locus control region (LCR) were co-introduced into the mouse germ line. In one of .... The founder SAD-1 mouse contained approximately one copy of the 3SAD ...
The EMBO Journal vol.10 no.11 pp.3157-3165, 1991
Towards a transgenic mouse model of sickle cell disease: hemoglobin SAD
Marie Trudel1' 3, Nacera Saadane2, Marie-Claude GareI2, Josiane Bardakdjian-Michau2, Yves Blouquit2, Jean-Luc Guerquin-Kern4, Philippe Rouyer-Fessard2, Dominique Vidaud2, Agathe Pachnis1, Paul-Henri Romeo2, Yves Beuzard2 and Frank Costantinil 'Department of Genetics and Development, Columbia University, 701 W. 168th Street, New York, NY 10032, USA, 2INSERM U.91 and CNRS UA 607, H6pital Henri-Mondor, 94010 Creteil, France, 3Institut de Recherches Cliniques de Montreal, Faculte de Medecine de l'Universite de Montreal, 110 Avenue des Pins Ouest, Montrdal, Quebec, Canada H2W 1R7 and 4INSERM U.219, Institut Curie, 91405 Orsay, France Communicated by M.F.Perutz
In order to obtain a transgenic mouse model of sickle Fell disease, we have synthesized a novel human (-globin gene, ,ISAD, designed to increase the polymerization of the transgenic human hemoglobin S (Hb S) in vivo. gSAD (WS-Antilles-D Punjab) includes the fl6Val substitution of the Os chain, as well as two other mutations, Antilles (823Ile) and D Punjab (3121Gln) each of which promotes the polymerization of Hb S in human. The SAD gene and the human a2-globin gene, each linked to the (-globin locus control region (LCR) were co-introduced into the mouse germ line. In one of the five transgenic lines obtained, SAD-1, red blood cells contained 19% human h Hb SAD (ai2I 32SAD) and mouse-human hybrids in addition to mouse hemoglobin. Adult SAD-1 transgenic mice were not anemic but had some abnormal features of erythrocytes and slightly enlarged spleens. Their erythrocytes displayed sickling upon deoxygenation in vitro. SAD-1 neonates were anemic and many did not survive. In order to generate adult mice with a more severe sickle cell syndrome, crosses between the SAD progeny and homozygous for (-thalassemic mice were performed. Hemoglobin SAD was increased to 26% in 0-thal/SAD-1 mice which exhibited: (i) abnormal erythrocytes with regard to shape and density; (ii) an enlarged spleen and a high reticulocyte count indicating an increased erythropoiesis; (iii) mortality upon hypoxia; (iv) polymerization of hemolysate similar to that obtained in human homozygous sickle cell disease; and (v) anemia and mortality during development. Key words: hemoglobin/transgenic mouse/animal model/ sickle cell disease
Introduction Sickle cell disease (Bunn and Forget, 1986) is an autosomal recessive disorder characterized by a chronic anemia and acute vascular occlusions in individuals homozygous for (D Oxford University Press
hemoglobin S (f30Val), or in compound heterozygotes for Os and other j-globin gene mutations (e.g. g0 Arab, OD Punjab, f3c and /-thalassemia). The erythrocytes become rigid and deformed (sickled) as a result of the intracellular polymerization of the deoxygenated hemoglobin S (Hb S) which is reversible upon reoxygenation of blood. The sickled cells are trapped in the microcirculation resulting in painful 'sickle cell crises', thrombosis and damage of various organs. Permanently elongated and dehydrated cells, the irreversible sickle cells (ISCs) result from repetitive sickling of cells. While the molecular pathogenesis of sickle cell disease has been characterized in great detail, relatively little is understood about the pathophysiology of the disease. Despite the identification of a number of agents that prevent molecular or cellular defects in vitro, a specific therapy that is both effective and safe has yet to be developed (Schechter et al., 1989). One impediment to the understanding of sickle cell disease and the development of effective treatments has been the lack of an animal model for pathophysiological studies or drug testing in vivo. Previously, the demonstration that high levels of human hemoglobin S or of the ,S-Antilles chain could be producted in transgenic mice (Greaves et al., 1990; Ryan et al., 1990; Rubin et al., 1991) indicated that an animal model might be generated by the introduction of mutant human globin genes into the mouse germ line. However, the high levels of Hb S or of the OS Antilles that were achieved in these studies were without clinical effect, in spite of sickling in vitro. In the present study, we have constructed and utilized a modified human O3s transgene (3SAD) to produce a new form of human hemoglobin, Hb SAD, designed to increase polymerization of transgenic human Hb when diluted by the endogenous mouse Hb. The /SAD gene carries two additional mutations that occur naturally in human, in Hb S-Antilles and Hb D Punjab (Los Angeles). The ,3Antiles mutation (f23Ile), which occurs in cis to the Os mutation (36Val 323Ile), increases the clinical severity of the Os mutation such that Hb A/Hb S-Antilles heterozygotes display a major sickle cell syndrome, in contrast to the asymptomatic A/S genotype (Monplaisir et al., 1986). This is due, first, to the decreased oxygen affinity of Hb S Antilles, which shifts the allosteric equilibrium towards the T state in which Hb S molecules polymerize and second to the creation of new contact sites in the polymer, as suggested by the low solubility of fully deoxygenated Hb S Antilles. The (3121Gln mutation of Hb D Punjab gives a very severe sickle cell syndrome in the compound heterozygous state, Hb S/Hb D Punjab (Milner et al., 1970). This is due to the greatly lowered solubility of the deoxygenated hybrid, Hb a(3S/af3D, which results from the creation of new contact sites that stabilize the polymer (Padlan and Love, 1985). In this paper, we report the co-expression of the ,BSAD and human a genes, and the consequent production of Hb SAD, in transgenic mouse erythrocytes. We show that the red cells 3157
M.Trudel et al. Mutations
S
D A
LCR D
SAD
LCR 3
-iH
a
I-4'
1 Kb
I
Fig. 1. The fiSAD and a-globin gene construct used to generate transgenic mice. Table I. Proportion of human and mouse globin chains in SAD-1 and fl-thal/SAD-i erythrocytes, determined by UT-PAGE Genotype
n
Hbbs/s SAD 1 Hbbs/Hbbthal SAD 1
19 5
SAD/fTotal 20.6 (±0.7) 26.6 (4 1.3) ,B
fminor/Total
human/UTotal 52.0 (±0.9) 53.2 (± 1.3)
21.5 (4 1.3)
of mice in the SAD- I transgenic line, which produce 19 % Hb SAD, is susceptible to extensive sickling when deoxygenated in vitro. Furthermore, 3-thal/SAD-1 mice, which are heterozygous for a murine j3-thalassemia, and produce an increased level (26%) of Hb SAD, display several characteristics of human sickle cell disease.
fTotal/CTotal
ca mouselflmouse
ahuman/f SAD
0.98 (± 0.4) 0.98 (± 0.03)
0.63 (± 0.03) 0.69 (4 0.03)
2.63 (± 0.10) 1.97 (+ 0.11)
-
Results Production of transgenic mice expressing Hb SAD To promote high expression of the /SAD and human aglobin genes in transgenic mice, we fused each gene to the locus control region (LCR) of the human 3-globin gene cluster, as shown in Figure 1. The two constructs were coinjected into fertilized mouse eggs, and five transgenic lines were derived, each of which expressed both the human and 13SAD genes. This paper describes the analysis of the SAD-1 line, the highest expressing line that was obtained. The founder SAD-1 mouse contained approximately one copy of the 3SAD gene and two copies of the human aglobin gene, which were co-transmitted to its progeny (data not shown).
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Fig. 2. UT-PAGE of globin chains from mice of various genotypes: Lanes 1, non-transgenic, homozygous Hbb5s1 (f-single); lanes 2, SAD-I transgenic, homozygous Hbb5s1; lanes 3, non-transgenic, heterozygous Hbbs/Hbbthal (f-single/f-thalassemic); lanes 4, fithal/SAD-1 transgenic, HbbS/Hbbthal. Cys indicates samples treated with cystamine to separate fl-minor and czhuman chains.
H)H 6
a
Globin chains and hemoglobins in SAD- 1 mice When the SAD-1 founder, a male homozygous for the Hbbs (single) haplotype at the ,B-globin locus, was mated to C57BL/6J females (also HbbS/S), the transgenic progeny all displayed similar levels of (SAD-globin, human at-globin and Hb SAD, indicating that expression of the transgenes was a stable trait. In erythrocytes of SAD-1 mice, human f3SAD-globin represented 20.6 + 0.7% of the total 3-globin chains, and human a-globin accounted for 52.0 0.9% of the total a-globin chains (Table I), as determined by urea-Triton polyacrylamide gel electrophoresis (UTPAGE) (Figure 2). Hemoglobin analysis by isoelectric focusing (Figure 3) showed the presence of three new hemoglobins in addition to the normal mouse 0single hemoglobin which represented 39.5 % of total Rb (Table II). One new fraction (19% of total Hb) was Hb SAD (a22human02SAD) with a pl of 7.4, as expected from the two charge differences introduced in the 13SAD chain (36 Glu - Val and 3121Glu - Gln). When analyzed by UT-PAGE, the purified Hb SAD contained only the human 3158
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m
2
3
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4
Siin gi -; Minor
-/ Hybrid *- Hybrid ;I .i'Hybridid SA
pH 9 Fig. 3. Isoelectric focusing of hemoglobin. Lane 1, non-transgenic, heterozygous HbbS/Hbbthal (f-single/fi-thalassemic); lane 2, nontransgenic, homozsygous Hbbs/s (fl-single); lane 3, SAD-1 transgenic, homozygous Hbb /; lane 4, f-thal/SAD-1 transgenic, Hbbs/Hbbthal. Cys indicates samples treated with cystamine to separate fl-minor and a uman chains. Hybrid I, a2humanf02single; hybrid II, C12mousef2; hybrid Ill, ce2human2minor
ae and f3SAD chains. The two other new hemoglobin fractions were hybrid I, formed by human a chains and mouse 3 chains (a 2human32SiOSI) (39.5 %) and a small amount of hybrid II (a2mouse/32 AD) (2%). The HPLC profile of tryptic peptides of the aminoethylated fSAD chain shown in Figure 4 indicated the disappearance of the normal peptides 3T 1, 3T3 and f3T13 and the presence of new fractions. The sequence determination of all peptides allowed us to assign the three expected mutations to the new peptides and the absence of other alteration in the sequence.
Transgenic mouse model for sickle cell disease Table II. Proportion of hemoglobins in transgenic mice and controls
Genotype
n
Hbbs/S
Hbbs's SAD
6 52 19 20
1
Hbbs/Hbbthal HbbS/Hbbthal SAD 1
Hb single
Hb minor
Hybrid I
Hybrid 1I
Hybrid III
(%)
(%)
(Z,human1 single)
(a2human02 SAD)
(%)
(%)
(2 human3 (%)
39.5 (= 3.3)
2
26.0 (:i 3.4)
3
100 39.5 (i 3.9) 75.3 (- 2.1) 29.0 (± 3.8)
24.6 (+ 1.9) 9.0 (i 1.1)
Hb SAD minor)
(%)
19.0
(+
2.7)
26.0 (± 1.5)
7.0 (+ 1.6)
PT6 PT5 PT8PT7~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~T 0.2
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5
10
15
20
25
30
35
40
45
TIME (minutes)
Fig. 4. HPLC profile of tryptic peptides of aminoethylated l3SAD globin chain. Table III. Hematological parameters of transgenic mice and of controls Genotype
n
Hbbs's Hbbs/s SAD 1
Hbbs/Hbbthal Hbbs/Hbbtha,
SAD 1
5 6 5 5
Hct (%)
45.4 44.0 47.8 39.4
(+ 0.6) (+ 0.7) (+ 0.8) (+ 1.2)c
Hb (g/dl)
14.8 16.5 15.3 15.2
(± 0.4) (+ 1.4) (± 0.2) ( 0.6)
RBC
MCV
MCH
(1061p1)
(fl)
(pg/cell) 15.3 (+ 0.8)
9.7 10.9 9.8 11.3
(+ 0.5) (+ 0.6) (+ 0.8) (± 0.6)
48.2 40.7 49.6 35.4
(+ 1.8) (+ 2.4) (+ 3.7) (+ 2.5)a
15.1 (± 0.9) 15.9 (± 1.4) 13.6 (+ 0.9)
MCHC (%)
Reticulocytes
32.6 37.3 32.1 38.6
2.3 0.1) 3.6 (+ 0.4) 2.0 (+ 0.08) 6.2 (+0.4)c
(± 0.9) (+ 2.9) (+ 0.7) ( 1.3)b
(%)
(O
Statistical significance ('P < 0.05; bp < 0.01; CP < 0.001) between HbbS/Hbbthal SADI mice and Hbbs/Hbbthal controls. Hct, hematocrit; Hb, hemoglobin; RBC, red blood cell count; MCV, mean cell volume cell; MCH, mean hemoglobin; MCHC, mean cell hemoglobin concentration.
The peptide profile of the a chain from Hb SAD was identical to that of the a chain obtained from human Hb A. It is interesting to note that the high proportion of human a chains determined by UT -PAGE was compensated by an equivalent reduction in mouse at chains, and that free or unpaired a or ,B chains were not detectable in solution by isoelectric focusing of hemolysate, or by UT -PAGE analysis of red cell ghosts (data not shown). In addition, the a/: ratio of globin chain synthesis was normal (1.04) for mouse SAD-l and identical to that of non-SAD littermates. The mechanism by which overall a and 3 chain expression is balanced, despite the expression of the a and ( human transgenes at different levels, remains to be determined. Hematology and erythrocyte morphology of SAD mice Adult mice in the SAD-I line displayed normal hematological indices with regard to hemoglobin, hematocrit, red cell
counts and reticulocytes (Table EII). However, the mean cell hemoglobin concentration (MCHC) was increased, suggesting cell dehydration. This hypothesis was confirmed by the density distribution curve of red blood cells (Figure 5) which shows a shift of the curve of SAD-1 erythrocytes towards high density. When fully oxygenated, a small percentage of red cells (0.5-2.5%) was elongated (Figure 6a) resembling irreversibly sickled human cells (ISCs). Upon deoxygenation with nitrogen, almost all cells became irregular in shape, sickled or with sharp elongations (Figure 6b). These changes were reversible upon reoxygenation, as is the deformation of most human sickled cells. Therefore, even the low percentage of the SAD hemoglobin (19%) in these mice is sufficient to cause sickle cell features. The spleens of adult SAD-1 mice were increased in size (0.15-0.26 g) in comparison to that of non-SAD transgenic littermates ( < 0. 1 g).
3159
M.Trudel et al. 0.1
SAD
100
a)
E
-
c
0.
0
n
0
w
50
C.)
z
n
.Te
m
01
C/)
0
L
'
1.07
1.08
1.09
1.10
1.11
1.12
Phthalate density
0 I1
5
Fig. 5. Density distribution curves of red blood cells of mice. 1, nontransgenic homozygous Hbb1s5; 2, non-transgenic, heterozygous Hbbs/Hbbthal (3-single/[-thalassemic); 3, SAD-1 transgenic, homozygous HbbS/S; 4, ,B-thal/SAD-I transgenic, Hbbs/Hbbthal.
a ..
.. .,
;f
;
......
*) ~~~~~~~~~~~~~~~~~~.1~~~~~~~~~~~~~~~
b
Fig. 6. Normarski optics microscopy of (a) oxygenated or (b) deoxygenated red blood cells of founder mouse SAD-1.
Polymerization and oxygen affinity To determine the effects of the Antilles (/32311e) and D Punjab (f32'Gln) mutations on the polymerization of Hb SAD, we compared the delay time of polymer formation upon deoxygenation of Hb SAD with that of Hb S, after a temperature jump. The delay time of polymerization for the hemolysate from mouse SAD-I was 8 min, while purified Hb S added to mouse hemolysate to the same proportion
3160
10 -
15 delay time
20
25
30
Minutes
35
40
-L
45
50
5
10
15
Hours
Fig. 7. Delay time of polymerization upon temperature jump of the deoxygenated hemolysate from mouse SAD-I founder (solid line) or normal mouse hemolysate containing 19% Hb S (discontinuous line).
(19% Hb S) and at the same concentration of total Hb (1.5 mg/ml), failed to polymerize after 16 h (Figure 7). Studies of purified and deoxygenated hemoglobins confirmed the highly decreased delay time of polymerization of deoxy-Hb SAD in comparison with that of deoxyHb S (Figure 8a). The probability factor of nucleation calculated according to Adachi et al. (Adachi et al., 1987) was much greater for Hb SAD (7.01) than for Hb S (1.00) as shown in Table IV, indicating that the early phase of the polymerization process, i.e. the nucleation step, of Hb SAD polymerization, was very fast. Equimolar mixtures of Hb SAD with mouse Hb single, hybrid U2human32single or Hb A displayed a probability factor of nucleation higher than that obtained with pure Hb S. All curves shown in Figure 8 are parallel, indicating a similar nucleation process of polymerization for Hb SAD, Hb S and their mixtures with Hb A, mouse Hb single or the hybrid a2humanfl2single The results shown in Figure 8b indicate that the hemolysate from SAD-I mice exhibited a polymerization process lower than that of pure Hb S but close to that observed for hemolysate from homozygous S blood cells. The oxygen affinity of transgenic Hb SAD containing erythrocytes was slightly lower (p50 = 44.6 mm Hg) than that of non-transgenic Hbbs/s erythrocytes (p50 = 39.1 mm Hg) (P < 0.05) (Table V). In contrast, purified Hb SAD at pH 7.02 had an oxygen affinity (p50 = 5.52 mm Hg) higher than mouse Hb single (p50 = 8.73 mm Hg) but lower than human Hb A or S (p50 = 4.26 mm Hg). This apparent paradox between the higher oxygen affinity of Hb SAD than mouse Hb and the lower oxygen affinity of SAD- 1 erythrocytes in comparison with that of normal mouse cells can be explained by the low oxygen affinity of the hybrid o22human 02Single (p5O = 12.30 mm Hg), present in high proportion (39.5%) in SAD-1 erythrocytes. The alkaline Bohr effect and the effect of 2,3-DPG (2 mM) on the oxygen dissociation curve were identical for purified Hb SAD, A or S. The concentration of 2,3-DPG was similar in SAD-l (mean: 28.6 ytmol/g Hb) and normal mouse erythrocytes (27.5 ytmol/g Hb) (Table V). These results indicate that Hb SAD had an intrinsically lower oxygen affinity than Hb S
Transgenic mouse model for sickle cell disease 0.5
a
-3.5 -2.5
-2.0
-1.5
-1.0
-0.5
b -0.5 E -1.0
E
-1.5
*0 o-2.0
o -2.5
-2.5
-2.0
-1.5
-1.0
-0.5
log C t (g/dl) Fig. 8. Relationship between the reciprocal delay time (log dt- ) and the hemoglobin concentration (log Ct) for purified Hb SAD, Hb S and mixtures with human Hb A, mouse Hb single and human/mouse hybrid I (a2humanl2single). a: +, SAD;), hemolysate homozygote S. Table IV. Probability factor for nucleation Hb Hb Hb Hb Hb Hb Hb Hb Hb
SAD SAD + Hb single SAD + Hb minor SAD + Hybrid I SAD + Hb A S S + Hb A S + Hb single S + Hybrid I
7.01 2.49 2.49 2.03 1.60 1.00 0.49 0.39 0.34
Hemolysate fl-thal/SAD-1 Hemolysate SS Hemolysate SAD- 1
0.70 0.55 0.44
and that SAD-1 erythrocytes exhibited a slightly decreased oxygen affinity, by 14%, due to the presence of the human mouse hybrid (a2human l2single) or to polymerization of Hb SAD as observed in human sickle cells for Hb S. An increased level of Hb SAD in,3-thal/SAD-1 mice To increase the proportion of Hb SAD and the severity of the observed phenotype, we attempted to increase the level of Hb SAD genetically by several means. Intercrosses among SAD-I hemizygotes have so far failed to yield any progeny
with the increased level of Hb SAD expected for SAD-1 homozygotes. Attempts to generate compound hemizygotes with other SAD transgenic lines are in progress. As an
alternative approach, we crossed the SAD- I mice with mice homozygous for a 0-thalassemia mutation [Hbbd3(th)], a deletion of the 13 major gene, which leaves the /3 minor globin gene intact (Skow et al., 1983). The SAD-1 positive progeny, which were heterozygous 3-thalassemic (animals of this genotype will be termed ,3thal/SAD-1), produced less mouse 1-globin and thus accumulated increased proportions of 13SAD_globin chains (27% of total 13 chains; Table I). and of Hb SAD (26% of total Hb; Table II), while the level of human a chains was essentially unchanged (53%; Table I). The delay time of polymerization of the 3-thal/SAD- 1 hemolysate (1.5 mg/ml) was considerably shortened in comparison with the SAD-I hemolysate (25 s versus 8 min) indicating that polymerization is greatly favored by this small increment in the proportion of Hb SAD. The apparent probability factor of nucleation for the ,B-thal/SAD-I hemolysate was 0.70, close to that of pure Hb S (1.0) and higher than that of homozygous Hb S state (0.55) in which Hb S makes up 90%, Hb F 6% and Hb A2 4%. Equimolar mixtures of Hb SAD and mouse minor hemoglobin or Hb single had identical delay times. The 02 affinity of the ,B-thal/SAD-1 erythrocytes (p50 = 44.8 mm Hg) and the 2,3-DPG value (26.5 ,tmol/g Hb) were similar to those of SAD-1 (Table V). When (-thal/SAD-1 mice were mated with homozygous ,B-thalassemic mice in an attempt to increase the level of Hb SAD further, none of the surviving offspring were SAD-1 transgenic and homozygous for the 1-thalassemia. Only one such animal was found among the neonatal progeny of these crosses; it was very small and severely anemic (hematocrit decreased by 63%). This genotype appears to be lethal. Phenotype of adult,3-thal/SAD-1 mice Erythrocytes of,-thal/SAD-1 mice were highly abnormal and heterogenous with regard to size, shape and hemoglobin content (Figure 9). A significant proportion (5-15%) of small, elongated cells was present, resembling the irreversibly sickled cells found in the human disease. The 13-thal/SAD-1 erythrocytes were also highly heterogenous with regard to cell density, a fraction of cells displaying abnormally high density as shown in Figure 5. Most cells became sickled upon deoxygenation in vitro (data not
shown). All adult 1-thal/SAD-1 mice had larger spleens (0.30-0.58 g) than their non-transgenic 13-thal littermates (
