U.S. patent application number 15/161603 was filed with the patent office on 2016-12-22 for methods and compositions for the treatment of beta-thalassemia.
The applicant listed for this patent is Assistance Publique - Hopitaux de Paris, CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE (CNRS), Fondation Imagine, Institut National de la Sante et de la Recherche Medicale (INSERM), Universite Paris Descartes - Paris V. Invention is credited to Jean-Benoit ARLET, Genevieve COURTOIS, Olivier HERMINE, Jean-Antoine RIBEIL.
Application Number | 20160368975 15/161603 |
Document ID | / |
Family ID | 53173910 |
Filed Date | 2016-12-22 |
United States Patent
Application |
20160368975 |
Kind Code |
A1 |
HERMINE; Olivier ; et
al. |
December 22, 2016 |
METHODS AND COMPOSITIONS FOR THE TREATMENT OF BETA-THALASSEMIA
Abstract
Methods and compositions for the treatment of .beta.-thalassemia
are provided. Methods and compositions restore or increase
erythrocyte maturation in individuals afflicted with .beta.-TM by
preventing proteolysis of GATA-1 protein. Screening methods for
identifying agents which bind heat shock protein 70 (HSP-70) and
inhibit HSP-70 .alpha.-globin binding, but which allow GATA-1
protein-HSP-1 binding in a manner that prevents GATA-1
proteolysis.
Inventors: |
HERMINE; Olivier; (Paris,
FR) ; COURTOIS; Genevieve; (Paris, FR) ;
ARLET; Jean-Benoit; (Paris, FR) ; RIBEIL;
Jean-Antoine; (Paris, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Institut National de la Sante et de la Recherche Medicale
(INSERM)
CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE (CNRS)
Universite Paris Descartes - Paris V
Assistance Publique - Hopitaux de Paris
Fondation Imagine |
Paris
Paris
Paris
Paris
Paris |
|
FR
FR
FR
FR
FR |
|
|
Family ID: |
53173910 |
Appl. No.: |
15/161603 |
Filed: |
May 23, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14547208 |
Nov 19, 2014 |
9377471 |
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15161603 |
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61906556 |
Nov 20, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/7088 20130101;
G01N 2333/805 20130101; G01N 2800/22 20130101; C07K 16/18 20130101;
G01N 2500/02 20130101; G01N 33/5094 20130101; A61K 31/70 20130101;
A61K 38/02 20130101; G01N 33/80 20130101; G01N 33/6893
20130101 |
International
Class: |
C07K 16/18 20060101
C07K016/18; G01N 33/68 20060101 G01N033/68; A61K 31/70 20060101
A61K031/70; G01N 33/50 20060101 G01N033/50; A61K 38/02 20060101
A61K038/02; A61K 31/7088 20060101 A61K031/7088 |
Claims
1. A method of restoring or increasing erythrocyte maturation in a
subject suffering from .beta.-thalassemia major (.beta.-TM) by
preventing proteolytic inactivation of GATA-1.
2. The method of claim 1, wherein said preventing is achieved by
administering to said subject a compound that inhibits binding of
.alpha. globin to Hsp70.
3. The method of claim 2, wherein said compound that inhibits
binding of .alpha. globin to Hsp70 is a small molecule that binds
to the .alpha. chain binding pocket of Hsp70.
4-5. (canceled)
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The invention generally relates to the treatment of
.beta.-thalassemia (.beta.-TM). In particular, the invention
provides methods and compositions for restoring or increasing
erythrocyte maturation in individuals afflicted with .beta.-TM by
preventing proteolysis of GATA-1 protein.
BACKGROUND OF THE INVENTION
[0003] Adult mammalian Hg is a multimeric protein that includes two
.alpha. and two .beta. globin chains which together form the
(.alpha./.beta.).sub.2 tetrameric hemoglobin (Hb) molecule.
Beta-thalassemias are a group of inherited blood disorders caused
by a quantitative defect in the synthesis of the .beta. chains of
hemoglobin. In individuals with this disorder, the synthesis of
.beta.-globin chains is reduced or absent. Three main forms of the
disease have been described: .beta.-thalassemia major (.beta.-TM or
.beta..sup.0-TM) in which no .beta. chain is produced, and
.beta.-thalassemia intermedia and .beta.-thalassemia minor, in
which .beta. chain is produced but in lower than normal amounts.
These conditions cause variable phenotypes ranging from severe
anemia to clinically asymptomatic individuals. Individuals with
.beta.-TM usually present within the first two years of life with
severe anemia, poor growth, and skeletal abnormalities during
infancy. Affected children will require regular lifelong blood
transfusions. .beta.-thalassemia intermedia is less severe than
.beta.-thalassemia major and may require episodic blood
transfusions. Transfusion-dependent patients will develop iron
overload and require chelation therapy to remove the excess
iron.
[0004] It is known that defective .beta.-globin chain synthesis
leads to the accumulation of free .alpha.-globin chains that form
toxic aggregates.sup.1,2. However, despite extensive knowledge on
the molecular defects causing .beta.-TM, little is known about the
mechanisms responsible for ineffective erythropoiesis (IE) in
.beta.-TM patients. In such individuals, erythropoiesis does not
result in the production of mature erythrocytes but instead is
characterized by accelerated erythroid differentiation, maturation
arrest and apoptosis at the polychromatophilic stage.sup.3-5. This
lack of understanding of the mechanism has prevented the
development of effective strategies for treating the disease.
[0005] Humans are capable of producing three types of Hb chains:
.alpha., .beta. and .gamma.. The main oxygen transport protein in
the human fetus during the last seven months of development in the
uterus and in the newborn until roughly 6 months of age is
(.alpha./.gamma.).sub.2Hb. As the nomenclature indicates, this type
of Hb tetramer contains two .alpha. globin subunits and two .gamma.
globin subunits. After about 6 months of age, humans shift from
production of (.alpha./.gamma.).sub.2 Hb toward production of
(.alpha./.beta.), Hb, and in non-.beta.-TM adult humans,
(.alpha./.gamma.).sub.2 Hb represents only about 1% or less of
hemoglobin. However, the amount of (.alpha./.gamma.).sub.2 Hb is
increased in individuals with .beta.-TM. Functionally, fetal
hemoglobin differs from adult hemoglobin in that it is able to bind
oxygen with greater affinity than the adult form, giving the
developing fetus better access to oxygen from the mother's
bloodstream. Thus, the production of (.alpha./.gamma.).sub.2 by
.beta.-TM cells could, in theory, be a boon for those suffering
from thalassemias. However, since .beta.-TM erythrocytes generally
fail to mature, the presence of the alternate form of Hb is not
especially useful to patients with this disease.
SUMMARY OF THE INVENTION
[0006] Individuals afflicted with the genetic disease .beta.-TM do
not produce hemoglobin .beta. chains. Thus, mature
(.alpha./.beta.).sub.2 Hb cannot be formed and the .alpha. globin
chains that are produced accumulate in the cytoplasm of immature
erythrocytes (erythroblasts). Up until the present invention, the
relationship between .alpha. chain accumulation and the etiology of
.beta.-TM was unknown. The lack of knowledge greatly hampered the
development of effective treatment regimes for .beta.-TM
patients.
[0007] The present inventors have elucidated the consequences of
.alpha. chain cytoplasmic accumulation and the cascade of failed
reactions that result therefrom which ultimately cause .beta.-TM
symptoms such as anemia. The discovery is based on the further
clarification of the roles of the chaperone protein Hsp70 and the
erythrocyte maturation protein GATA-1. The inventors have
discovered that Hsp70 has important functions in both the cytoplasm
and the nucleus of erythroblasts. A primary function of Hsp70 in
the nucleus is binding to the GATA-1 protein and preventing its
cleavage and proteolytic degradation (by the protease caspase-1).
GATA-1 thus prevents inactivation of GATA-1 and preserves its
function as a key factor in erythrocyte maturation. A secondary
function of Hsp70 is binding to .alpha. globin in the cytoplasm and
ensuring that the protein chains are properly folded and can form
tetrameric (.alpha./.beta.).sub.2 Hb. Ordinarily, there is
sufficient Hsp70 available in the cell to carry out both of these
functions. However, in .beta.-TM cells, the Hsp70 is monopolized by
the excess free .alpha. chains which accumulate in the cytoplasm.
Thus, a disproportionate amount of the Hsp70 is sequestered in the
cytoplasm, and there is not sufficient Hsp70 available for binding
and protecting GATA-1 in the nucleus. Unprotected GATA-1 is
proteolytically cleaved and inactivated, and proper erythrocyte
maturation does not occur. Rather, the absence of active GATA-1
results in maturation arrest and apoptosis of immature erythrocytes
at the polychromatophilic stage. This sequence of events is thus
initially triggered by a lack of hemoglobin .beta. chains and
ultimately results in low (or no) erythrocyte production, causing
anemia.
[0008] The present invention provides methods and pharmaceutical
compositions designed to intervene in this defective process and to
promote or restore erythrocyte maturation in individuals suffering
from .beta.-TM. It is noted that because .beta. globin is not
formed in .beta.-TM erythrocytes, the type of erythrocytes that are
produced in individuals treated with the methods and compositions
described herein contain (.alpha./.gamma.).sub.2 Hb, and the
invention provides methods and compositions for increasing the
production of (.alpha./.gamma.).sub.2 Hb erythrocytes in .beta.-TM
cells and .beta.-TM individuals. The methods involve maintaining
the activity of GATA-1 by preventing its proteolysis, e.g. by
preventing sequestration of Hsp70 in the cytoplasm.
[0009] Accordingly, it is an object of this invention to provide
methods of restoring or increasing erythrocyte maturation in a
subject suffering from .beta.-thalassemia major (.beta.-TM) by
preventing proteolytic inactivation of GATA-1. In some embodiments,
preventing is achieved by administering to the subject a compound
that inhibits binding of .alpha. globin to Hsp70. In an exemplary
aspect, the compound that inhibits binding of .alpha. globin to
Hsp70 is a small molecule that binds to the .alpha. chain binding
pocket of Hsp70. The invention also includes screening methods to
identify such agents.
[0010] Other features and advantages of the present invention will
be set forth in the description of invention that follows, and in
part will be apparent from the description or may be learned by
practice of the invention. The invention will be realized and
attained by the compositions and methods particularly pointed out
in the written description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIGS. 1A-B. Hsp70 and GATA-1 expression in fresh bone marrow
of .beta.-thalassemia major patients (.beta.-TM). (A)
Representative confocal microscopy analysis of .alpha.-globin,
Hsp70 and GATA-1 expression in fresh bone marrow (BM) from 3 adult
.beta.-TM patients and 3 healthy donors. Scale bar, 5 .mu.m. Gray
arrows indicate Hsp70 cytoplasmic sequestration into mature
.beta.-TM cell, and the white arrow shows immature cell. (B) Hsp70
cytoplasmic/nuclear mean fluorescence intensity (MFI) ratio (top)
and GATA-1 nuclear MFI (bottom) of .beta.-TM and controls. Bars
indicate the median (IQR). p values were calculated using the
Mann-Whitney U-test. **p<0.01.
[0012] FIGS. 2A-D. The characteristics of ineffective
erythropoiesis in .beta.-TM and the kinetics of Hsp70 and GATA-1
expression during in vitro erythroid differentiation. CD36.sup.+
cells derived from CD34.sup.+ adult .beta.-TM peripheral blood
cells (n=16 independent experiments, 7 patients) or healthy
peripheral blood cells (controls, n=8) were cultured with a
two-phase amplification liquid culture system, as described in
Methods section. Day 0 of differentiation is the start of
CD36.sup.+ cell culture and represents the differentiation phase of
the culture system. (A) May-Grunwald-Giemsa staining at day 8. Left
panel. A representative morphological analysis (.times.40) of
erythroid differentiation is shown. Solid arrows indicate
reticulocytes, dashed arrows represent acidophilic cells, and
dotted arrows represent polychromatophilics cells. Upper right
panel: graph represents proportion of each erythroid stage in
.beta.-TM or control-derived cells. Proerythtroblast
(ProE)+basophilic (baso), polychromatophilics (Poly) cells and very
mature cells-acidophilic cells (Acido)+reticulocytes (Retic) are
expressed as a percentage of the total erythroid cells. Bars
indicate the mean .+-.SEM for 14 independent experiments. p values
were calculated using t-tests. **p<0.01; ***p<0.001. Lower
right panel: graph represents the terminal maturation index of
.beta.-TM and control-derived cells, as calculated by (acidophilic
cells+reticulocytes per slide).times.100/polychromatophilic cells
per slide. The box and whiskers indicate the median extremes.
**p<0.01. (B) Upper: apoptosis curves assessed daily by flow
cytometry analysis of Annexin-V (AV) staining (mean
percentage.+-.SEM of AV positive, Propidium Iodide (PI) negative
cells). Lower: differentiation curves assessed by benzidine
staining. The results are the means of 17 independent experiments
(mean percentage.+-.SEM of positive cells). (C) Left:
representative confocal microscopy analysis of Hsp70, GATA-1 and
.alpha.-globin at day 8.The merged panel shows Hsp70 and
.alpha.-globin co-localisation in the cytoplasm. Bar graphs at the
right show: upper, Hsp70 cytoplasmic/nuclear MFI ratio and; lower,
nuclear GATA-1 MFI of .beta.-TM and control-derived cells. Bars
indicate the median (IQR) of 15 independent experiments. p values
15 were calculated using the Mann-Whitney U-test. ***p<0.001.
(D) Kinetics of Hsp70 and GATA-1 expression in vitro. Hsp70
cytoplasmic/nuclear MFI ratio and GATA-1 nuclear MFI were analysed
at days 3, 5 and 8 of culture: right, 3 .beta.-TM patients; and
left, 3 controls. Bars indicate the median (IQR). p values were
calculated using the Maim-Whitney U-test. *p<0.05, **p<0.01;
***p<0.001.
[0013] FIG. 3A-C: Hsp70 and .alpha.-globin interaction. (A)
DUOLINK.RTM. in situ proximity ligation assay (PLA). Interactions
were analysed at day 8 in CD36.sup.+ .beta.-TM or control-derived
cell cultures by PLA, according to the manufacturer's instructions
using Hsp70 (1:200) and .alpha.-globin (1:200) antibodies. A
FITC-conjugated Annexin-V antibody (1:200) was added at the end of
the assay. The spots indicate close proximity (<40 nm) between
cellular bound antibodies. A representative experiment is shown
(n=3). Nuclei are stained with DAPI, and Annexin-V staining is also
shown. Scale bar, 5 .mu.m. (B) Yeast two-hybrid assay detected, in
upper panel, a direct interaction of .alpha.- and .beta.-globin
chains with Hsp70 (diploid yeast cells), but not, in lower panel,
with the Hsp70 deletion mutants, Hsp70-Nucleotide Binding Domain
(NBD) or Hsp70-Substrate Binding Domain (SBD). Each section
contains diploid yeast cells resulting from an independent yeast
mating experiment with the corresponding bait protein and prey
protein. The empty vectors, pGADT7 and pGBKT7, are used as negative
controls. SV40 and p53 coding sequences are used as positive
controls. (C) Structural modelling of Hsp70 and the
Hsp70.sup.1-701/.alpha.-globin complex. In the upper panel, from
left to right, are the molecular surface and cartoon representation
of the generated models of Hsp70, the Hsp70/.alpha.-globin complex
and their superimposition. The different Hsp70 domains are labelled
and highlighted. The lid, the SBD, the NBD, the hinge region
between the NBD and the SBD, and .alpha.-globin are shown. The
superimposed models are presented in transparent (Hsp70) and
contrast (Hsp70/.alpha.-globin complex) modes. The lid and SBD
movements are shown by arrows. The haem group and ADP are shown by
space-filling models. In the lower panel, from left to right, are
the electrostatic potential of the molecular surfaces of the
Hsp70.sup.1-701/.alpha.-globin complex, where Hsp70 and
.alpha.-globin are shown. The .alpha.-globin in the
Hsp70.sup.1-701/.alpha.-globin complex is shown for clarity.
.alpha.-globin is rotated 180.degree. respectively to its position
in the Hsp70.sup.1-701/.alpha.-globin complex, and its image is
flipped horizontally to show the charge distribution on the surface
interacting with Hsp70.
[0014] FIG. 4A-D. The transduction of a nuclear Hsp70 mutant
(Hsp70-S400A) or a caspase resistant GATA-1 mutant (.mu.GATA-1)
rescues cell terminal maturation and cell survival in
.beta.-thalassemia major (.beta.-TM). .beta.-TM CD34+ cells were
transduced with a nuclear-targeted Hsp70 lentiviral mutant
(Hsp70-S400A), a retroviral mutant of GATA-1 uncleavable by
activated caspase-3 (.mu.GATA-1), and their appropriate empty
vectors as controls and were cultured as described in the methods.
CD36.sup.+/GFP.sup.+ cells were then purified, cultured, and
differentiated in Epo-containing medium. All data presented here
were analysed at day 7 of the CD36.sup.+ culture. (A) A confocal
microscopy image of Hsp70 and GATA-1 is shown in the left panel.
The image is representative of three experiments. Graphs in the
right panel represent the nuclear mean fluorescence intensity (MFI)
of Hsp70 and GATA-1 (.+-.95% CI) in transduced and control cells
(top panel: Hsp70-S400A transduced cells; bottom panel: .mu.GATA-1
transducted cells) (20 cells were analysed in three different
fields/slide, n=3 patients). Scale bar, 5 .mu.m. P values were
calculated using Mann-Whitney U-test. ***p<0.001. (B)
May-Grunwald-Giemsa (MGG) staining at day 7. Left panel: Graphs
represent proportion of each erythroid stage in transduced and
control .beta.-TM cells (upper panel, Hsp70-5400A; lower panel,
.mu.GATA-1). Proeryhtroblast (ProE)+basophilic (baso),
polychromatophilics (Poly) cells and very mature cells-acidophilic
cells (Acido)+reticulocytes (Retic) are expressed as a percentage
of the total erythroid cells. Bars indicate the mean.+-.SEM for 3
independent experiments. p values were calculated using t-tests.
Right panel: one representative morphological analysis of the
erythroid differentiation for each transduction (n=3) is shown
(.times.100). Solid arrows indicate reticulocytes, dashed arrows
indicate acidophilic cells, and dotted arrows indicate
polychromatophilic cells. (C) Apoptosis in the GFP.sup.+/propidium
iodide-cell population was assessed by Annexin V binding by flow
cytometry. A representative experiment of each transduction is
shown (n=3 patients/transduction). (D) The percentage of HbFhigh
cells was assessed by flow cytometry on mature cells (GFP.sup.+ and
low forward light scatter-FSC). A representative experiment of each
transduction is shown (n=3 patients/infection).
[0015] FIG. 5A and B. The kinetics of differentiation of .beta.-TM
and control progenitors. CD36 cells derived from
CD34.sup.+.beta.-TM peripheral blood cells (n=16 independent
experiments, 7 patients) or healthy peripheral blood cells
(controls, n=8) were cultured with a two-phase amplification liquid
culture system, as described. Day 0 of differentiation is the 18
start of CD36.sup.+ cell culture, which represents the
differentiation phase of the culture system. Differentiation
staining was assessed daily by flow cytometry. (A) Upper panel:
differentiation curves assessed by Glycophorin A (GpA) mean
fluorescence intensity (MFI). Lower panel: differentiation curves
assessed by c-KIT/CD 117 staining mean fluorescence intensity
(MFI). The results are means (.+-.SEM). p values compare .beta.-TM
and control-derived cells differentiated at day 8 and were
calculated using t-tests. *p<0.05, ***p<0.001. (B) One
representative flow cytometry image of the stage of erythroid
differentiation at day 8 of culture is shown for .beta.-TM (upper
panel) and control-derived cells (lower panel).
GpA.sup.+/CD117.sup.- cells represent mature erythroid cells.
[0016] FIG. 6. The lentiviral transduction of .beta.-TM CD34.sup.+
cells with the .beta.-globin gene rescues Hsp70 nuclear
localisation and GATA-1 protection. .beta.-TM CD34+ cells derived
from peripheral blood cells were infected with a lentivirus
encoding the .beta.-globin gene or a control virus. They were then
cultured with a two-phase amplification liquid culture system, as
described. Confocal microscopy analysis at day 8 of the CD36.sup.+
cell cultures shows Hsp70 nuclear localisation and GATA-1
expression in .beta.-globin gene transduced cells (top) and control
transduced cells (bottom). Scale bar, 10 .mu.m.
DETAILED DESCRIPTION
[0017] Methods and compositions for increasing erythrocyte
maturation in individuals suffering from .beta. thalassemia are
provided. The methods involve preventing the otherwise untoward
effects of Hsp70 sequestration in the cytoplasm of maturing
erythrocytes. The invention takes advantage of the new
understanding of the mechanism behind the .beta.-TM disease process
as described herein, in order to provide methods and compositions
for increasing the maturation of erythrocytes containing HbF in
.beta.-TM subjects.
[0018] The inventors have found that symptoms of .beta.-TM can be
alleviated by compositions and methods which slow or prevent (e.g.
interfere with, impede, stop, decrease, etc.) cleavage and
inactivation of GATA-1, or conversely, which preserve or promote
the erythrocyte maturation activity of GATA-1. Several avenues of
doing so are provided.
[0019] In one aspect, GATA-1 inactivation is prevented by
disrupting formation of the Hsp70/.alpha. globin complex in the
cytoplasm, thereby increasing the concentration of Hsp70 in the
nucleus of .beta.-TM cells, increasing nuclear localization of
Hsp70, reducing maturation arrest and increasing the number of HbF
cells. Using both wet and in silico chemistries, the Hsp70 .alpha.
globin binding site has been elucidated, and it has been
surprisingly discovered that the binding site may be blocked or
altered in ways that prevent .alpha. globin binding but that do not
impair the ability of Hsp70 to fulfill other functions in the cell,
such as protecting the GATA-1 protein from proteolysis.
[0020] Thus, in some aspects, the invention provides compositions
and methods for blocking the .alpha. Hb binding site of Hsp70,
without interfering significantly with the ability of Hsp70 to
enter or access the nucleus, bind GATA-1 and protect it from
proteolytic degradation, e.g. by caspase-1. Blockage of the a Hb
binding site may be carried out by contacting Hsp70 in .beta.-TM
cells with a ligand that binds with relatively high affinity to the
.alpha. globin binding site, the affinity usually being at least
equal to or greater than that of .alpha. globin i.e. the Kd of the
ligand is approximately equal to or lower than that of .alpha.
globin. Binding of the ligand may be competitive so that .alpha.
globin is outcompeted and the equilibrium distribution of Hsp70
between the cytoplasm and nucleus is shifted, and so that at least
a portion of the Hsp70 that is present in the cell is not complexed
to .alpha. globin in the cytoplasm. Alternatively, ligand binding
may be irreversible so that Hsp70 that binds the ligand cannot bind
.alpha. globin, but can still access and bind GATA-1 protein in the
nucleus.
[0021] Ligands which may be used in the practice of the invention
include but are not limited to various so-called "small molecules".
A "small molecule" is generally a low molecular weight (<900
Daltons organic compound with a size on the order of 10.sup.-9 m.
In general, the upper molecular weight limit for a small molecule
is approximately 900 Daltons which allows for the possibility to
rapidly diffuse across cell membranes and reach intracellular sites
of action. In addition, this molecular weight cutoff is a favorable
(although insufficient) condition for oral bioavailability. In some
aspects, lower molecular weight compounds may be used, e.g.
compounds of about 100, 200, 300, 400, 500, 600, 700 or 800 Daltons
or less. The compound may, but does not always, obey "Lipinski's
rule", which states that, in general, an orally active drug has no
more than one violation of the following criteria: [0022] 1. Not
more than 5 hydrogen bond donors (nitrogen or oxygen atoms with one
or more hydrogen atoms) [0023] 2. Not more than 10 hydrogen bond
acceptors (nitrogen or oxygen atoms) [0024] 3. A molecular mass
less than 500 daltons [0025] 4. An octanol-water partition
coefficient [5] log P not greater than 5. Such "small molecule"
drugs (active agents) are generally designed to interact with amino
acid residues and/or with functional groups thereof, located in the
.alpha. chain binding pocket of Hsp70. By the ".alpha. chain
binding pocket" of Hsp70, we mean the highly electronegative cavity
formed by the N-terminal nucleotide binding domain (NBD), the
C-terminal substrate binding-domain (SBD) and the lid (a C-terminal
10 kDa helical subdomain of the SBD) of Hsp70. Suitable small
molecules may be, for example, small proteins or peptides, nucleic
acids, carbohydrates, antibodies, and suitable fragment thereof.
Alternatively, the small molecules may be a chemically synthesized
organic or inorganic molecule that is purposefully designed to fit
the .alpha. chain binding pocket of Hsp70.
[0026] In further embodiments, the invention provides methods
identifying compounds or agents (e.g. small molecules) that inhibit
binding of .alpha. globin to Hsp70, e.g. methods of designing,
generating and screening groups of agents in order to identify
those which bind to, in, at or near the .alpha. chain binding
pocket of Hsp70 in a manner that prevents Hsp70 from binding to
.alpha. globin, or that lessens the affinity of Hsp70 for .alpha.
globin. Those of skill in the art are familiar with techniques for
so-called rational drug design, e.g. designing, synthesizing, and
screening libraries of compounds that may have a desired activity,
and then analyzing results obtained in order to select suitable
ligands for use. Typically, information concerning the binding
properties of the molecule to which the agent will bind is used to
design, e.g. in silico, one or more suitable molecular "skeletons"
or "frameworks" or "pharmacores" a possessing minimal properties
required to bind to the target designed to "fit" a binding pocket,
or a site adjacent to a binding pocket, or an allosteric site
distant from the binding pocket but which communicates structural
changes across the molecule to the binding pocket when an agent is
bound thereto, etc. Various atoms or atomic groups are then added,
in silico, to the basic framework in a systematic fashion, e.g. by
first adding first a H atom, then a methyl group, then an ethyl
group, etc. to increase the length of a variable group at one or
more positions by one CH.sub.2 group at a time; or a battery of
positively or negatively charged groups may be placed at one or
more positions, etc. Once candidate compounds or families thereof
are designed in silico, various computer implemented programs can
be used to identify the most likely candidates or families of
molecular candidates, e.g. those which appear to possess
statistically suitable binding affinities. In the present
invention, such ligands must also appear to not prevent the ability
of Hsp70 to bind to and protect GATA-1 from caspase-1, or at least
to still allow sufficient binding to Hsp70 to GATA-1 to provide a
suitable positive outcome when administered to a patient by
lessening disease symptoms.
[0027] Alternatively, or in addition to (e.g. before, after or
during) the process of drug design, high throughput screening (HTS)
data may be used to rapidly identify active compounds of interest
that bind to HSP-70. The results of HTS experiments may provide
starting points for drug design, and/or confirmation of previous in
silico drug design results, and/or may provide additional
understanding of the interaction of HSP-70 and the candidate
ligands.
[0028] Once suitable candidate ligands or families of candidate
ligands are identified, synthesis of larger quantities of compounds
of interest is accomplished by methods known in the art, e.g. by a
suitable chemical synthetic routes. Such molecules are then tested
(e.g. in vitro, in vivo using animal models, and during clinical
trials) by methods known to those of skill in the art, e.g. further
HTS using HSP-70 as the target, or further testing to elucidate
specific attributes of the compounds (binding affinity,
bioavailability, toxicity, stability, etc.).
[0029] The invention thus also provides method of screening
candidate compounds in order to select compounds which inhibit
binding of .alpha. globin to Hsp70 but which do not inhibit binding
of Hsp70 to GATA-1. The methods may comprise, for example steps
such as: i) providing a plurality of candidate compounds which may
inhibit binding of .alpha. globin to Hsp70; ii) exposing Hsp70 to
said plurality of candidate compounds in the presence of a globin
and under conditions which allow .alpha. globin to bind to Hsp-70;
iii) identifying Hsp70-compound complexes which do not contain
bound .alpha. globin; iv) exposing Hsp70-compound complexes
identified in said identifying step iii) to GATA-1 under conditions
that permit Hsp70 to bind to GATA-1; and v) identifying
Hsp70-compound-GATA-1 complexes formed in said exposing step iv);
and vi) selecting compounds identified in said identifying step v)
as compounds which inhibit binding of .alpha. globin to Hsp 70 but
which do not inhibit binding of Hsp70 to GATA-1. The method may
also comprise a step of exposing Hsp70-compound-GATA-1 complexes to
caspase-1, and identifying Hsp70-compound-GATA-1 complexes in which
GATA-1 is not proteolytically cleaved. In some aspects, the first
exposing step may be carried out in two steps such as first
exposing HSP70 to candidate compounds and identifying complexes
formed between HSP70 and compounds, and then exposing the
HSP70-compound complexes to .alpha. globin and selecting
HSP70-compound complexes that do not bind .alpha. globin, or from
which the compound is not displaced by .alpha. globin. Whatever the
order of the screening steps, the compounds that are selected for
clinical use (positive "hits") must interfere with or prevent or
inhibit or decrease or compete with the ability of HSP70 to bind
.alpha. globin, and yet not interfere with or prevent or inhibit or
decrease or compete with the ability of HSP70 to bind to
Hsp70-compound-GATA-1, and also confer protection from proteolysis
of Hsp70-compound- GATA-1 by caspase-1. Those of skill in the art
are familiar with various statistical tools that can be used to
assess the significance of such data, compared to that obtained
with suitable controls.
[0030] Steps of the screening method (e.g. identifying, selecting)
may be carried out by attaching to or incorporating into one or
more of the substances being tested (e.g. HSP-70 and/or .alpha.
globin and/or the compound being tested and/or GATA-1 protein) a
detectable label including but not limited to: a radioactive
moiety; a bioluminescent, chemiluminescent or fluorescent label; an
affinity label or tag; etc. Further, one or more of the substances
may be immobilized on a substrate such as a plate or bead and the
screening assays may include suitable steps of washing to remove
unreacted substances, separation via size exclusion or affinity
chromatography or by filtering, centrifugation, etc.
Characterization of identified complexes of interest or
confirmation of the identity may be carried out by known
techniques, e.g. sequencing, reaction with antibodies, mass spec,
etc.
[0031] The steps of the screening assays are carried out using
suitable concentrations of each reactant. Viable candidates for
further testing and for clinical use will typically have binding
affinities (Kd values) for HSP-70 in the range of at least about
25%, and usually 30, 35, 40, 45, 50, 55, 60, 65, 70,. 75, 80, 85,
90, 95% or more of that of .alpha. globin, compared to a suitable
control, and the binding affinity may equal or exceed that of
.alpha. globin. When bound to HSP-70, a selected compound will
typically reduce the binding of .alpha. globin to HSP-70 by at
least about 25%, and usually by 30, 35, 40, 45, 50, 55, 60, 65,
70,. 75, 80, 85, 90, 95% or more, compared to a suitable control,
and binding to may be completely prevented. When bound to an
HSP-70-compound complex, GATA-1 will typically be protected so as
to reduce proteolysis by at least 25%, and usually by 30, 35, 40,
45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95% or more, compared to a
suitable control, and the binding of .alpha. globin to HSP-70 may
be completely prevented.
[0032] The present invention provides compositions for use in
methods of increasing erythrocyte maturation in individuals or
subjects in need thereof, particularly those with .beta.-TM. The
compositions include one or more substantially purified active
agents that promote erythrocyte maturation as described herein, and
a pharmacologically suitable carrier. The preparation of such
compositions for administration to a mammal is well known to those
of skill in the art. Typically, such compositions are prepared
either as liquid solutions or suspensions, however solid forms such
as tablets, pills, powders and the like are also contemplated.
Solid forms suitable for solution in, or suspension in, liquids
prior to administration may also be prepared. The preparation may
also be emulsified. The active ingredients may be mixed with
excipients which are pharmaceutically acceptable and compatible
with the active ingredients. Suitable excipients are, for example,
water, saline, dextrose, glycerol, ethanol and the like, or
combinations thereof. In addition, the composition may contain
minor amounts of auxiliary substances such as wetting or
emulsifying agents, pH buffering agents, and the like. If it is
desired to administer an oral form of the composition, various
thickeners, flavorings, diluents, emulsifiers, dispersing aids or
binders and the like may be added. The composition of the present
invention may contain any such additional ingredients so as to
provide the composition in a form suitable for administration. The
final amount of active agent in the formulations may vary. However,
in general, the amount in the formulations will be from about
1-99%.
[0033] The active agent compositions (preparations) of the
invention may be administered by any of the many suitable means
which are well known to those of skill in the art, including but
not limited to: by injection, (e.g. intravenous [IV],
intraperitoneal, intramuscular, subcutaneous), by inhalation,
orally, intravaginally, intranasally, by ingestion of a food or
probiotic product containing the agent, etc. In preferred
embodiments, the mode of administration is orally or by injection
or IV. In addition, the compositions may be administered in
conjunction with other treatment modalities such as various
chemotherapeutic agents, iron supplements, blood transfusions,
agents that activate .gamma. chain expression (e.g. that cause or
promote transcription or translation of Hb .gamma. chain), and the
like.
[0034] For administration of genes encoding one or more (at least
one) active agent(s) as described herein, various options may be
implemented. In some asepcts, nucleic acids comprising sequences
encoding an active agent of the invention or functional derivatives
thereof, are administered to prevent, manage, treat and/or
ameliorate .beta. thalassemia by way of gene therapy. Gene therapy
refers to therapy performed by the administration to a subject of
an expressed or expressible nucleic acid which encodes the active
agent. Generally, the encoding region is operably linked to one or
more expression control sequences, e.g. promoters, enhancers, etc.
The sequence may be linked to other sequences such as STOP codons,
and the like, in order to enable transcription of the gene into
funcation mRNA, or, if RNA is administered, to enable translation
thereof into an active form (or possibly a precursor of an active
form) of the active agent. The active agent, once fully expressed,
mediates a prophylactic or therapeutic effect.
[0035] Any of the methods for gene therapy available in the art can
be used according to the present invention. Exemplary methods are
described below. For general review of the methods of gene therapy,
see Goldspiel et al., 1993, Clinical Pharmacy 12:488-505; Wu and
Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev.
Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science
260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem.
62:191-217; May, 1993, TIBTECH 11(5):155-215. Methods commonly
known in the art of recombinant DNA technology which can be used
are described in Ausubel et al. (eds.), Current Protocols in
Molecular Biology, John Wiley & Sons, N.Y. (1993); and
Kriegler, Gene Transfer and Expression, A Laboratory Manual,
Stockton Press, N.Y. (1990).
[0036] In some embodiments, a composition of the invention
comprises nucleic acids encoding an active agent of the invention,
said nucleic acids being part of an expression vector that
expresses the active agent in the host to whom it is administered.
In particular, such nucleic acids have promoters, e.g. heterologous
promoters, operably linked to the coding region, the promoter being
inducible or constitutive, and, optionally, tissue- or
cell-specific. In other embodiments, nucleic acid molecules are
used in which the coding sequences and any other desired sequences
are flanked by regions that promote homologous recombination at a
desired site in the genome, thus providing for intrachromosomal
expression of the antibody encoding nucleic acids (Koller and
Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra
et al., 1989, Nature 342:435-438).
[0037] Delivery of the nucleic acids into a subject may be either
direct, in which case the subject is directly exposed to the
nucleic acid or nucleic acid-carrying vectors, or indirect, in
which case, cells are first transformed with the nucleic acids in
vitro, then transplanted into the subject. These two approaches are
known, respectively, as in vivo or ex vivo gene therapy.
[0038] In some embodiments, the nucleic acid sequences are
administered in vivo, where the sequences are expressed to produce
the encoded product. This can be accomplished by any of numerous
methods known in the art, e.g., by constructing them as part of an
appropriate nucleic acid expression vector and administering the
vector so that the sequences become intracellular, e.g., by
infection using defective or attenuated retrovirals or other viral
vectors (see U.S. Pat. No. 4,980,286), or by direct injection of
naked DNA, or by use of microparticle bombardment (e.g., a gene
gun; Biolistic, Dupont), or coating with lipids or cell surface
receptors or transfecting agents, encapsulation in liposomes,
microparticles, or microcapsules, or by administering them in
linkage to a peptide which is known to enter the nucleus, by
administering it in linkage to a ligand subject to
receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol.
Chem. 262:4429-4432) (which can be used to target cell types
specifically expressing the receptors), etc. In other embodiments,
nucleic acid-ligand complexes can be formed in which the ligand
comprises a fusogenic viral peptide to disrupt endosomes, allowing
the nucleic acid to avoid lysosomal degradation. In yet another
embodiment, the nucleic acid can be targeted in vivo for cell
specific uptake and expression, by targeting a specific receptor
(see, e.g., PCT Publications WO 92/06180; WO 92/22635; WO 92/20316;
WO93/14188, WO 93/20221). Alternatively, the nucleic acid can be
introduced intracellularly and incorporated within host cell DNA
for expression, by homologous recombination (Koller and Smithies,
1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; and Zijlstra et al.,
1989, Nature 342:435-438).
[0039] In some embodiments, viral vectors that contain nucleic acid
sequences encoding an active agent of the invention are used. For
example, a retroviral vector can be used (see Miller et al., 1993,
Meth. Enzymol. 217:581-599). These retroviral vectors contain the
components necessary for the correct packaging of the viral genome
and integration into the host cell DNA. The nucleic acid sequences
encoding the active agent to be used in gene therapy can be cloned
into one or more vectors, thereby facilitating delivery of the gene
into a subject. More detail about retroviral vectors can be found
in Boesen et al., 1994, Biotherapy 6:291-302, which describes the
use of a retroviral vector to deliver the mdr 1 gene to
hematopoietic stem cells in order to make the stem cells more
resistant to chemotherapy. Other references illustrating the use of
retroviral vectors in gene therapy are: Clowes et al., 1994, J.
Clin. Invest. 93:644-651; Klein et al., 1994, Blood 83:1467-1473;
Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141; and
Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel.
3:110-114.
[0040] Adenoviruses are other viral vectors that can be used in
gene therapy. Adenoviruses are especially attractive vehicles for
delivering genes to respiratory epithelia. Adenoviruses naturally
infect respiratory epithelia where they cause a mild disease. Other
targets for adenovirus-based delivery systems are liver, the
central nervous system, endothelial cells, and muscle. Adenoviruses
have the advantage of being capable of infecting non-dividing
cells. Kozarsky and Wilson, 1993, Current Opinion in Genetics and
Development 3:499-503 present a review of adenovirus-based gene
therapy. Bout et al., 1994, Human Gene Therapy 5:3-10 demonstrated
the use of adenovirus vectors to transfer genes to the respiratory
epithelia of rhesus monkeys. Other instances of the use of
adenoviruses in gene therapy can be found in Rosenfeld et al.,
1991, Science 252:431-434; Rosenfeld et al., 1992, Cell 68:143-155;
Mastrangeli et al., 1993, J. Clin. Invest. 91:225-234; PCT
Publication WO94/12649; and Wang et al., 1995, Gene Therapy
2:775-783. In a preferred embodiment, adenovirus vectors are used.
Adeno-associated virus (AAV) has also been proposed for use in gene
therapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med.
204:289-300; and U.S. Pat. No. 5,436,146).
[0041] Another approach to gene therapy involves transferring a
gene to cells in tissue culture by such methods as electroporation,
lipofection, calcium phosphate mediated transfection, or viral
infection. Usually, the method of transfer includes the transfer of
a selectable marker to the cells. The cells are then placed under
selection to isolate those cells that have taken up and are
expressing the transferred gene. Those cells are then delivered to
a subject. In the present invention, such cells may be
erythrocyles, e.g. immature erythroblasts. In this embodiment, the
nucleic acid is introduced into a cell prior to administration in
vivo of the resulting recombinant cell. Such introduction can be
carried out by any method known in the art, including but not
limited to transfection, electroporation, microinjection, infection
with a viral or bacteriophage vector containing the nucleic acid
sequences, cell fusion, chromosome-mediated gene transfer,
microcellmediated gene transfer, spheroplast fusion, etc. Numerous
techniques are known in the art for the introduction of foreign
genes into cells (see, e.g., Loeffler and Behr, 1993, Meth.
Enzymol. 217:599-618; Cohen et al., 1993, Meth. Enzymol.
217:618-644; Clin. Pharma. Ther. 29:69-92 (1985)) and may be used
in accordance with the present invention, provided that the
necessary developmental and physiological functions of the
recipient cells are not disrupted. The technique should provide for
the stable transfer of the nucleic acid to the cell, so that the
nucleic acid is expressible by the cell and preferably heritable
and expressible by its cell progeny. The resulting recombinant
cells can be delivered to a subject by various methods known in the
art. Recombinant blood cells (e.g., hematopoietic stem or
progenitor cells) are preferably administered intravenously. The
amount of cells envisioned for use depends on the desired effect,
patient state, etc., and can be determined by one skilled in the
art.
[0042] Cells into which a nucleic acid can be introduced for
purposes of gene therapy encompass any desired, available cell
type, and include but are not limited to epithelial cells,
endothelial cells, keratinocytes, fibroblasts, muscle cells,
hepatocytes; blood cells such as T lymphocytes, B lymphocytes,
monocytes, macrophages, neutrophils, eosinophils, megakaryocytes,
granulocytes; various stem or progenitor cells, in particular
hematopoietic stem or progenitor cells, e.g., as obtained from bone
marrow, umbilical cord blood, peripheral blood, fetal liver, etc.
In a preferred embodiment, the cell used for gene therapy is
autologous to the subject.
[0043] In an embodiment in which recombinant cells are used in gene
therapy, nucleic acid sequences encoding an active agent of the
invention are introduced into the cells such that they are
expressible by the cells or their progeny, and the recombinant
cells are then administered in vivo for therapeutic effect. In a
specific embodiment, stem or progenitor cells are used. Any stem
and/or progenitor cells which can be isolated and maintained in
vitro can potentially be used in accordance with this embodiment of
the present invention (see e.g., PCT Publication WO 94/08598;
Stemple and Anderson, 1992, Cell 7 1:973-985; Rheinwald, 1980,
Meth. Cell Bio. 21A:229; and Pittelkow and Scott, 1986, Mayo Clinic
Proc. 61:771).
[0044] In some embodiments, the nucleic acid to be introduced for
purposes of gene therapy comprises an inducible promoter operably
linked to the coding region, such that expression of the nucleic
acid is controllable by controlling the presence or absence of the
appropriate inducer of transcription.
[0045] In other embodiments, the active agent of the invention is
not a translatable peptide or protein but is e.g. an organic
molecule that is designed and synthesized in a manner that confers
on the molecule the features that are necessary or desirable to
promote its interaction with and binding to the negatively charged
.alpha. chain binding pocket/cleft as described herein. Methods of
synthesizing molecules with e.g. charged surface atoms and/or
surface atoms capable of hydrogen bonding are known. For example,
various condensation reactions are used to join functional groups
of interest, with or without the use of protecting groups, and in
the presence of suitable solvents, to generate linked functional
groups presenting desired atoms or groups of atoms in a desired
location in the molecule. The molecules can be designed to include
areas of rigidity and/or flexibility to accommodate the binding
pocket. Positioning and spacing of the atoms is such that the
groups or atoms with which they are intended to interact in binding
pocket will be contacted, or at least presented within bonding
distance, when introduced into the binding pocket. Typically, at
least 2, and usually more (e.g. 3, 4, 5, 6, 7, 8, 9, 10 or more,
e.g. 15, 20, 25, 30 or more) specific binding interactions are
planned, e.g. one positively charged group to interact with one
formal negative charge in the targeted binding site, 1-3 hydrogen
bond donors to interact with 1-3 other hydrogen bond donors, etc.
Suitable dimensions or ranges of dimensions are generally
determined in silico. Candidate active agents can be manufactured
and screened for activity using known methodology, e.g. via high
throughput screening.
[0046] In some embodiments, the active agent is a small molecule
that is a peptidomimetic. Peptidomimetic are small protein-like
chains designed to mimic a peptide of interest. For the present
invention, a peptide of interest is generally .alpha. globin or a
portion thereof, e.g. the portion of .alpha. globin that contacts
and mediates binding to Hsp70. Peptidomimetics typically arise
either from modification of an existing peptide, or by designing
similar systems that mimic peptides, such as peptoids and
.beta.-peptides. Irrespective of the approach, the altered chemical
structure is designed to advantageously adjust desirable molecular
properties such as stability, biological activity, ligand binding,
etc. The modifications generally involve changes to the peptide
sequence that do not occur naturally, e.g. altered backbones,
reduced peptide bonds, acylation of reactive groups, amidation of
reactive groups, incorporation of nonnatural (non-proteinogenic or
non-standard) amino acids (e.g. D-amino acids, norleucine,
lanthionine, 2-aminoisobutyric acid, dehydroalanine,
gamma-aminobutyric acid, ornithine, citrulline, beta alanine
(3-aminopropanoic acid), carnitine, hydroxyproline,
selenomethionine, homocysteine, homoserine, and homophenyalanine,
S-benzyl cysteine, etc. In addition, modifications such as
sulfoniation, phosphorylation, etc. may be used to create the
desired binding motif.
[0047] Herein, where a range of values is provided, it is
understood that each intervening value, to the tenth of the unit of
the lower limit unless the context clearly dictates otherwise,
between the upper and lower limit of that range and any other
stated or intervening value in that stated range, is encompassed
within the invention. The upper and lower limits of these smaller
ranges may independently be included in the smaller ranges and are
also encompassed within the invention, subject to any specifically
excluded limit in the stated range. Where the stated range includes
one or both of the limits, ranges excluding either or both of those
included limits are also included in the invention.
[0048] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention, representative illustrative methods and materials are
now described.
[0049] All publications and patents cited in this specification are
herein incorporated by reference as if each individual publication
or patent were specifically and individually indicated to be
incorporated by reference and are incorporated herein by reference
to disclose and describe the methods and/or materials in connection
with which the publications are cited. The citation of any
publication is for its disclosure prior to the filing date and
should not be construed as an admission that the present invention
is not entitled to antedate such publication by virtue of prior
invention. Further, the dates of publication provided may be
different from the actual publication dates which may need to be
independently confirmed.
[0050] It is noted that, as used herein and in the appended claims,
the singular forms "a", "an", and "the" include plural referents
unless the context clearly dictates otherwise. It is further noted
that the claims may be drafted to exclude any optional element. As
such, this statement is intended to serve as antecedent basis for
use of such exclusive terminology as "solely," "only" and the like
in connection with the recitation of claim elements, or use of a
"negative" limitation.
[0051] As will be apparent to those of skill in the art upon
reading this disclosure, each of the individual embodiments
described and illustrated herein has discrete components and
features which may be readily separated from or combined with the
features of any of the other several embodiments without departing
from the scope or spirit of the present invention. Any recited
method can be carried out in the order of events recited or in any
other order which is logically possible.
[0052] Before exemplary embodiments of the present invention are
described in greater detail, it is to be understood that this
invention is not limited to particular embodiments described, as
such may, of course, vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular
embodiments only, and is not intended to be limiting, since the
scope of the present invention will be limited only by the appended
claims.
EXAMPLES
Example 1
Cytoplasmic Sequestration of Hsp70 by Excess of Free .alpha.-Globin
Promotes Ineffective Erythropoiesis in .beta.-Thalassimia
[0053] It has been previously demonstrated that normal human
erythroid cell maturation requires a transient activation of
caspase-36. Although GATA-1, the master transcriptional factor of
erythropoiesis, is a caspase-3 target, it has been shown that
during human erythroid differentiation, it is protected from
cleavage through its association with the chaperone heat shock
protein 70 (Hsp70) in the nucleus.sup.7. Hsp70 is constitutively
highly expressed in normal human erythroid cells.sup.7. The
best-known role of this ubiquitous chaperone is to participate in
proteins folding and refolding of proteins denatured by cytoplasmic
stress, thus preventing their aggregation.sup.8. Here, evidence is
provided showing that during the maturation of human .beta.-TM
erythroblasts, Hsp70 is sequestrated in the cytoplasm by the excess
of free .alpha.-globin chains, resulting in nuclear GATA-1 cleavage
and, in turn, end-stage maturation arrest and apoptosis. A
molecular modelling shows that .alpha.-globin binds to a highly
electronegative cavity formed by all Hsp70 domains. Additionally,
the transduction of a nuclear-targeted Hsp70 mutant (Hsp70-S400A)
or caspase-3 uncleavable GATA-1 mutant (.mu.GATA-1) corrects
.beta.-thalassemia major IE in human cultured .beta.-TM cells.
Methods
[0054] Erythroid cells were generated in vitro from the peripheral
blood circulating CD34.sup.+ cells from 7 adult patients with
.beta..sup.0-TM or 8 healthy donors. Fresh normal bone marrow cell
smears were obtained from 3 adult patients with .beta.-TM who had
undergone cholecystectomy or splenectomy, or 3 healthy controls
(allogenic bone marrow donors), after they had given written
informed consent. This study was performed according to the
Helsinki Declaration with the approval from the ethics committee of
our institution. Erythroid cells in culture were generated as
previously described.sup.6,7. Statistical analyses were performed
using GraphPad PRIAM.TM. software. Data are expressed as the
mean.+-.standard deviation (SD) or median (interquartile
range-IQR), unless noted otherwise. Student's paired t-test or
Mann-Whitney Utest was used as appropriate. *p value <0.05, **p
value <0.01, ***p value <0.001
Erythroid Liquid Culture
[0055] The circulating peripheral blood of .beta.-TM patients
contains a small number of hematopoietic progenitor cells.sup.19.
Erythroid cells were also generated in vitro from peripheral blood
circulating CD34.sup.+ cells from adult patients with .beta..sup.0
thalassemia major (.beta.-TM), which were collected before routine
transfusion, or from control patients who were healthy donors
treated with G-CSF to induce hematopoietic stem cell mobilisation.
This study was done according to the Helsinki Declaration with the
approval from the ethics committee of the Comite de Protection des
personnes (CPP) "Ile de France II". All patients gave written
informed consent. In the first step of culture ("cell expansion"),
isolated CD34.sup.+ progenitors (Miltenyi CD34 Progenitor Cell
Isolation Kit) were grown in the presence of 100 ng/ml IL-6, 10
ng/ml IL-3 and 100 ng/ml SCF for 7 days. At day 7,
CD36.sup.+erythroid progenitors were isolated by magnetic isolation
(Miltenyi Biotec). In the second phase of culture, which allows the
"differentiation and maturation of erythroblasts", CD36.sup.+ cells
were cultured in the presence of 10 ng/ml IL-3, 100 ng/ml SCF and 2
U/ml Epo in IMDM (Gibco cell culture) supplemented with 15% BIT
9500 (Stem Cell Technologies), as previously described.sup.6,7.
Apoptosis Assay and Cell Differentiation
[0056] Apoptosis was assessed by Annexin V binding and propidium
iodide (PI) staining (ebioscience). Early apoptotic cells were
defined as Annexin-V positive and PI negative. Differentiation was
assessed by various methods. First, morphological analysis after
May-Grunwald-Giemsa (MGG) staining was used. Cells were examined
under a Leica DMRB microscope with a PLFluotar 40.times. oil
objective in a blinded fashion. The number of proerythroblasts,
basophilic, polychromatic, acidophilic erythroblasts and
reticulocytes was assessed in each experiment by counting
approximately 300 cells in consecutive oil immersion fields and
expressed as a percentage of total cells. Additionally,
differentiation was assessed by calculating a terminal maturation
index on MGG, defined by the number of acidophils+reticulocytes per
slide.times.100/number of polychromatophilic cells per slide. This
allowed better characterization of maturation arrest at the
polychromatophilic stage, which is known to be a hallmark of
ineffective erythropoiesis.sup.4, and its modulation. Haemoglobin
content was also measured, as assessed by benzidine staining. Flow
cytometry analysis was also performed each day after double
labelling with a phycoerythrin (PE)-conjugated anti-GPA (BD
Pharmingen) antibody and an APC-conjugated anti-CD117 (c-kit)
(e-bioscience) antibody. The percentage of GPA+/CD117-cells
represented mature erythroid cells20. F cells were evidenced by
flow cytometry analysis. The cultured cells were fixed and
permeabilised, washed with 1.times. PBS/1% BSA and then stained
with PE-conjugated anti-human haemoglobin F (HbF) (BD Pharmingen)
for 30 min at room temperature. The cells were then analysed by
fluorescence-activated cell sorting (FACS).
Analysis of the Hsp70/Alpha-Globin Complex
Yeast Two-Hybrid Assay
[0057] The bait vector used was pGBKT7, and the prey vector was
pGADT7 (Clontech). The human .alpha.- and .beta.-globin coding
sequences were cloned into the EcoRI/BamHI and NdeI/ClaI sites,
respectively, of the pGADT7. The coding sequence of the human Heat
Shock Protein 70 (Hsp70) was cloned into the EcoRI/BamHI sites of
the pGBKT7. An N-terminal nucleotide binding domain (NBD) (residues
1-380) and a C-terminal substrate binding-domain (SBD) (residues
394-615) were cloned in pGBKT7. The pGBKT7-p53 plasmid was used as
a positive bait control. This plasmid encodes the GAL4 DNA-BD fused
with murine p53. As a positive control prey plasmid, pGADT7-SV40,
which encodes the GAL4 activation domain fused with SV4021 large
T-antigen, was used. The empty vector, pGADT7, was used as a
negative control prey plasmid. All pGADT7- and pGBKT7-derived
vectors were transformed into Y187 and Y2HGold yeast strains,
respectively (Clontech Yeastmaker Yeast Transformation System 2).
After growth at 30.degree. C. for 3 to 5 days, the transformants
were selected on Leu.sub.-- and Trp.sub.-- minimal media plates,
respectively. Each prey strain was mated with the bait strain to
generate diploid yeast cells (Clontech Matchmaker Gold Yeast
2-Hybrid system). Diploid yeast cells selected on Leu_Trp.sub.--
minimal media plates were then patched onto Leu_Trp.sub.-- minimal
media plates with X-.alpha.-Galactosidase (40 .mu.g/mL) and
Aureobasidin A (70 ng/mL). Blue diploid cells appeared after 3 to 5
days at 30.degree. C., indicating the interaction between the bait
and the prey proteins. To confirm these results, the diploid yeast
cells were then patched onto higher stringency
His_Ade_Leu_Trp.sub.-- minimal media plates supplemented with 40
.mu.g/mL X-.alpha.-Galactosidase and 70 ng/mL Aureobasidin A.
Confocal Analysis
Cell Permeabilisation and Labelling for Fluorescence
Microscopy.
[0058] The cells were washed, spun onto slides, fixed with acetone,
hydrated with cold 1.times. PBS/1% BSA for 30 minutes, treated with
formaldehyde (Sigma) for 15 minutes, and then with methanol
(Prolabo) for 10 minutes at room temperature. Next, the cells were
permeabilised with 1.times. PBS/0.2% Triton X100 (Sigma) for 10
minutes at 4.degree. C., washed with 1.times. PBS/1% BSA and
incubated in 3% BSA for 30 min. They were then sequentially
incubated with the antibodies as follows: anti-GATA-1 overnight at
4.degree. C., anti-rat-Cy3 for 45 minutes at room temperature,
rabbit anti-Hsp70 or anti-caspase-3 for 1 hour at room temperature,
anti-rabbit Alexa 647 for 45 minutes at room temperature and
anti-haemoglobin-FITC (Abcam) for 60 minutes at room temperature.
All antibodies were diluted in 1.times. PBS/1% BSA/0.1% Tween
(Sigma). Nuclei were stained with DAPI, and the slides were
examined with a confocal laser microscope (LSM 510 Carl Zeiss).
Fresh, normal, bone-marrow cell smears were fixed with acetone.
Permeabilisation and labeling with anti-GATA-1, anti-Hsp70 and
anti-.alpha. globin antibodies were performed as above. To more
precisely analyse the Hsp70/.alpha.-globin interaction, the
Duolink.RTM. II technology (Olink.RTM. Bioscience), which is an in
situ proximity ligation assay technology, was used. In this assay,
a pair of secondary antibodies labelled with oligonucleotides (PLA
probes) only generates a signal when the two probes are bound in
close proximity (<40 nm). The signal from each detected pair of
PLA probes is visualised as an individual fluorescent spot.sup.21 .
Slides were incubated with primary antibodies as described above
and with secondary antibodies conjugated with oligonucleotides (PLA
probe MINUS anti-mouse and PLA probe PLUS anti-rabbit). Ligation
and amplification reactions were performed according to the
manufacturer's instructions.
Molecular Modelling, Docking and Molecular Dynamics (MD)
Simulations
[0059] All calculations were carried out on a PC station and on the
Meso Center of ENS de Cachan running Linux CentOs 5.2. Figures were
produced with the PyMOL molecular graphics system22, and R was used
for statistical computing.sup.23. The template structures for
homology modelling were retrieved from the Protein Data Bank
(PDB)24.
Molecular Modelling
[0060] Hsp70: The 3D model of the full-length human
Hsp70.sup.1-701was generated from partial structures (X-ray or
RIVIN) by homology modelling of the separate Hsp70 domains and
molecular complex Hsp110Hsp70 (PDB codes: 3C7N 25, 2QWL 26, 3FE1 27
and 2LMG 28). The sequence of Hsp70.sup.1-701was aligned to the
template sequences with ClustalW29 and Modeller30. The interdomain
linkers were constructed by ab-initio loop generation using
Modeler..sup.23 [0061] .alpha.-globin: The initial structural data
of .alpha.-globin was taken from the X-ray structure (PDB code:
3S66 31) and was docked onto the generated model of Hsp70.sup.1-701
using three algorithms: Zdock32, FlexDock33 and HingeProt34. The
minimised structure (CHARMM)35 of the
Hsp70.sup.1-701/.alpha.-globin complex was analysed for hydrophobic
and electrostatic complementarities and Hbond interactions on the
interface of the proteins. The ADP-Mg.sup.2+ and Haem Fe.sup.2+
were incorporated into proteins.
Molecular Dynamics (MD) Simulations
[0062] Molecular Dynamics (MD) simulations were performed using
GROMACS 4.5.4 36 with CHARMM 27 force field. Each generated model,
Hsp70 or the Hsp70.sup.1-70/.alpha.-globin complex, was solvated in
a TIP3P water box with a minimum distance of 15 .ANG. from the edge
of the box to any protein atom. The charges of the system were
neutralised by adding counterions (Na.sup.+ or Cl.sup.-). The
solvated systems were first minimised for 1,000 steps with the
protein atoms restrained, followed by another 3,000 steps of
minimisation with all atoms allowed to move. The temperature of
each system was then increased to 300 K by increments of 0.001 K
during 2 ps. The system was further equilibrated under constant
volume and temperature (NRT) conditions for 100 ps constraining
protein backbone atoms, followed by 500 ps equilibration without
constraints under constant pressure and temperature (NPT) at 300 K
and 1 bar. Production simulations were performed for 20 ns in the
NPT ensemble. Short-range interactions employed a switch function
with a 12 .ANG. cut-off and a 10 .ANG. switch distance, and the
long-range electrostatic interactions were calculated with the
Particle Mesh Ewald protoco137. During production simulations, the
time step was 2 fs, with a SHAKE constraint on all bonds containing
hydrogen atoms.
Viral Transduction
[0063] Lentiviral Production
[0064] The nucleus-targeted Hsp70 mutant (Hsp70-S400A) was cloned
in the pTrip_U3EF1 lentiviral vector upstream of an IRESECMV--Green
Fluorescent Protein (GFP) cassette. Infectious vector particles
were produced in 293T cells by cotransfection of the vector with
the encapsidation plasmid psPAX2 and the expression plasmid
pHCMV-G, using the jetPRIME.TM. transfection reagent (Polyplus).
Supernatants were collected 48 hours and 72 hours after
transfection and were pooled and concentrated by
ultracentrifugation. Virus stocks were kept frozen at -80.degree.
C. For the lentivirus production of the .beta.-globin gene,
vesicular stomatitis virus glycoprotein pseudotyped lentiviral
supernatant was produced by transient transfection of HEK293T cells
with the 5-plasmid system (LentiGlobin construct, HPV 275-gag-pol
plasmid, TN 15-vsvG env plasmid, p633-rev plasmid, HPV601-tat
plasmid) by calcium phosphate coprecipitation in Dulbecco's
modified Eagle's media supplemented with 5% foetal bovine serum
(Invitrogen), followed by harvest in CellGro SCGM serum-free media
(CellGenix) after 48 h. Concentrated virus was then frozen and
stored at -80.degree. C.
Retroviral Production
[0065] Uncleavable mutant GATA-1 (.mu.GATA-1) was cloned in PINCO
vector upstream of a CMV promoter-Green Fluorescent Protein (GFP)
cassette. These were used to produce vector particles by
cotransfection of 293EBNA cells with the vector plasmid, an
encapsidation plasmid (gag-pol) lacking all accessory HIV-1
proteins, and an expression plasmid (pHCMV-G) encoding the
vesicular stomatitis virus (VSVg) envelope, using jetPRIME.TM.
transfection reagent.
Infection of Erythroid Cells
[0066] Hsp70-S400A and .mu.GATA-1 transduction: CD34+cells isolated
from .beta.-TM peripheral blood mononuclear cells were cultured for
5 days, as described above. They were then infected by lentiviruses
or retroviruses, in the presence of 4.mu.g/ml protamine sulphate. A
second round of infection was performed 24 hours later, upon
changing to fresh medium with cytokines. After an additional 24
hours, cells were extensively washed in PBS and stained with the
anti-CD36-APC mAb (BD Pharmingen). The CD36.sup.+/GFP.sup.+ cell
population was purified by cell sorting and cultured for 7 to 10
additional days in serum-free medium in the presence of
IL3.sup.+SCF.sup.+EPO, as described above.
.beta.-Globin Gene Lentivirus Transduction
[0067] CD34.sup.+ cells were isolated from .beta.-TM peripheral
blood mononuclear cells by magnetic sorting (Miltenyi Biotec).
Sorted cells were prestimulated for 24 h in CellGro SCGM media
supplemented with 100 ng/mL hSCF, 100 ng/mL hTPO, 100 ng/mL hFIt3L
and 60 ng/mL hIL-3 at 37.degree. C. and 5% CO2. Then, prestimulated
cells were transduced for 22 h with the LentiGlobin vector at an
MOI of 50 in CellGro SCGM media supplemented with 100 ng/mL hSCF,
100 ng/mL hTPO, 100 ng/mL hFlt3L, 60 ng/mL hIL-3 and 4 .mu.g/mL
protamine sulphate, or mock transduced in the same conditions.
Two-phase liquid culture was then performed as described above.
Statistical Analyses
[0068] Statistical analyses were performed with GraphPad Prism.TM.
(version 5.0; GraphPad Software). The data are expressed as the
mean standard deviation (SD) or median (interquartile range-IQR),
unless noted otherwise. Student's paired t-test or Mann-Whitney
U-test was used as appropriate. *p value <0.05, **p value
<0.01, ***p value <0.001
RESULTS
[0069] To investigate the hypothesis that Hsp70 can be sequestrated
in the cytoplasm of mature .beta.-TM erythroblasts by binding to
free .alpha.-globin chains of haemoglobin or aggregates, its
subcellular localisation was analyzed in fresh bone marrow samples
from adult TM patients (n=3) and healthy donors (n=3) by confocal
microscopy. Erythroid maturation was evaluated by cell size and by
.alpha.-globin staining intensity. The results showed that Hsp70
was mainly localised in the cytoplasm and that GATA-1 was poorly
expressed in the nucleus of mature haemoglobinised erythroblasts
from .beta.-TM patients, in contrast to controls (FIG. 1A).
[0070] Next, to decipher the role of Hsp70 in ineffective
erythropoiesis in .beta.-TM, an in vitro two-phase amplification
liquid culture was performed, allowing the proliferation, survival
and erythroid differentiation of .beta.-TM (n=16) or control
CD34+(n=8) progenitors towards the formation of acidophilic
erythroblasts and reticulocytes. During the first phase of
amplification, cell proliferation did not differ between
thalassemic and control cells. In contrast, during the second
phase, corresponding to erythroid terminal differentiation and
maturation, at day 8, we observed, in .beta.-TM, an accelerated
differentiation characterised by a higher percentage of
polychromatophilic cells (26.2%*8.4 vs. 9.0%.+-.2.7; p=0.003) (FIG.
2A), an accelerated down regulation of the early erythroid marker
KIT/CD117 (mean fluorescence intensity (MFI) 25.7.+-.17 vs.
115.1.+-.47.1; p=0.0001) and an up regulation of GPA (MFI
243.8.+-.87.8 vs. 178.9.+-.56, p=0.04) (FIGS. 5A and B). At the
time of intense haemoglobinisation (d8-d10), in .beta.-TM cells,
apoptosis was increased (at d10, 38.2%.+-.15.1 vs 18.5%.+-.8.7;
p=0.01) (FIG. 2B) and terminal maturation was arrested at the
polychromatophilic stage. To quantify this maturation arrest, an
index of terminal maturation was defined as the number of
(acidophilic cells+reticulocytes per slide).times.100/number of
polychromatophilic cells per slide. At day 8, this index was
significantly decreased in .beta.-TM cells, to 16% (IQR 8.8-27.8)
compared to 37.6% in control cells (IQR 24.4-70.7; p=0.009) (FIG.
2A). Taken together, this system of cell culture reproduced the
characteristics of IE observed in .beta.-TM, namely accelerated
differentiation, maturation arrest and the death of mature
haemoglobinised cells.sup.3-5. Next, the subcellular localisation
of Hsp70 was analysed at several time intervals in .beta.-TM
patients (n=7) and healthy donors (n=7) by confocal microscopy. In
agreement with what we observed in fresh primary bone marrow
erythroblasts, Hsp70 was detected in the nucleus of control cells
but was absent or only weakly expressed in mature haemoglobinised
.beta.-TM cells. Thus, at day 8, the ratio of cytosoplasmic/nuclear
Hsp70 MFI in .beta.-TM erythroblast cells was significantly
increased, with a median ratio of 2.3 (IQR 1.6-3) compared to 1.1
in control cells (IQR 0.7-1.6; p<0.0001) (FIG. 2C). As a result,
GATA-1 was poorly expressed in the nucleus of mature
haemoglobinised .beta.-TM erythroblasts (FIG. 2C), thus supporting
the hypothesis. To further analyse and understand the link between
haemoglobinisation, Hsp70 localisation and the decrease in GATA-1
expression, the changes in the expression of these proteins during
differentiation and maturation was studied. In .beta.-TM derived
cells, the intensity of nuclear Hsp70 and GATA-1 staining decreased
with erythroid differentiation and maturation, while these
increased in controls (FIG. 2D).
[0071] To demonstrate that Hsp70 could act as a molecular chaperone
of free .alpha.-globin chains, the subcellular localisation of both
proteins was first analyzed by co-immunofluorescence experiments
(n=7). From day 6 of culture, Hsp70 and .alpha.-globin were
co-localised in the cytoplam of .beta.-TM erythroblasts. Features
of aggregates were sometimes observed in differentiated,
haemoglobinised cells, with one typical picture shown in FIG. 2C.
Co-localisation was assessed using the average Pearson's
correlation coefficient (PCC). At day 8, an apparent high level of
co-localisation between Hsp70 and .alpha.-globin was detected, both
in .beta.-TM (PCC=0.4.+-.0.13) and in controls (PCC=0.31.+-.0.09).
This finding was confirmed by Van Steensel's approach (data not
shown). Similar findings were observed in fresh bone marrow
experiments from patients and healthy donors (data not shown). To
further characterise this co-localisation, a close in situ
proximity ligation assay we used (Duolink.RTM.), which allows the
identification of interacting proteins by fluorescent spots. At day
8, spots we detected in .beta.-TM mature haemoglobinised cells
(n=3) but much less so in controls. Additionally, cells containing
abundant Hsp70/.alpha.-globin complexes were apoptotic (FIG.
3A).
[0072] Next, using a yeast two-hybrid system, additional evidence
for the biochemical interaction of Hsp70 with the human
.alpha.-globin chains was provided. In the assays, the entire
coding sequence of human Hsp70 was used as bait. Blue diploid
transformants could be detected on a high stringency minimal
medium, indicating a direct interaction between Hsp70 and the
.alpha.-globin chains Similar results were obtained when the
.beta.-globin coding sequence was used as prey, indicating that
both the .alpha.- and .beta.-globin chains could interact with
Hsp70 (FIG. 3B). To identify the Hsp70 domains involved in this
interaction, we tested the binding of .alpha.-globin chains to
deletion mutants of Hsp70 that expressed either the Nucleotide
Binding Domain (NBD) or the Substrate Binding Domain (SBD) of Hsp70
(FIG. 3B). Interestingly, neither of these two deletion mutants
interacted with the .alpha.-globin chain, suggesting that the
entire structure of Hsp70 is required for the recognition of
.alpha.-globin. To better characterise this interaction, an in
silico study involving molecular modelling, docking and molecular
dynamics simulations was performed. The .alpha.-globin was docked
onto a homology-generated model of Hsp70.sup.1-70. It was found
that .alpha.-globin binds to a highly electronegative cavity formed
by the NBD, SBD and lid (a C-terminal 10 kDa helical subdomain of
the SBD) (FIG. 3C). The resulting Hsp70.sup.1-70/.alpha.-globin
complex is stabilised by extensive protein-protein interactions
mediated mainly by multiple hydrogen bonds engaging the three
structural domains of Hsp70 (data not shown). Thus, the binding of
.alpha.-globin to Hsp70 crucially modulates the chaperone structure
and the interdomains interaction between NBD-lid and NBD-SBD.
Altogether, these findings indicate that, in addition to Alpha
Haemoglobin Stabilising Protein (AHSP), which stabilises the a
-globin chains.sup.9, Hsp70 could act as a novel chaperone of
.alpha.-globin chains. However, this apparent cytoprotective
function of Hsp70 might be detrimental during stages of high
haemoglobinisation and globin chain imbalance in .beta.-TM by
preventing the nuclear localisation of Hsp70 and, consequently, its
function in protecting GATA-1 from cleavage by caspase-3.
[0073] To investigate the contribution of Hsp70 cytoplasmic
sequestration to the pathophysiology of .beta.-TM IE, lentiviral
transduction we used to restore Hsp70 expression in the nucleus of
.beta.-TM erythroblasts. For this purpose, .beta.-TM CD34+ cells
(n=3) were transduced with lentiviruses expressing a
nuclear-targeted Hsp70 mutant (Hsp70-S400A) 10 wtHsp70, or an empty
lentivector. As expected, at day 7 of the differentiation phase of
culture, Hsp70-S400A (FIG. 4A) and wtHsp70 (not shown) lentivectors
increased nuclear Hsp70 localisation and rescued GATA-1 expression
inn-TM erythroid cells. The restoration of nuclear Hsp70
localisation efficiently improved the terminal maturation of
.beta.-TM erythroblasts. At day 7, in Hsp70-S400A transduced
.beta.-TM erythroblasts, the percentage of mature cells
(acidophilic cells and reticulocytes) was increased when compared
to the empty vector control (10.6.+-.3.2% vs 1.1.+-.0.7%, p=0.01
FIG. 4B). Similarly, the terminal maturation index was increased
(28.1% (IQR 28.1-51.3) vs 4.6% (IQR 1.7-6.7); p=0.01). In addition,
rescuing nuclear Hsp70 localisation induced a dramatic two-fold
decrease in apoptosis (9.9.+-.2.8% vs 20.7.+-.5.6%; p<0.001)
(FIG. 4C). Next, to analyse the consequences of GATA-1 cleavage on
the maturation arrest and apoptosis observed in cultured .beta.-TM
erythroblasts, we transduced .beta.-TM CD34+ cells with a GATA-1
mutant that was uncleavable by caspase-3 (.mu.GATA-1).sup.11 or a
GFP+ empty vector. The .mu.GATA-1 mutant had a positive effect on
erythroid terminal maturation that was similar to that of
Hsp70-S400A (FIG. 4A-D). Conversely, apoptosis was not corrected
indicating that cleavage of GATA-1 contributes to impair the
erythroid maturation but to a less extent to apoptosis of .beta.-TM
cells, as previously reported in low grade myelodysplastic
syndromes.sup.10.
[0074] Foetal haemoglobin (HbF, cop), which is replaced after birth
by the adult haemoglobin (HbA, .alpha..sub.2.beta..sub.2), is
concentrated in a few F cells and represents less than 1% of the
haemoglobin content in healthy adults.sup.12. In .beta.-TM
patients, there is an elevation in the proportion of F cells to
compensate for the lack of .beta.-chain synthesis, and the only
surviving mature erythroblasts are F cells. GATA-1 has a major role
in regulating the haemoglobin gene expression; it is sometimes
described as a transcription repressor or activator of the human
.gamma.-globin chains gene.sup.14-16. As such, the effect of GATA-1
nuclear restoration on HbF expression was studied by flow
cytometry. At day 7, it was observed that while the number of F
cells decreased with maturation (data not shown and .sup.17), the
percentage of HbF.sup.high cells, as assessed by flow cytometry,
was significantly increased concomitantly with the protection of
GATA-1 by Hsp70-S400A (54.8%.+-.12 vs 45.9%.+-.10.5; p<0.004)
(n=3) and in GATA-1 transduced erythroblasts (51.4.+-.8.2% vs
40.5.+-.9.4%; p<0.002) (n=3) (FIG. 4D). Finally, to ensure the
specificity of these findings, .beta.-TM CD34.sup.+ cells were
lentivirally transduced with the .beta.-globin gene. This led to
Hsp70 nuclear re-localisation, GATA-1 protection (FIG. 6), and the
restoration of normal erythroid maturation (data not shown).
[0075] Taken together, our data demonstrate that the cytoplasmic
sequestration of .alpha.-globin chains by Hsp70 prevents their
nuclear localisation. This is a key mechanism inducing the IE
observed in .beta.-TM patients. The modelling studies suggest that
Hsp70 could have been selected during evolution to serve as a
specific chaperone of globin chains to protect early erythroblasts
during erythroid differentiation.
[0076] The structural model of the Hsp70.sup.1-70/.alpha.-globin
complex provides a new rationale for a targeted therapy in
.beta.-thalassemia major IE. Small compounds disrupting the
Hsp70/.alpha.-globin complex in the cytoplasm may increase the
nuclear localisation of Hsp70 and may thus reduce maturation arrest
and increase the number of F cells. Ultimately, these outcomes may
decrease the patients' requirement for a blood transfusion and the
associated complications, including iron overload.
REFERENCES
[0077] 1. Khandros, E. & Weiss, M. J. Hematology/Oncology
Clinics of North America 24, 1071-1088 (2010). [0078] 2. Ginzburg,
Y. & Rivella, S. Blood 118, 4321-4330 (2011). [0079] 3. Yuan,
J. et al. Blood 82, 374-377 (1993). [0080] 4. Mathias, L. A. et al.
Experimental Hematology 28, 1343-1353 (2000). [0081] 5. Centis, F.
et al. Blood 96, 3624-3629 (2000). [0082] 6. Zermati, Y. et al. J
Exp Med 193, 247-254 (2001).11 [0083] 7. Ribeil, J.-A. et al.
Nature 445, 102-105 (2007). [0084] 8. Hartl, F. U., Bracher, A.
& Hayer-Hartl, M. Nature 475, 324-332 (2011). [0085] 9. Kihm,
A. J. et al. Nature 417, 758-763 (2002). [0086] 10. Frisan, E. et
al. Blood 119, 1532-1542 (2012). [0087] 11. De Maria, R. et al.
Nature 401, 489-493 (1999). [0088] 12. Dover, G. J. & Boyer, S.
H. Blood 69, 1109-1113 (1987). [0089] 13. Yao, X. et al. Exp.
Hematol. 37, 889-900 (2009). [0090] 14. Woon Kim, Y., Kim, S., Geun
Kim, C. & Kim, A. Nucleic Acids Res 39, 6944-6955 (2011).
[0091] 15. Zhu, J. et al. Blood 117, 3045-3052 (2011). [0092] 16.
Sankaran, V. G. & Orkin, S. H. Cold Spring Harb Perspect Med 3,
(2013). [0093] 17. Bhanu, N. V. et al. Blood 105, 387-393 (2005).
[0094] 18. Zermati, Y. et al. Exp. Hematol. 28, 885-894 (2000).
[0095] 19. De Franceschi, L. et al. Haematologica 92, 1319-1326
(2007).26 [0096] 20. Gabet, A.-S. et al. Cell Death Differ. 18,
678-689 (2011). [0097] 21. Schallmeiner, E. et al. Nat. Methods 4,
135-137 (2007). [0098] 22. DeLano, W. L. The PyMOL Molecular
Graphics System (2002). Website at www.pymol/org [0099] 23. R
Development Core Team R: A language and environment for statistical
computing. (2011). Website at www.R-project.org/24. [0100] 24.
Berman, H. M. et al. Nat. Struct. Biol. 7 Suppl, 957-959 (2000).
[0101] 25. Sousa, R. & Lafer, E. M. Traffic 7, 1596-1603
(2006). [0102] 26. Jiang, J. et al. Mol. Cell 28, 422-433 (2007).
[0103] 27. Wisniewska, M. et al. PLoS ONE 5, e8625 (2010). [0104]
28. Gao, X.-C. et al. J. Biol. Chem. 287, 6044-6052 (2012). [0105]
29. Larkin, M. A. et al. Clustal W and Clustal X version 2.0.
Bioinformatics 23, 2947-2948 (2007). [0106] 30. Eswar, N. et al.
Nucleic Acids Res. 31, 3375-3380 (2003). [0107] 31. Shibayama, N.,
Sugiyama, K. & Park, S.-Y. J. Biol. Chem. 286, 33661-33668
(2011). [0108] 32. Chen, R., Li, L. & Weng, Z. Proteins 52,
80-87 (2003). [0109] 33. Mashiach, E. et al. Proteins 78, 3197-3204
(2010). [0110] 34. Emekli, U., Schneidman-Duhovny, D., Wolfson, H.
J., Nussinov, R. & Haliloglu, T. Proteins 70, 1219-1227 (2008).
[0111] 35. Brooks, B. R. et al. J Comput Chem 30, 1545-1614 (2009).
[0112] 36. Van Der Spoel, D. et al. J Comput Chem 26, 1701-1718
(2005). [0113] 37. Batcho PF, Case DA & Schlick T Journal of
chemical physics 115, 4003-4018 (2001).
[0114] While the invention has been described in terms of its
preferred embodiments, those skilled in the art will recognize that
the invention can be practiced with modification within the spirit
and scope of the appended claims. Accordingly, the present
invention should not be limited to the embodiments as described
above, but should further include all modifications and equivalents
thereof within the spirit and scope of the description provided
herein.
* * * * *
References