; DOI 10.1182/blood-2006-02-004812.
Supported by grants from the National Heart, Lung, and Blood Institute (T.M.T.
and T.M.R.).
Reprints: Tim M. Townes, Department of Biochemistry and Molecular
Genetics, University of Alabama at Birmingham, Schools of Medicine and
Dentistry, Birmingham, AL 35294; e-mail: ttownes@uab.edu.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.

L.-C.W., C.-W.S., and T.M.R. contributed equally to this work. © 2006 by The American Society of Hematology

BLOOD, 15 AUGUST 2006 䡠 VOLUME 108, NUMBER 4 1183

1184 WU et al
cells. The results demonstrate successful correction of the hemo-
lytic anemia and organ pathology that characterize sickle cell
disease in humans.
Materials and methods
Production of the ⴚ383 ␥-␤A-globin gene construct
A plasmid containing the human A␥ globin gene with 383 bp of 5⬘ flanking
sequence (4.6 kb total) and a human ␤A globin gene with 815 bp of 5⬘
flanking sequence (4.1 kb total) was digested with SalI to produce an 8.7-kb
⫺383 ␥-␤A DNA fragment. The SalI sites were changed to ClaI by adding
adaptors and the 8.7-kb ClaI fragment was subcloned into pBlueScript. A
herpes simplex virus (HSV) thymidine kinase (TK) gene, which is driven
by a phosphoglycerate kinase (PGK) promoter, was digested with XbaI and
a 2.0-kb XbaI fragment was isolated. This fragment was subcloned into the
plasmid pTR18 that contains XhoI and SalI sites on either side of XbaI. This
plasmid was digested with XhoI and SalI to generate a 2.0-kb XhoI/SalI
fragment. Another plasmid, containing 1.7 kb of mouse 5⬘ homology, a
floxed PGK/Hygromycin (Hygro) gene, and 7 kb of mouse 3⬘ homology,
was linearized with SalI, and the 13.3-kb DNA fragment was isolated from
an agarose gel. The 2.0-kb XhoI/SalI fragment containing PGK/TK was
inserted into the SalI site of pTR401. The resulting plasmid was then
digested with ClaI, which cuts between the mouse 5⬘ homology and the
floxed PGK/Hygro gene, and the 8.7-kb ␥-␤A ClaI fragment was inserted.
This final plasmid containing TK/mouse 5⬘ homology/⫺383 ␥-␤A/floxed
PGK/Hygro/mouse 3⬘ homology was digested with multiple enzymes to
verify that the 24-kb plasmid was correct.
ES cell culture and homologous recombination to repair
DNA lesion
The production of a novel, knock-in mouse model of sickle cell disease and
the derivation of ES cells from these mice will be described elsewhere
(T.M.R. and T.M.T., unpublished data, November 2003). The ⫺383 ␥-␤A
DNA construct was linearized by NotI digestion and electroporated into the
knock-in sickle ES cells (h␣/h␣, ⫺1400 ␥-␤S/⫺1400 ␥-␤S). The cells were
plated onto mitomycin C–treated mouse embryonic fibroblasts (MEFs) in
Dulbecco modification of Eagle medium (DMEM) containing 16.7% fetal
bovine serum (FBS; HyClone, Logan, UT), 1⫻ nucleosides, 2 mM
L-glutamine, 1⫻ nonessential amino acids, 50 IU/mL penicillin, 50 ␮g/mL
streptomycin, 0.1 mM ␤-mercaptoethanol, and 1000 U/mL leukemia
inhibitory factor (LIF). Positive/negative selection in hygromycin (125 ␮g/mL)
and gancyclovir (2 ␮M) was used to enrich for homologous recombinants.25
DNA isolated from individual ES cell colonies was analyzed by polymerase
chain reaction (PCR). Homologous recombinants were identified with primer 1
and primer 2 to identify correct 5⬘ sequences (primer 1 is outside of the vector
homology region) and with primers 5 and 6 to identify correct 3⬘ sequences
(primer 6 is outside of the vector homology region). PCR with primers 3 and 4
followed by Bsu36I digestion was used to distinguish ␤S and ␤A alleles. Primer
sequences are as follows: primer 1, 5⬘-CTCCTGACTCGGTATCCTGC-3⬘;
primer 2, 5⬘-GAAGTTCTCAGGATCCACATGC-3⬘; primer 3, 5⬘-GATATATCT-
TAGAGGAGGGC-3⬘; primer 4, 5⬘-CCAACTTCATCCACGTTCAC-3⬘; primer
5, 5⬘-CAGAGCTTGGTTGACGGCAATTTCG-3⬘; and primer 6, 5⬘-TGA-
GCTCCGGAGGTACCCAGG-3⬘.
Positive colonies were electroporated with a cytomegalovirus (CMV)/
Cre plasmid, and individual colonies containing a correctly deleted marker
gene were identified by PCR.
Blastocyst injection and globin protein analysis
Correctly targeted ES cell lines were injected into C57BL/6 blastocysts.
Chimeric males obtained from these injected blastocysts were mated with
h␣/h␣, ⫺1400 ␥-␤S/m␤ females, and offspring were screened for the
corrected genotypes (⫺383 ␥-␤A/⫺1400 ␥-␤S and ⫺1400 ␥-␤A/⫺1400
␥-␤S; “Results”) by PCR of tail DNA and Bsu36I digestion.
Fifty microliters of whole blood was washed in 500 ␮L PBS. Cell
pellets were resuspended in 100 ␮L lysis solution (5 mM sodium
BLOOD, 15 AUGUST 2006 䡠 VOLUME 108, NUMBER 4
phosphate, 0.5 mM EDTA, pH 7.4) and incubated on ice for 15 minutes.
One tenth volume of 10% NaCl was added and the sample was centrifuged
at maximum speed in an Eppendorf microfuge (Hamburg, Germany).
Approximately 1 ␮L of the supernatant was analyzed on an isoelectric
focusing (IEF) gel. IEF was performed using the Isothermal Controlled
Electrophoresis system (Fisher Scientific, Pittsburgh, PA) with precast
agarose IEF gels (RESOLVE from PerkinElmer, Helsinki, Finland).
Hemoglobin bands were quantitated by densitometry with a BioRad
(Hercules, CA) GS-800 scanner using Quantity One software (BioRad).
Hematologic indices and histopathology
Blood was collected from anesthetized animals into Microtainer EDTA
collection tubes (Becton Dickinson, Franklin Lakes, NJ). RBC count was
measured on a HemaVet 1700 (CDC Technology, Oxford, CT) hematology
analyzer. Hemoglobin concentration was determined spectrophotometri-
cally after conversion to cyanmethemoglobin with Drabkin reagent (Sigma,
St Louis, MO). Before determining the hemoglobin concentration, red cell
membranes were pelleted at 16 000g for 5 minutes in an Eppendorf
microfuge. Packed cell volume (PCV) was measured with a JorVet J503
(Jorgenson Laboratories Systems, Loveland, CO) microhematocrit centri-
fuge. Reticulocyte counts were determined by flow cytometry after staining
with thiazole orange. Urine osmolality was measured with the Wescor
Vapro Vapor Pressure Osmometer 5520 (Logan, UT) after food and water
were withheld from the mice for 16 hours. Tissue preparations from
the spleen, liver, and kidney were fixed in 70% alcoholic formalin,
embedded in paraffin, sectioned, and stained with hematoxylin-eosin stain
by standard methods.
Results
Production of a human ␤A-globin gene targeting construct
We recently produced a knock-in mouse model of sickle cell
disease by replacing the mouse ␣-globin genes with a human
␣-globin gene (h␣/h␣) and by replacing the mouse ␤-globin genes
with human A␥- and ␤S-globin genes (⫺1400 ␥-␤S/⫺1400 ␥-␤S;
T.M.R. and T.M.T., unpublished data, November 2003). We also
derived ES cells from these knock-in sickle mice. The ES cells are
homozygous for the human ␣-globin gene (not shown) and
homozygous for the A␥- and ␤S-globin genes as indicated in Figure
1A. The A␥-globin gene in the knock-in sickle ES cells contains
1400 bp of 5⬘ flanking sequence; therefore, the genotype is
designated as ⫺1400 ␥-␤S. The targeting construct that we
produced for gene replacement contained a human ⫺383 ␥-␤A-
globin gene fragment that is similar to ⫺1400 ␥-␤S except that the
␥ gene has only 383 bp of 5⬘ flanking sequence. This-24 kb
replacement vector contained 1.7 kb of mouse 5⬘ flanking se-
quence, the ⫺383 ␥-␤A fragment (8.7 kb), a floxed PGK/Hygro
gene, 7 kb of mouse 3⬘ flanking sequence, and a PGK/TK gene
(Figure 1B).
Replacement of the human ␤S-globin gene with the human
␤A-globin gene in ES cells derived from knock-in sickle mice
The targeting construct was electroporated into knock-in sickle ES
cells, and the cells were grown in hygromycin and gancyclovir for
2 weeks. One hundred thirteen colonies were picked and homolo-
gous recombinants were identified by PCR of genomic DNA. In 16
(14.2%) of the 113 colonies, the ␤S-globin gene was successfully
replaced with the ␤A-globin gene. In 1 of the 16 colonies,
recombination at the 5⬘ end occurred upstream of the ⫺383 ␥
sequence; therefore, the replacement allele contained the
BLOOD, 15 AUGUST 2006 䡠 VOLUME 108, NUMBER 4 SICKLE GENE REPLACEMENT 1185

Figure 1. Replacement of the ␤S-globin gene with a ␤A-globin gene in knock-in sickle ES cells. (A) Schematic representation of the human ␤-globin locus, mouse
␤-globin locus, and the human ⫺1400 A␥-␤S knock-in locus in ES cells derived for this study. Arrows indicate DNase I hypersensitive sites (HSs) that mark the locus control
region (LCR). Red and yellow boxes represent functional human and mouse genes, respectively. White boxes represent pseudogenes. Black circles indicate loxP sites. (B)
Schematic representation of gene replacement in knock-in sickle ES cells. The 24-kb replacement vector contains a 2.1-kb PGK/TK marker gene, 1.7 kb of mouse 5⬘ flanking
sequence, a ⫺383 ␥-␤A fragment (8.7 kb), a 1.8-kb floxed PGK/Hygro gene, and 7 kb of mouse 3⬘ flanking sequence. Homologous recombinants were identified by PCR with
primers 1 and 2 to identify correct 5⬘ sequences (primer 1 is outside of the vector homology region) and with primers 5 and 6 to identify correct 3⬘ sequences (primer 6 is outside
of the vector homology region). PCR with primers 3 and 4 followed by Bsu36I digestion was used to distinguish ␤S and ␤A alleles. (C) 5⬘ PCR (primers 1 and 2) and 3⬘ PCR
(primers 5 and 6) from 2 positive homologous recombinant ES cell lines (clones 1 and 2). In clone 1, recombination at the 5⬘ end occurred downstream of the ⫺383 ␥ sequence;
therefore, the replacement allele maintained the ⫺1400 ␥ promoter (2.8-kb PCR product). In clone 2, recombination at the 5⬘ end occurred upstream of the ⫺383 ␥ sequence;
therefore, the replacement allele contained the ⫺383 A␥ promoter (1.8-kb PCR product). Bsu36I digestion of PCR fragments derived with primers 3 and 4 is presented in the
second panel of panel C. ␤A fragments are digested, but ␤S fragments are resistant to digestion.

⫺383 A␥-globin gene promoter (Figure 1B-C, 5⬘ PCR, lane 2; 1-3 and 1-4 are ⫺1400 ␥-␤A/⫺1400 ␥-␤S and animals 2-3 and 2-4
1.8-kb PCR product). In the remaining 15 colonies, recombination are ⫺383 ␥-␤A/⫺1400 ␥-␤S. The 1-1, 1-2, 2-1, and 2-2 animals are
at the 5⬘ end occurred downstream of the ⫺383 ␥ sequence; homozygous sickle mice derived from the same matings. The last 2
therefore, the replacement allele maintained the ⫺1400 ␥ promoter lanes are controls obtained from a human sickle patient (␤S/␤S) and
(Figure 1B-C, 5⬘ PCR, lane 1; 2.8-kb PCR product). Genomic an unaffected individual (␤A/␤A).
DNA from all 16 colonies was analyzed by PCR with primers 3 and Blood samples were obtained from the animals, and hemoly-
4, which amplify both ␤S and ␤A alleles producing 340-bp sates were analyzed on IEF gels. The results are illustrated in
amplicons. These amplicons were digested with Bsu36I, which Figure 2B. The last 3 lanes are control hemolysates from a sickle
does not cut the ␤S allele but digests the ␤A allele into 260- and
80-bp fragments (Figure 1B-C). Homologous recombination at the
3⬘ end of all 16 colonies was correct (Figure 1B-C, 3⬘ PCR; 7.7-kb
PCR product). Cell lines containing the ⫺1400 ␥-␤A replacement
allele or the ⫺383 ␥-␤A replacement allele were transiently
transfected with a CMV/Cre plasmid, and colonies containing a
deletion of the PGK/Hygro marker gene were identified by PCR.
Mice derived from corrected sickle ES cells express high levels
of human HbA

After removal of the marker, ⫺1400 ␥-␤A and ⫺383 ␥-␤A ES cell
lines were injected into C57BL/6 blastocysts. Chimeric males
obtained from these blastocysts were mated with females that were
homozygous for human ␣-globin gene knock-in and heterozygous
for human ⫺1400 ␥-␤S knock-in (h␣/h␣, ⫺1400 ␥-␤S/m␤), and
offspring were screened for the corrected genotypes (⫺383 ␥-␤A/
⫺1400 ␥-␤S and ⫺1400 ␥-␤A/⫺1400 ␥-␤S) by PCR of tail DNA
and Bsu36I digestion. Figure 2A demonstrates that animals with
corrected genotypes were obtained from both cell lines. Animals
Figure 2. Genomic DNA and hemoglobin analysis of mice derived from targeted
ES cell lines 1 and 2. (A) After removal of the PGK/Hygro marker from corrected ES
cell clone 1 (⫺1400 ␥-␤A/⫺1400 ␥-␤S) and clone 2 (⫺383 ␥-␤A/⫺1400 ␥-␤S), cells
were injected into C57BL/6 blastocysts. Chimeric males obtained from these
blastocysts were mated with h␣/h␣, ⫺1400 ␥-␤S/m␤ females and offspring were
screened for the corrected genotypes (⫺1400 ␥-␤A/⫺1400 ␥-␤S and ⫺383 ␥-␤A/
⫺1400 ␥-␤S) by PCR of tail DNA and Bsu36I digestion. (B) IEF gel of hemolysates
from mice identified as sickle and corrected animals in panel A. The last 3 lanes are
human control hemolysates from a sickle patient, an individual with sickle trait, and an
unaffected individual. Of interest, the ratio of HbA to HbS in corrected animals mimics
the ratio in humans with sickle trait.
1186 WU et al
patient, an individual with sickle trait, and an unaffected individual.
The results in Figure 2B demonstrate that high levels of human
HbA are synthesized in red blood cells from 1-3, 1-4, 2-3, and 2-4
animals. Of interest, all 4 of these animals mimic the HbA to HbS
ratio in humans with sickle trait. Individuals with sickle trait
present little if any pathology; therefore, these results suggest that
the level of HbA achieved by gene replacement is sufficient to
correct the disease.
Correction of abnormal RBC morphology and hematologic
parameters in mice derived from targeted ES cells
Blood smears from knock-in sickle (␤S/␤S), corrected (␤A/␤S), and
control (␤A/␤A) animals are illustrated in Figure 3. Many rigid,
elongated cells are observed in the sickle mice (Figure 3B);
however, no sickled cells are observed in ␤A/␤S-corrected mice or
in ␤A/␤A controls (compare 3A and 3C). The blood smears of
corrected animals also lack the anisocytosis, poikilocytosis, and
polychromasia characteristics of erythrocytes in the sickle mice.
The red cells in corrected mice are nearly identical to the cells in
control animals, and this phenotype is similar to human red cells
from individuals with sickle trait.
We also measured hematologic values in the corrected mice and
compared these values to the numbers obtained for sickle and
control animals. These data are presented in Table 1. Compared
with sickle animals, the corrected mice have marked increases in
RBC counts (6.7 ⫾ 1.1 to 11.3 ⫾ 1.0 ⫻ 1012/L [6.7 ⫾ 1.1 to
11.3 ⫾ 1.0 ⫻ 106/␮L]), Hb levels (71 ⫾ 9 to 108 ⫾ 10 g/L
[7.1 ⫾ 0.9 to 10.8 ⫾ 1.0 g/dL]), and hematocrit levels (.351 ⫾ .033
to .417 ⫾ .025 as a proportion of 1 [35.1% ⫾ 3.3% to
41.7% ⫾ 2.5%]) and a significant reduction in reticulocyte counts
(.754 ⫾ .039 to .084 ⫾ .026 as a proportion of RBCs
[75.4% ⫾ 3.9% to 8.4% ⫾ 2.6%]). All of the values in the
corrected mice (␤A/␤S) are similar to values in controls (␤A/␤A).
These data demonstrate that the hematologic defects in sickle cell
disease can be corrected by replacing one ␤S allele with a ␤A allele
in ES cells.
Table 1. Correction of hematologic pathology and urine concentration defect in mice by homologous recombination
RBC,
Mice ⴛ 106/␮L Hb, g/dL PCV, %
h␤A/h␤A control 10.7 ⫾ 0.5 10.5 ⫾ 0.6 41.1 ⫾ 1.9
h␤S/h␤S sickle 6.7 ⫾ 1.1 7.1 ⫾ 0.9 35.1 ⫾ 3.3
h␤A/h␤S corrected 11.3 ⫾ 1.0* 10.8 ⫾ 1.0* 41.7 ⫾ 2.5*
All animals were analyzed at 6 to 8 weeks after birth. Values represent the mean ⫾ SD. Statistical significance was determined for h␤A/h␤S-corrected mice compared with
h␤S/h␤S sickle animals; P values were calculated using a 2-tailed Student t test; n ⱖ 6.
RBC indicates red blood cell; Hb, hemoglobin; PCV, packed cell volume or hematocrit; RDW, red cell distribution width; MCV, mean corpuscular volume; fL, femtoliter; and
MCH, mean corpuscular hemoglobin. To convert RBC counts to SI units, multiply by 106; for Hb, multiply by 10; and for reticulocytes, divide by 100.
*P ⱕ .001.
BLOOD, 15 AUGUST 2006 䡠 VOLUME 108, NUMBER 4
Figure 3. Correction of abnormal RBC morphology in
ⴚ383 ␥-␤A–corrected mice. (A) Blood smear of an
h␤A/h␤A control. (B) Blood smear of an h␤S/h␤S sickle
animal with characteristic sickled erythrocytes and a
pronounced reticulocytosis. (C) Blood smear of an h␤A/
h␤S-corrected mouse. No sickled cells were observed in
any field examined. Blood smears were stained with
Wright-Giemsa and the magnification is 100⫻/1.40 NA oil
objective (Nikon Eclipse E800 inverted microscope [Ni-
kon, Tokyo, Japan], Hamamatsu C5810 Color Chilled
3CCD camera [Hamamatsu City, Japan]; Adobe Photo-
shop CS version 8.0 imaging software [San Jose, CA]).
Data from ⫺1400 ␥-␤A–corrected mice are identical to
⫺383 ␥-␤A–corrected animals.
Amelioration of spleen, liver, and kidney pathology, and
restoration of kidney function in mice derived from corrected
ES cells
␤A/␤S-corrected mice developed little spleen, liver, and kidney
pathology compared with knock-in ␤S/␤S sickle mice. Histologic
sections of control (␤A/␤A), sickle (␤S/␤S), and corrected (␤A/␤S)
animals are presented in Figure 4A. The spleens of knock-in sickle
mice are characterized by a massive expansion of red pulp,
dramatic pooling of sinusoidal erythrocytes, vasoocclusion, and a
complete loss of lymphoid follicular structure. In corrected mice,
normal splenic red and white pulp is observed, and virtually no
pools of sickle erythrocytes or infarcts are evident. In addition,
splenomegaly is substantially diminished in corrected mice (Figure
4B). The spleens of ␤A/␤S mice weigh approximately 7-fold less
than the spleens of ␤S/␤S animals and are almost the same size as
spleens of ␤A/␤A control animals.
The livers of knock-in sickle mice are characterized by focal
areas of necrosis and pronounced congestion of the intrahepatic
vasculature with aggregates of sickled RBCs. Erythroid progeni-
tors are evident in the sinusoids, and this extramedullary hematopoi-
esis is indicative of severe anemia. There is also abundant
hemosiderin deposition subsequent to Kupffer cell erythrophagocy-
tosis. In ␤A/␤S-corrected animals, focal areas of necrosis and
aggregation of sickled erythrocytes are not observed. In addition,
extramedullary hematopoiesis and hemosiderin deposition are
absent. These results demonstrate that organ pathology is signifi-
cantly ameliorated in mice derived from corrected ES cells.
In the kidneys of knock-in sickle mice, engorgement and
occlusion of blood vessels with sickled erythrocytes results in
vascular, tubular, and glomerular changes. Sequestration and
occlusion are most obvious at the corticomedullary junctions where
dilated capillaries are easily observed in this region of reduced
oxygen tension. Reduced medullary blood flow in HbS patients
causes extensive tubular damage that results in hyposthenuria, and
this same loss of urine-concentrating ability is observed in the
knock-in ␤S/␤S sickle mice. In contrast, the kidneys of ␤A/␤S-
Urine
Reticulocytes, concentration,
% RDW, % MCV, fL MCH, pg mOsm
7.7 ⫾ 2.9 21.7 ⫾ 2.0 38.4 ⫾ 2.0 9.8 ⫾ 0.8 2129 ⫾ 281
75.4 ⫾ 3.9 32.5 ⫾ 2.1 52.4 ⫾ 3.5 10.6 ⫾ 1.4 768 ⫾ 102
8.4 ⫾ 2.6* 22.6 ⫾ 1.2* 36.9 ⫾ 2.7* 9.6 ⫾ 1.4 2388 ⫾ 397*
BLOOD, 15 AUGUST 2006 䡠 VOLUME 108, NUMBER 4
Figure 4. Amelioration of spleen, liver, and kidney pathology in ⴚ383 ␥-␤A–
corrected mice. (A) Spleen, liver, and kidney sections were analyzed at low
(10⫻/0.45 NA objective for spleen and kidney, 40⫻/0.75 NA objective for liver) and
high (100⫻/1.40 NA oil objective) magnification. In ⫺383 ␥-␤A–corrected mice,
normal splenic red and white pulp is observed, and virtually no pools of sickle
erythrocytes or infarcts are evident. In livers of ⫺383 ␥-␤A animals, focal areas of
necrosis and aggregation of sickled erythrocytes are not observed; also, extramedul-
lary hematopoiesis and hemosiderin deposition are absent. Kidneys of ⫺383 ␥-␤A
mice appear normal and free of the disruptive vascular RBC pooling. All sections
were stained with hematoxylin-eosin. (B) Correction of splenomegaly in ⫺383
␥-␤A–corrected mice. Data from ⫺1400 ␥-␤A–corrected mice are identical to ⫺383
␥-␤A–corrected animals.
corrected mice appear normal and free of the disruptive vascular
RBC pooling and hemosiderin deposits observed in the sickle mice.
Most importantly, urine-concentrating ability is completely re-
stored in ␤A/␤S-corrected mice (Table 1). Urine concentrations are
increased from 768 ⫾ 102 mOsm in ␤S/␤S animals to 2388 ⫾ 397
mOsm in ␤A/␤S mice; urine concentrations in ␤A/␤A controls are
2129 ⫾ 281. These data demonstrate that kidney function is
restored in mice derived from corrected ES cells.
Discussion
We have recently produced a new mouse model of sickle cell
disease by replacing the mouse ␤-globin genes with a 9.7-kb DNA
SICKLE GENE REPLACEMENT 1187
fragment containing the human A␥-globin gene (5.6 kb) and a
human ␤S-globin gene (4.1 kb). The ␥- and ␤S-globin genes contain
1400 bp and 815 bp of 5⬘ flanking sequence, respectively. Mouse
␣-globin genes were also replaced with a human ␣-globin gene. At
birth, these knock-in sickle mice continue to synthesize significant
amounts of human HbF. During the first week after birth, the
animals switch to more than 99% HbS and develop severe disease.
We linked the human ␥- and ␤S-globin genes in a relatively small
fragment because we previously found that this design resulted in
an HbF to HbS switch that mimicked the switch in human sickle
patients.11-13 Larger fragments derived from cosmids, YACs, and
BACs switch hemoglobins in utero,14-19 and this early switch from
HbF to high levels of HbS results in a high rate of perinatal
mortality. The ␥-␤S fragment in the knock-in sickle mice is
identical to the configuration of the transgene in our previous
knock-out/transgenic sickle mice, and globin gene switching is
similar in these 2 models.
We derived ES cells from these animals and tested a gene
replacement protocol for correcting the disease. This approach
avoids random insertions that are characteristic of viral gene
therapy approaches. Although relatively few insertional mutations
have been observed in viral gene therapy studies, the SCID gene
therapy trials in France demonstrated that leukemic cell clones can
arise from insertional activation of LMO-2,24 and experiments in
nonhuman primates have suggested that insertional inactivation of
BCL-2A1 can result in acute myeloid leukemia.26 The risk of
mutagenesis is a consequence of random insertion of one or more
copies of the viral vector in a large number of cells. If 2 to 3 million
CD34⫹ cells per kilogram of body weight are transduced and
transplanted, a 50-kg patient would receive 100 million cells and,
potentially, 100 million different viral insertions. Although the
number of reported insertional mutations after viral gene therapy
has been low, the large number of insertion sites remains a concern.
The gene replacement strategy described in this paper avoids
the risk of insertional mutagenesis. The defective ␤-globin gene
(␤S) was replaced with a normal copy of the gene (␤A) in ES cells,
and cells derived from a single corrected clone were fully
characterized before transplantation. In the present study, the cells
were transplanted into blastocysts, and hematopoietic stem cells
(HSCs) derived from these cells in vivo produced corrected red
blood cells that did not sickle in recipients; consequently, the
anemia and organ pathology of the disease was cured. Of course,
translation of this approach to human patients would involve in
vitro derivation of HSCs27-29 from corrected, patient-specific ES
cells. This approach would provide corrected, fully characterized,
syngeneic HSCs for intravenous transplantation, and red blood
cells derived from these HSCs in vivo would not sickle.
The application of this approach to patients with sickle cell
disease requires successful derivation of patient-specific ES cells
by somatic cell nuclear transfer (SCNT). The recent retractions of 2
papers30,31 describing the derivation of human ES cells by this
technique are a significant setback for the field. However, the
feasibility of the approach has been demonstrated in animal
models.32,33 Therefore, with additional basic research, there is a
reasonable expectation that patient-specific, nuclear transfer ES
(ntES) cells can be derived in the future. Correction of the sickle
mutation by gene replacement in ntES cells derived from skin
fibroblasts (or in skin fibroblasts before nuclear transfer) should
provide a means to produce corrected cells that can be differenti-
ated into HSCs in vitro and transplanted into patients. Red blood
cells derived from these HSCs in vivo will not sickle, and this
treatment should cure the disease.
1188 WU et al
References
1. Ingram VM. A specific chemical difference be-
tween the globins of normal human and sickle-
cell anaemia haemoglobin. Nature. 1956;178:
792-794.
2. Ingram VM. Gene mutations in human haemoglo-
bin: the chemical difference between normal and
sickle cell haemoglobin. Nature. 1957;180:326-
328.
3. Wishner BC, Ward KB, Lattman EE, Love WE.
Crystal structure of sickle-cell deoxyhemoglobin
at 5 A resolution. J Mol Biol. 1975;98:179-194.
4. Padlan EA, Love WE. Refined crystal structure of
deoxyhemoglobin S: II, molecular interactions in
the crystal. J Biol Chem. 1985;260:8280-8291.
5. Hofrichter J, Ross PD, Eaton WA. Kinetics and
mechanism of deoxyhemoglobin S gelation: a
new approach to understanding sickle cell dis-
ease. Proc Natl Acad Sci U S A. 1974;71:4864-
4868.
6. Crepeau RH, Dykes G, Garrell R, Edelstein SJ.
Diameter of haemoglobin S fibres in sickled cells.
Nature. 1978;274:616-617.
7. Dykes GW, Crepeau RH, Edelstein SJ. Three-
dimensional reconstruction of the 14-filament fi-
bers of hemoglobin S. J Mol Biol. 1979;130:451-
472.
8. Noguchi CT, Schechter AN. The intracellular poly-
merization of sickle hemoglobin and its relevance
to sickle cell disease. Blood. 1981;58:1057-1068.
9. Brittenham GM, Schechter AN, Noguchi CT. He-
moglobin S polymerization: primary determinant
of the hemolytic and clinical severity of the sick-
ling syndromes. Blood. 1985;65:183-189.
10. Eaton WA, Hofrichter J. Hemoglobin S gelation
and sickle cell disease. Blood. 1987;70:1245-
1266.
11. Ryan TM, Townes TM, Reilly MP, et al. Human
sickle hemoglobin in transgenic mice. Science.
1990;247:566-568.
12. Behringer RR, Ryan TM, Palmiter RD, Brinster
RL, Townes TM. Human gamma- to beta-globin
gene switching in transgenic mice. Genes Dev.
1990;4:380-389.
13. Ryan TM, Ciavatta DJ, Townes TM. Knockout-
transgenic mouse model of sickle cell disease.
Science. 1997;278:873-876.
BLOOD, 15 AUGUST 2006 䡠 VOLUME 108, NUMBER 4
14. Paszty C, Brion CM, Manci E, et al. Transgenic
knockout mice with exclusively human sickle he-
moglobin and sickle cell disease. Science. 1997;
278:876-878.
15. Peterson KR, Clegg CH, Huxley C, et al. Trans-
genic mice containing a 248-kb yeast artificial
chromosome carrying the human beta-globin lo-
cus display proper developmental control of hu-
man globin genes. Proc Natl Acad Sci U S A.
1993;90:7593-7597.
16. Strouboulis J, Dillon N, Grosveld F. Developmen-
tal regulation of a complete 70-kb human beta-
globin locus in transgenic mice. Genes Dev.
1992;6:1857-1864.
17. Greaves DR, Fraser P, Vidal MA, et al. A trans-
genic mouse model of sickle cell disorder. Nature.
1990;343:183-185.
18. Gaensler KM, Kitamura M, Kan YW. Germ-line
transmission and developmental regulation of a
150-kb yeast artificial chromosome containing the
human beta-globin locus in transgenic mice. Proc
Natl Acad Sci U S A. 1993;90:11381-11385.
19. Kaufman RM, Pham CT, Ley TJ. Transgenic anal-
ysis of a 100-kb human beta-globin cluster-con-
taining DNA fragment propagated as a bacterial
artificial chromosome. Blood. 1999;94:3178-
3184.
20. Levasseur DN, Ryan TM, Pawlik KM, Townes
TM. Correction of a mouse model of sickle cell
disease: lentiviral/antisickling beta-globin gene
transduction of unmobilized, purified hematopoi-
etic stem cells. Blood. 2003;102:4312-4319.
21. Pawliuk R, Westerman KA, Fabry ME, et al. Cor-
rection of sickle cell disease in transgenic mouse
models by gene therapy. Science. 2001;294:
2368-2371.
22. Puthenveetil G, Scholes J, Carbonell D, et al.
Successful correction of the human beta-thalas-
semia major phenotype using a lentiviral vector.
Blood. 2004;104:3445-3453.
23. Imren S, Fabry ME, Westerman KA, et al. High-
level beta-globin expression and preferred intra-
genic integration after lentiviral transduction of
human cord blood stem cells. J Clin Invest. 2004;
114:953-962.
24. Hacein-Bey-Abina S, Von Kalle C, Schmidt M, et
al. LMO2-associated clonal T cell proliferation in
two patients after gene therapy for SCID-X1. Sci-
ence. 2003;302:415-419.
25. Mansour SL, Thomas KR, Capecchi MR. Disrup-
tion of the proto-oncogene int-2 in mouse em-
bryo-derived stem cells: a general strategy for
targeting mutations to non-selectable genes. Na-
ture. 1988;336:348-352.
26. Seggewiss R, Pittaluga S, Adler RL, et al. Acute
myeloid leukemia associated with retroviral gene
transfer to hematopoietic progenitor cells in a rhe-
sus macaque. Prepublished on January 26, 2006,
as DOI 10.1182/blood-2005-10-4108. (Now avail-
able as Blood. 2006;107:3865-3867.)
27. Zambidis ET, Peault B, Park TS, Bunz F, Civin CI.
Hematopoietic differentiation of human embry-
onic stem cells progresses through sequential
hematoendothelial, primitive, and definitive
stages resembling human yolk sac development.
Blood. 2005;106:860-870.
28. Wang Y, Yates F, Naveiras O, Ernst P, Daley GQ.
Embryonic stem cell-derived hematopoietic stem
cells. Proc Natl Acad Sci U S A. 2005;102:19081-
19086.
29. Willey S, Ayuso-Sacido A, Zhang H, et al. Accel-
eration of mesoderm development and expansion
of hematopoietic progenitors in differentiating ES
cells by the mouse Mix-like homeodomain tran-
scription factor. Prepublished on January 10,
2006, as DOI 10.1182/blood-2005-10-4120. (Now
available as Blood. 2006;107:3122-3130.)
30. Hwang WS, Roh SI, Lee BC, et al. Patient-spe-
cific embryonic stem cells derived from human
SCNT blastocysts. Science. 2005;308:1777-
1783.
31. Hwang WS, Ryu YJ, Park JH, et al. Evidence of a
pluripotent human embryonic stem cell line de-
rived from a cloned blastocyst. Science. 2004;
303:1669-1674.
32. Rideout WM III, Hochedlinger K, Kyba M, Daley
GQ, Jaenisch R. Correction of a genetic defect by
nuclear transplantation and combined cell and
gene therapy. Cell. 2002;109:17-27.
33. Brambrink T, Hochedlinger K, Bell G, Jaenisch R.
ES cells derived from cloned and fertilized blasto-
cysts are transcriptionally and functionally indis-
tinguishable. Proc Natl Acad Sci U S A. 2006;103:
933-938.