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Hematopoietic Stem Celltargeted Neonatal Gene Therapy
Reverses Lethally Progressive
Osteopetrosis in oc/oc Mice
MARIA K. JOHANSSON, TEUN J. DE VRIES, TON SCHOENMAKER, MATS EHINGER, ANN
C.
M. BRUN, ANDERS FASTH, STEFAN KARLSSON, VINCENT EVERTS AND JOHAN RICHTER
ANNA MIENKO
WHAT IS INFANTILE MALIGNANT
OSTEOPETROSIS (IMO)?
• a rare, congenital disease caused by lack of functional osteoclasts
• fatal if left untreated
• caused by a mutation in the TCIRG1 gene, which codes for the α3
subunit of a proton pump essential for bone resorption
• impaired bone resorption leads to an increase in bone density
• there is currently only one available treatment! HSC
transplantation
• Cons: high mortality, lack of suitable stem cell donors
Question: Can gene therapy reverse lethally
progressive osteopetrosis in oc/oc mice?
Hypothesis: the osteopetrotic phenotype in oc/
oc mice can be reversed by transplantation of
fetal liver–derived oc/oc HSCs that have been
retrovirally transduced to express a nonmutated
form of tcirg1.
MATERIALS AND METHODS
Mice:
• 2 pairs of heterozygous F1 oc/+ mice were obtained
• They were bred to produce homozygous F2 oc/oc mice,
which were identified by their dental abnormalities
(due to lack of tooth eruption)
• oc/+ mice appeared phenotypically normal and were thus
indistinguishable from WT littermates
FIGURE 1. Vector design and
experimental setup.
•
•
•
•
•
•
SFFV- Spleen FocusingForming Virus
wPRE- woodchuck posttranscriptional response
element
IRES- internal ribosomal
entry site
GFP- green fluorescence
protein
FL- fetal liver
FACS- fluorescent activated
cell sorting
FIGURE 2. Multilineage
reconstitution of transduced cells in
oc/oc+tcirg1 mice and reversal of
peripheral blood B-cell deficiency in
oc/oc+tcirg1 mice.
Goal: to see if transplantation of
transduced cells into oc/oc mice can
reverse PB B-cell deficiency
Results: significant rise in the mature
B220+ B-cell population in oc/oc+tcirg1
mice 12 to 18 weeks after
transplantation (compared to untreated
oc/oc mice)
FIGURE 3. BM cells harvested from
oc/oc+tcirg1 mice can be in
vitro–differentiated to
osteoclast-like cells in normal
numbers.
Goal: to investigate if
transduced oc/oc FL cells can
differentiate into boneresorbing osteoclasts
Results: the number of
osteoclasts that formed was
similar between genotypes (WT
and oc/oc+tcirg1) and the 2
sorted cell populations (from
oc/oc+tcirg1 mice)
FIGURE 4. In vitro–
differentiated cells from oc/
oc+tcirg1 mice are capable of
bone resorption.
Goal: to further assess invitro bone resorbing activity
Results: after 6 days only WT
mice showed bone resorption,
whereas after 14 days, both WT
and oc/oc+tcirg1 mice showed
bone resorption
FIGURE 5. Expression of WT tcirg1 is up-regulated during osteoclast differentiation,
while vector-mediated expression in oc/oc+tcirg1 mice is down-regulated.
GOAL: To observe gene expression of tcrig1 as the BM cells differentiated toward
osteoclast-like cells
RESULTS: Quantitative RT-PCR reveals that lower bone-resorption activity (Figure 4)
in oc/oc + tcirg1 mice may be due to downregulation of tcirg1 in the retroviral vectors
FIGURE 6. Reversal of
osteopetrotic phenotype in
oc/oc+tcirg1 mice as
demonstrated by x-ray and
histology.
(a,e): 3wk untreated WT control
(b,f): 3wk untreated oc/oc
mouse
(c,g): 8wk oc/oc+tcirg1 mouse,
partial remodeling of the bone
(d,h): 18wk oc/oc+tcirg1
mouse, bone structure and bone
marrow almost normalized
bone marrow volume increases at
the expense of bone volume in
oc/oc+tcirg1.
left: x-ray images, right: corresponding
DISCUSSION
• 8/15 treated oc/oc animals survived past the normal
life span of oc/oc mice and exhibited signs of
osteoclast activity both in vivo and in vitro.
• signs of slow bone resorption and the fact that not all
treated mice survive!
need to enhance osteoclast
function early after transplantation
• Can be done by a) improving vectors, b) differentiating part of
the transduced cells toward the osteoclastic lineage before
transplantation
DISCUSSION (CONT.)
• Fairly low level of correction of osteoclast function
observed in vitro, but an almost complete normalization of
skeletal phenotype
• Therefore, only a small portion of the bone-resorbing capacity of
osteoclasts is needed to restore the balance between production and
destruction of bone in oc/oc mice
• Reasons for low level osteoclast activity: impaired
development of osteoclasts from transduced cells, low level
of expression of the transgene in the transduced cells.
IN CONCLUSION…
• osteopetrosis in oc/oc mice can be reversed by neonatal
transplantation of gene-modified HSCs and progenitor
cells.
• These findings represent a first but significant step
toward gene therapy for osteopetrosis
REPORTS
2368
construction of the MFA1pr-HIS3 reporter; P. Jorgensen
for discussions concerning SGA mapping of gain-offunction mutations; and G. Brown, N. Davis, D. Durocher, S. Gasser, S. Fields, T. Hughes, and T. Roemer for
comments on the manuscript. Supported by grants
from the Canadian Institute of Health Research (B.A.,
C.B., C.H., and M.T.), an operating grant from the National Cancer Institute of Canada (C.B.), and an
operating grant from the Natural Sciences and
Engineering Research Council of Canada (H.B.).
28 August 2001; accepted 13 November 2001
Correction of Sickle Cell
Disease in Transgenic Mouse
Models by Gene Therapy
Robert Pawliuk,1,2 Karen A. Westerman,1,2 Mary E. Fabry,3
Emmanuel Payen,4 Robert Tighe,1,2 Eric E. Bouhassira,3
Seetharama A. Acharya,3 James Ellis,5 Irving M. London,1,6
Connie J. Eaves,7 R. Keith Humphries,7 Yves Beuzard,4
Ronald L. Nagel,3 Philippe Leboulch,1,2,4,8*
Sickle cell disease (SCD) is caused by a single point mutation in the human “A
globin gene that results in the formation of an abnormal hemoglobin [HbS
(#2″S2 )]. We designed a “A globin gene variant that prevents HbS polymerization and introduced it into a lentiviral vector we optimized for transfer to
hematopoietic stem cells and gene expression in the adult red blood cell lineage.
Long-term expression (up to 10 months) was achieved, without preselection,
in all transplanted mice with erythroid-specific accumulation of the antisickling
protein in up to 52% of total hemoglobin and 99% of circulating red blood cells.
In two mouse SCD models, Berkeley and SAD, inhibition of red blood cell
dehydration and sickling was achieved with correction of hematological parameters, splenomegaly, and prevention of the characteristic urine concentration defect.
Sickle cell disease (SCD) is one of the most
prevalent autosomal recessive disorders worldwide. In 1957, SCD became the first genetic
disorder for which a causative mutation was
identified at the molecular level: the substitution of valine for glutamic acid in human “Aglobin codon 6 (1). In homozygotes, the abnormal hemoglobin (Hb) [HbS (#2″S2 )] polymerizes in long fibers upon deoxygenation within
red blood cells (RBCs), which become deformed (“sickled”), rigid, and adhesive, thereby
triggering microcirculation occlusion, anemia,
infarction, and organ damage (2, 3).
Human $-globin is a strong inhibitor of
HbS polymerization, in contrast to human
“A-globin, which is effective only at very
1
Harvard-MIT, Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. 2Genetix Pharmaceuticals,
Cambridge, MA 02139, USA. 3Division of Hematology,
Albert Einstein College of Medicine, Bronx, NY 10461,
USA. 4INSERM EMI 0111, Hôpital Saint-Louis, 75010
Paris, France. 5Department of Genetics, Hospital for
Sick Children, Toronto, ON M5G1X8, Canada. 6Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. 7The Terry Fox
Laboratory and the University of British Columbia,
Vancouver, BC V5Z3L6, Canada. 8Harvard Medical
School and Department of Medicine, Brigham and
Women’s Hospital, Boston, MA 02115 USA.
*To whom correspondence should be addressed. Email: pleboulch@mit.edu
high concentrations (4). Hence, gene therapy
of SCD was proposed by means of forced
expression of human $-globin or $/” hybrids
in adult RBCs after gene transfer to hematopoietic stem cells (HSCs) (5–11).
Although the discovery of the human “-globin locus control region (LCR) held promise to
achieve high globin gene expression levels (12,
13), the stable transfer of murine onco-retroviral vectors encompassing minimal core elements of the LCR proved especially challenging (14–20). To allow the transfer of larger
LCR and globin gene sequences, we proposed
the use of RNA splicing and export controlling
elements that include the Rev/R responsive element (RRE) components of human immunodeficiency virus (HIV) (21), and an RRE-bearing HIV-based lentiviral vector recently resulted in substantial amelioration of “-thalassemia
in transplanted mice (22). However, gene expression remained heterocellular, and the
amount of human “A-globin found incorporated in Hb tetramers in a nonthalassemic background is unlikely to be therapeutic for SCD
(22). Here, a lentiviral vector was optimized to
express an antisickling protein at therapeutic
levels in virtually all circulating RBCs of SCD
mouse models.
We constructed a human “A-globin gene
variant mutated at codon 87 to encode the
amino-acid residue believed to be responsible
14 DECEMBER 2001 VOL 294 SCIENCE www.sciencemag.org
Downloaded from www.sciencemag.org on September 2, 2010
MIPS (www.mips.biochem.mpg.de/). Yeast genetic
and protein-interaction data sets were obtained from
YPD and MIPS databases. Cellular role and biochemical functions were obtained from YPD.
19. G. D. Bader et al., Nucleic Acids Res. 29, 242 (2001);
www.bind.ca.
20. http://vlado.fmf.uni-lj.si/pub/networks/pajek/.
21. A. H. Y. Tong et al., Science, in preparation.
22. Because both Bbc1 (Mti1) and Vrp1 bind yeast type I
myosins (Myo3 and Myo5), and a BBC1 deletion
mutation (bbc1!) suppresses the temperature sensitivity and endocytosis defects of vrp1 mutants, Bbc1
and Vrp1 may antagonistically control the function
of type I myosins (33).
23. D. C . Winter, E. Y. Choe, R. Li, Proc. Natl. Acad. Sci.
U.S.A. 96, 7288 (1999).
24. M. J. Cope, S. Yang, C. Shang, D. G. Drubin, J. Cell Biol.
144, 1203 (1999).
25. K. Bloom, Nature Cell Biol. 2, E96 (2000).
26. H. Debrauwere, S. Loeillet, W. Lin, J. Lopes, A. Nicolas,
Proc. Natl. Acad. Sci. U.S.A. 98, 8263 (2001).
27. E. S. Kroll, K. M. Hyland, P. Hieter, J. J. Li, Genetics 143,
95 (1996).
28. Because double mutants are created by meiotic
crossover, a set of gene deletions that are linked to
the query gene, which we refer to as the “linkage
group,” form double mutants at reduced frequency. For example, the gene-deletion mutations of 13
different genes that are linked to BNI1 and contained on the array [YNL253W, YNL254C, YNL255C,
YNL257C, YNL259C, YNL264C, YNL265C, YNL266W,
YNL268W, YNL270W, BNI1 (YNL271W), YNL273W,
and YNL275W] appeared to be synthetically lethal
with the bni1! query mutation. A list of the gene
deletions that were linked to the query gene and
appeared to be synthetically lethal with the query
gene is provided as supplementary material (16).
Thus, systematic random spore analysis with the
set of gene-deletion mutants provides a method
for genetically mapping mutations that are linked
to a dominant selectable marker. Similarly, alleles
that are associated with a gain-of-function phenotype can also be mapped. For example, through
identification of linkage groups that are defective
for the gain-of-function phenotype, we should be
able to map mutations that result in filamentous
growth or high-temperature growth, neither of
which are associated with the S288C strains within
the deletion array, or suppressors of the lethality
associated with temperature-sensitive alleles
linked to a dominant selectable marker.
29. L. H. Hartwell et al., Science 278, 1064 (1997).
30. Each of the deletion mutations is marked with two
unique oligonucleotide bar codes that were integrated along with common flanking primer sites for
PCR amplification (34). Because the bar codes allow the growth rate of all the deletion mutants to
be followed within a population of cells, the steps
for creating double mutants outlined in Fig. 1 could
be carried out with a pool of deletion mutants. In
this scheme, synthetic lethality or slow growth of
the resulting double mutants would be analyzed by
PCR amplification of the bar codes and subsequent
hybridization to a bar-code microarray, such that
the intensity of the signal observed for an element
on the bar-code array reflects the representation
of the double-mutant meiotic progeny (35). As
another means of large-scale synthetic lethal analysis, a collection of deletion mutant strains can be
transformed en masse with a gene-deletion cassette and the lethal double-mutant transformants
predicted by bar-code microarray analysis (36).
31. R. Barstead, Curr. Opin. Chem. Biol. 5, 63 (2001).
32. T. R. Hughes et al., Nature Genet. 25, 333 (2000).
33. J. Mochida, T. Yamamoto, K. Fujimura-Kamada, K.
Tanaka, personal communication.
34. D. D. Shoemaker, D. A. Lashkari, D. Morris, M. Mittmann, R. W. Davis, Nature Genet. 14, 450 (1996);
www-sequence.stanford.edu/group/yeast_deletion_
project/deletions3.html.
35. M. Maiolatesi, P. Bieganowski, D. Shoemaker, C. Brenner, personal communication.
36. S. L. Ooi, et al., personal communication.
37. We thank B. Drees, S. Fields, and J. Friesen for advice on
the robotic manipulation of yeast arrays; C. Udell for
for most of the antisickling activity of $-globin
[“A87 Thr:Gln (“A-T87Q)] (23). To assess first
its antisickling capacity and oxygen-binding affinity, we generated transgenic mice expressing
both human “A-T87Q- and #-globins, but neither
mouse #- nor mouse “-globin. These mice had
normal hematological parameters and viability,
and the “A-T87Q-globin variant extracted from
their RBCs was found to be almost as potent an
inhibitor of HbS polymerization as $-globin in
vitro and much more so than “A-globin (Supplementary fig. 1A) (24). Whole-blood analysis
of p50, the pO2 at which 50% of the Hb molecules are oxygenated, showed that the oxygenbinding affinity of “A-T87Q Hb was well within
the range observed with wild-type “A Hb in
mice: 31.1 % 0.2 mm Hg (standard error & SE)
versus 32.7 % 1.8 mm Hg (SE), respectively
(24).
The “A-T87Q-globin gene variant was then
inserted in a lentiviral vector we optimized
for transfer to HSCs and erythroid-specific
expression. The central polypurine tract/
DNA flap of HIV-1 (25) was incorporated in
the construct to increase viral titers and transduction of HSCs after pseudotyping with the
vesicular stomatitis virus glycoprotein G
(VSV-G) and concentration (Supplementary
fig. 1C) (24). Specific LCR elements were
chosen on the basis of results of single integrants in erythroid cells assessed by recombinase-mediated cassette exchange (26).
The “A-T87Q-globin lentivirus was first
analyzed in lethally irradiated normal syngeneic
C57BL/6 recipient mice in the absence of any
selection (24). Proviral transfer was stable with
an average copy number of 3.0 % 0.5 (SE) per
genome of peripheral nucleated blood cells 3
months after transplantation (Supplementary
fig. 1B) (24). At 10 months after transplantation, all mice expressed human “A-T87Q-globin
protein with up to 99% [mean 96 % 0.9% (SE)]
of their RBCs staining positive with an antibody that specifically recognizes human
“-globin, in this case, the “A-T87Q variant
(Fig. 1A) (24 ). No “A-T87Q-globin expression was detected in other blood lineages
by antibody staining. Human “A-T87Q-globin mRNA reached up to 107% [mean 71 %
15% (SE)] of endogenous mouse “-single
globin mRNA transcripts (Fig. 1B) (24 ).
Human “A-T87Q-globin protein represented
up to 22.5% [mean 16 % 3.1% (SE)] of
endogenous mouse “-chains in recipients
of “A-T87Q-globin lentivirus-transduced
bone marrow, as determined by high-performance liquid chromatography (HPLC)
(Fig. 1C) (24 ). The fourfold discrepancy
between human “A-T87Q-globin mRNA and
protein levels is consistent with differences
observed in mice transgenic for the “Aglobin gene (27 ).
Long-term secondary transplants were also
performed with bone marrow from a representative primary recipient killed 5 months after
Fig. 1. Analysis of human “A-T87Q-globin gene
expression in C57BL/6 recipient mice 5 months
after transplantation. (A) Circulating RBCs from
recipient mice were fixed, permeabilized,
stained with a FITC-labeled antibody that specifically recognizes human “-globin (PerkinElmer Wallac, Norton, Ohio), and subsequently
analyzed by FACS (24). Top: representative
mouse transplanted with mock-transduced
bone marrow cells. Bottom: representative
mouse transplanted with bone marrow transduced with the “A-T87Q-globin lentivirus. (B)
Primer extension analysis of peripheral blood
RNA (24). Lanes 1, 3, 5, 7, and 9: amplification
with primers specific for the endogenous murine “-single globin mRNA generating a 53–
base pair (bp) DNA fragment. Lanes 2, 4, 6, 8,
and 10: amplification with primers specific for
the human “A-T87Q-globin mRNA generating a
90-bp DNA fragment. Lanes 1 and 2: mocktransduced mouse. Lanes 3 and 4: transgenic
control mouse expressing 86% of human
“-globin mRNA. Lanes 5 to 10: three C57BL/6
recipients of “A-T87Q-globin–transduced bone
marrow cells (lanes 5 and 6, mouse #1; 7 and 8,
mouse #2; 9 and 10, mouse #3). (C) HPLC
profiles of globin chains extracted from RBCs of
a mock-transduced mouse (top) and a recipient
of human “A-T87Q-globin–transduced bone
marrow (bottom) (24).
Downloaded from www.sciencemag.org on September 2, 2010
REPORTS
Fig. 2. HPLC profiles of Hb extracted from RBCs of mouse recipients of (A) mock-transduced SAD,
(B) mock-transduced BERK, (C) “A-T87Q-globin–transduced SAD, and (D) “A-T87Q-globin–transduced BERK bone marrow cells (24).
www.sciencemag.org SCIENCE VOL 294 14 DECEMBER 2001
2369
transplantation (24). Fluorescence-activated
cell sorting (FACS) analysis of peripheral blood
samples of secondary recipients 4 months after
transplantation showed that 87 % 2.3 (SE) of
RBCs expressed high levels of human “A-T87Qglobin protein, thus demonstrating that transduction of true HSCs was achieved. Analysis of
position effect variegation suggested that pancellular expression was the result of balanced
expression from polyclonal stem cell reconstitution with multiple chromosomal integration
sites rather than true position-independent expression (24).
Because no transgenic mouse model perfectly recapitulates the exact disease characteristics of human SCD patients (28–33), we
investigated the efficacy of the “A-T87Q-globin lentiviral vector in two different SCD
transgenic mouse models: SAD (29) and
Berkeley (BERK) (31). SAD mice express
human #-globin together with a “super S”
globin resulting from two point mutations
added to the human “S gene (29), whereas
BERK mice, which express human #- and
human “S-globulins, do not express any murine globins because of complete disruption
of both mouse #- and “-globin gene loci (31).
The phenotype of BERK mice is overall more
severe than that of SAD mice, although some
of the hematological abnormalities in BERK
mice are caused by an associated “-thalassemic syndrome due to suboptimal expression
of the transgenic human “S gene (28).
SAD and BERK bone marrow was transduced with the “A-T87Q-globin lentiviral vector and transplanted into lethally irradiated
syngeneic C57BL/6 mouse recipients (24).
Transduced SAD marrow was also transplanted into lethally irradiated syngeneic
SAD recipients. Three months after transplantation, reconstitution of recipient C57BL
mice with donor BERK or SAD bone marrow
was essentially complete for all mice, as determined by quantification of murine “-single
Hb by HPLC (Fig. 2) (24).
Isoelectric focusing electrophoresis of
blood samples from mice 3 months after
transplantation showed all of the expected
species of Hb (Supplementary fig. 2) (24 ).
The amount of “A-T87Q-globin expressed in
the transplanted mice, as measured by Hb
HPLC, was up to 108% [mean 75.5 %
17.1% (SE)] and 51% [mean 42.5 % 5.5%
(SE)] of the transgenic HbS for recipients
of “A-T87Q-globin lentivirus–transduced
BERK and SAD bone marrow, respectively
(Fig. 2, C and D) (24 ). These values correspond to up to 52% and 12% of the total
Hb of BERK and SAD mice, respectively.
The greater amount of “A-T87Q-globin–containing Hb observed in erythrocytes derived from transduced bone marrow cells of
BERK mice as compared to SAD mice may
be explained by the absence of the murine
“-globulin mRNA and the associated
2370
Fig. 3. Correction of SCD pathology. (A) Nomarski optics microscopy of RBCs from mice transplanted
with either (top) mock- or (bottom) “A-T87Q-globin lentivirus–transduced BERK bone marrow cells
under 5% pO2 3 months after transplantation (24). (B) Quantification of the percentage of sickle RBCs
from recipients of mock-transduced and “A-T87Q-globin–transduced BERK or SAD bone marrow under
5% or 13% oxygen conditions, respectively (24). Error bars indicate SE; *, P & 0.01; †, P & 0.03. (C)
Relationship between log of reciprocal delay time (dt) of HbS polymerization and Hb concentration of
RBC lysates. Time courses of Hb polymerization in lysates were performed at various concentrations by
the temperature jump method (24). ‚, lysate from a homozygote SS patient; Œ, lysate from an
asymptomatic AS sickle cell trait patient; !, lysate from a mouse recipient of mock-transduced SAD
marrow; â– , lysate from a mouse recipient of “()T87Q-globin–transduced SAD marrow; E, lysate from
a mouse recipient of mock-transduced BERK marrow; F, lysate from a mouse recipient of “()T87Qglobin–transduced BERK marrow. (D) Percoll-Larex continuous density gradients from blood of recipient
mice (24). Lane 1, density marker beads; lanes 2 and 6, C57BL/6 controls; lanes 3 and 7, SAD and BERK
controls, respectively; lanes 4 and 5, C57BL/6 recipients of mock-transduced or “A-T87Q-transduced SAD
bone marrow, respectively; lane 8, C57BL/6 recipient of “A-T87Q-transduced BERK bone marrow; lane 9
transgenic BERK mouse expressing human $-globin at ‘100% of “S-globin. (E) Spleens from nontransplanted (1) BERK and (2) C57BL/6 mice, or C57BL/6 mice transplanted with either (3) “A-T87Qtransduced or (4) mock-transduced BERK bone marrow.
thalassemic phenotype of BERK mice,
which favors translation of the added
“A-T87Q-globin mRNA species (28).
To determine whether “A-T87Q-globin was
capable of inhibiting HbS polymerization in
vivo in transplanted SCD mouse models, the
morphology of RBCs from transplanted mice
was analyzed as a function of oxygen pressure
in vitro (24). Examination of the obtained sigmoid sickling curves showed a marked change
in the proportion of sickled cells (Fig. 3, A and
B). For recipients of “A-T87Q-globin lentivirus–
transduced BERK marrow, the greatest differ-
ence occurred at 5% pO2, with 80 % 1.7% (SE)
versus 26 % 7.5% (SE) (P & 0.01) sickle cells
for mock-transduced and “A-T87Q-globin lentivirus–transduced marrow, respectively. In comparison, analysis of RBCs from humans with
sickle trait, who are heterozygous for the “S
allele and asymptomatic, showed ‘40% sickled cells at 5% pO2. For SAD marrow, the
greatest difference occurred at 13% pO2, with
81 % 3% (SE) versus 46 % 11% (SE) (P &
0.03) sickle cells for mock-transduced and
“A-T87Q-globin lentivirus–transduced marrow,
respectively. Examination of peripheral blood
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REPORTS
REPORTS
Table 1. Correction of hematological abnormalities and urine concentrating defect in recipients of “A-T87Q-globin–transduced BERK bone marrow. RBCs, red
blood cells; Hb, hemoglobin; ISCs, irreversibly sickled cells. Values shown with SE and statistical significance established by Student’s t test.
Mice*
RBCs
(106/*l)
Hb (g/dl)
Reticulocytes
(%)
ISCs†
(‰)
Urine concentrations
(mOsM) (number of
mice)
C57Bl/6 controls (n & 3)
BERK controls (n & 3)
BERK “A-T87Q (n & 3)
SAD controls (n & 4)
SAD “A-T87Q (n & 3)
10.1 % 0.3
7.4 % 0.6
10.1 % 1.1§
8.4 % 0.6
8.7 % 0.1
15.0 % 0.6
9.4 % 0.9
13.0 % 0.4!
13.0 % 0.6
13.7 % 0.2
4.1 % 0.6
17.8 % 0.6
5.8 % 1.8¶
3.4 % 1.2
2.8 % 0.1
)
16.0‡
2.0#
2.6#
0
3247 % 500 (n & 22)
1452 % 331 (n & 4)
3600 % 381 (n & 2)**
3840 % 175 (n & 3)
3920 % 326 (n & 3)
smears at ambient pO2 showed an eightfold
decrease in the proportion of irreversibly
sickled cells (ISCs) in mice transplanted
with “A-T87Q-globin lentivirus–transduced
BERK marrow with complete disappearance of highly dehydrated ISCs. For SAD
mice, no ISCs could be detected after
lentivirus–transduction
“A-T87Q-globin
( Table 1).
Kinetic studies of HbS polymer formation
by turbidimetry of RBC lysates from transplanted mice showed delayed HbS polymerization in lysates from mice transplanted with
either SAD or BERK marrow transduced
with the “A-T87Q-globin lentivirus (Fig. 3C)
(24). The change in kinetics paralleled what
was observed with RBC lysates from homozygote SS patients versus asymptomatic
AS heterozygotes (Fig. 3C).
We next examined the density of RBCs
from transplanted SCD mouse models, since
HbS polymerization causes an abnormally high
cell density (11, 28). Whereas RBCs from control and mock-transduced SAD mice had a
higher density than those of syngeneic
C57BL/6 mice, mice completely reconstituted
with “A-T87Q-globin lentivirus–transduced
SAD marrow showed a clear shift toward normal (Fig. 3D) (24). In BERK RBCs, the phenomenon was reversed, because the associated
thalassemic phenotype decreases the mean corpuscular Hb concentration, resulting in lower
cell density. The addition of “A-T87Q-globin
partially cured the thalassemia and resulted in
higher cell density (Fig. 3D).
Unlike SAD mice, BERK mice have major alterations of their hematological parameters, as a consequence of both SCD and the
associated thalassemia (28, 31). In mice
transplanted with “A-T87Q-globin lentivirus–
transduced BERK marrow, RBC and reticulocyte counts were corrected with amelioration of Hb concentration, anisocytosis, and
poikilocytosis (Table 1) (24).
We finally examined whether the SCDassociated splenomegaly and characteristic
urine concentration defect in BERK mice
(28, 31) could be ameliorated by gene therapy. Following transplantation of “A-T87Q-globin lentivirus–transduced BERK bone mar-
row, both pathological features were corrected, whereas no effect was observed for recipients of mock-transduced BERK marrow
(Table 1 and Fig. 3E) (24).
These data demonstrate that chromosomal
integration of an antisickling globin gene variant in HSCs can result in its pancellular, erythroid-specific expression at levels sufficiently
high to correct the main pathological features of
SCD. In contrast to “-thalassemia, gene therapy
of SCD requires expression of the therapeutic
gene in most RBCs to prevent untoward vasoocclusion by even a small fraction of sickle
cells (2, 3). This criterion presented a major
obstacle, since the LCR, even in its largest
structural form, does not completely shield cislinked genes from position-effect variegation in
most settings in the absence of chromatin insulators (34). Here, structural optimization of the
“A-T87Q-globin gene/LCR lentivirus by recombination-mediated cassette exchange and incorporation of the central polypurine tract–DNA
flap of HIV-1 resulted in very high viral titers
yielding multiple events of chromosomal integration per HSC. This led to a state of balanced
expression sufficiently high and homogeneous
enough to surmount this hurdle and provide an
overall protection similar to that observed in
asymptomatic human AS heterozygotes.
Before gene therapy of SCD may be proposed to human patients on the basis of these
preclinical results, achieving large-scale lentiviral production devoid of replication-competent retrovirus and bone marrow reconstitution with transduced stem cells in the absence of toxic myeloablation regimens remain desirable objectives.
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28 August 2001; accepted 30 October 2001
www.sciencemag.org SCIENCE VOL 294 14 DECEMBER 2001
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*n is the number of mice for RBCs, Hb, and reticulocytes.
†A total of 2000 RBCs were examined from BERK control and BERK “A-T87Q mice (n & 2) and 3000 RBCs were examined
‡Mostly dehydrated ISCs.
§P & 0.15 with substantial correction of anisocytosis and poikilocytosis.
!P & 0.01.
¶P &
from SAD control and SAD “A-T87Q mice (n & 2).
0.05.
#Only hydrated ISCs.
**P & 0.01.
2371

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