Sickle Cell Anemia and ß-Thalassemia

Adult Hemoglobin

Heme

Mutations that reduce the synthesis

Alpha/Heme

Aggregates or

hemichromes

Heme

Mutations that alter the structure

(Glutamic acid to Valine at position 6)

Sickling Hb

and red cells

Falciparum malaria

Epidemiology of Sickle Cell Disease

~ 100,000,000 – 150,000,000 carriers worldwide

Highest incidence in Africa and developing countries

Lack of confirmed data

US > 2,500,000

In Nigeria, 1/3 population of US, 45,000 - 90,000 babies w SCD

born each year

β6 Glu. Val.

Global Epidemiology of Hgb Disorders

~7% of global population ~500,000,000

carries an abnormal Hemoglobin (Hgb)

gene

Globally, only 100,000 patients w

thalassemia major are registered and

treated regularly

DO YOU KNOW THAT…

• ~300,000-500,000 affected children are

born w Hgb disorders annually

• ~80% of affected children are born in

developing countries, mainly in Africa

• ~70% (200,000-350,000) are born w

sickle cell disease while the rest

(90,000-150,000) w thalassemia

disorders

• The majority, ~50-80%, of affected

children w SCD and thalassemia, die

each year, and do not survive early

childhood, in low & middle income

countries

2013 Global population ~ 7,120,000,000~7%, ~500,000,000 carriers of Hgb mutations

Thalassemia: Epidemiology

Papua Guinea

Indonesia

Malaysia

Thailand

Cambodia

Laos

Vietnam

South

China

India

Nepal

Bangladesh

Seri Lanka

Italy

Spain

Guinea

Sierra Leone

Liberia

Egypt

SudanIsrael

Lebanon

Syria

Jordan

Algeria

Iran

Iraq

Kuwait

Qatar

Uzbekistan

Azerbaijan

Armenia

Turkmenistan

Turkey

Balkans

THALASSEMIA-

+ Talassemia

o Talassemia

1-15%

5-40%

60%

40-80%

5-15%

5-80%

-Thalassemia -

-Thalassemia

1-25%

15-30%

1-3%

3-7%

4-8%

4-8%

1-3%

Nonsense and frameshift mutations;

mutations affecting transcription, splicing,

polyadenylation, and translation

Over 300 Mutations Cause ß-Thalassemia

Human ß-Globin Gene

5’ 3’Exon 2 Exon 3Exon 1

Deletions of the ß-globin gene

Clinical Classification and

Management of Thalassemia

Seve

rity

of

dis

eas

e

• Homozygous disorder

• Significant imbalance of α / β globin chains

• Severe anemia presenting early in life

• Requires lifelong RBC transfusions

• If untreated, leads to death usually in first decade

Thalassemia major

• Various genetic interactions

• Globin chain production moderately impaired

• Mild anemia, diagnosed usually in late childhood

• Occasional blood transfusions may be required

Thalassemia

intermedia

Thalassemia

minor

• Heterozygous condition

• Asymptomatic

• May require genetic counseling

Clinical Classification and

Management of Thalassemia

Seve

rity

of

dis

eas

e

• Homozygous disorder

• Significant imbalance of α / β globin chains

• Severe anemia presenting early in life

• Requires lifelong RBC transfusions

• If untreated, leads to death usually in first decade

Thalassemia major

• Various genetic interactions

• Globin chain production moderately impaired

• Mild anemia, diagnosed usually in late childhood

• Occasional blood transfusions may be required

Thalassemia

intermedia

Thalassemia

minor

• Heterozygous condition

• Asymptomatic

• May require genetic counseling

Transfusion

Independent

Transfusion

Dependent

Seve

rity

of

dis

eas

e

Transfusion requirement

Occasional

transfusions required

(e.g. surgery,

pregnancy, infection)

More frequent transfusions

required (e.g. poor growth

and development, specific

morbidities)

Lifelong regular

transfusions required

for survival

β-Thalassemia major

Severe hemoglobin E/β-thalassemia

Hemoglobin H Constant Spring

α-Thalassamia major (hemoglobin Bart’s hydrops fetalis)

Transfusions

seldom required

α-Thalassamia trait/minor

β-Thalassemia trait/minor

Non-transfusion-dependent thalassemias (NTDT)

β-Thalassemia intermedia

Mild/moderate hemoglobin E/β-thalassemia

α-Thalassemia intermedia (hemoglobin H disease)

Figure 1

Mortality in Thalassemia

Modell. J Cardiovasc MR 2008: 42

UK Thalassaemia Register. Causes of death by 5-year interval

0

5

10

15

20

25

30

35

40

45

50

1950-

1954

1955-

1959

1960-

1964

1965-

1969

1970-

1974

1975-

1979

1980-

1984

1985-

1989

1990-

1994

1995-

1999

2000-

2004

Death

s in

5 y

r

Unknow n

Other

Malignancy

Iron overload

Infection

BMT complication

Anaemia

Death by heart failure in 70%

Median age at death 35 years

Mechanism of denaturation of

α or β hemoglobin changes and

of sickle hemoglobin

Vinci F. et al. ASH 2016

AHSP inhibits ROS production by Hb

Oxidative status in RBC

Andrews N. C., 1999

?TfR1 DMT1TfR2

Fe(III)Fe(II)

transferrin Fe(II)

Dcyb

Fe(II)

ferritin

Iron is taken up by cells

from circulating transferrin

via transferrin receptors

TfR1 and TfR2

Following various steps iron

is delivered into the cytosol

labile iron pool (LIP) of Fe(II)

Iron is used mostly by

mitochondria for heme and

ISC synthesis

Most cells have no iron release mechanisms

All cells take up iron for metabolic needs but maintain a steady pool of labile iron (LIP)

Excess iron is stored or

withdrawn into ferritin

PLASMA

PLASMA

Fe(II)

Fe(II)LIP

ZIC 09

Cell iron homeostasis

?

Iron Overload in Thalassemia

Caused by:

• Hb instability enhances intracellular iron release

• Increased uptake of dietary iron

• Frequent blood transfusions

0

20

40

60

80

100

120

1 3 5 7 9 11 13 15 17 19

Age (years)

Iro

n (

g)

Hepatic Fibrosis --> Cirrhosis

Cardiac arrhythmia

Hypogonadism

Diabetes

Hypothyroidism

Hypoparathyroidism

Cardiac Failure

Transfusional Iron Overload in Thalassemia

Thalassemia Centre, Dept. of Pediatrics

University of Turin, Italy

Death

Labile plasma iron

Tissue ironoverload

Fe(II) Normally all the iron is absorbed by Tf whether it is taken upby the gut or recycled by the RE system from digested RBC

rbcR.E.

Transferrin

In iron overload the Tf binding capacity is surpassed

37

• When plasma iron rises and surpasses transferrin’s iron binding

capacity (at >70% saturation), it appears as NTBI (non transferrin bound

iron) .

• Forms of NTBI that are redox-active, chelatable and permeant to cells

are referred as labile plasma iron (LPI)

• Excessive ingress of LPI into cells leads to a rise in labile iron pool

(LIP) (Fe3+ and Fe2+) reacting with ROS - reactive O2 species - (O2÷ and

H2O2) forming OH∙ radicals (Haber Weiss cycle)

• Fe3+ + O2÷ → Fe2+ + O2

• Fe2+ + H2O2 → Fe3+ + OH∙ + OH-

(Fenton reaction)

NTBI/LPI in serum/plasma

Courtesy of Professor I. Cabantchik

Intracellular Iron Homeostasis: Ferritin functions as a ferroxidase, converting Fe2+ to Fe3+ as iron is

internalized and sequestered in the ferritin mineral core. Reactive species (shown as yellow spheres)

can directly damage DNA and proteins. DMT1 = divalent metal ion transporter 1,

Tf = Transferrin, TfR = Transferrin receptor.

M.A. Knovich et al. Blood reviews 23 (2009);95-104

mitochondrion DNAProteinLysosomes

H2O2 O2÷

1. 33 g of ROI= reactive O intermediates produced per day* Oxidations:

CO, met, tyr

Lipidperoxidation

baseoxidation

endoplasmic reticulum

OH.

ROS

2. LPI present in systemic iron overload leads to accumulation of labile iron pool (LIP)

LCI

LPI Fe

FeFe

Where and how does labile iron cause cell damage ?

ROI are normally converted to

water by resident enzymes

SOD and GPX

* Up to 3Kg ROI /d in inflammation!

4. OH∙ radicals are highly reactive and they can modify DNA, proteins and lipid

components of cells

3. ROI react with LPI producing noxious ROS, e.g. OH∙ radicals

O2

LIP

Liver cirrhosis/ fibrosis/cancer

Diabetes mellitus

Endocrine disturbances

→ growth failure

Cardiac failure

Infertility

Excess iron is deposited in multiple organs, resulting in organ damage

Diagnostic Tools for theEvaluation of Body Iron Status

DisadvantagesDiagnostic tool

Unreliable in patients with bleeding or chelationtherapy

Transfusion iron burden

Unreliable in patients with inflammation, liverfunction deficiency, and ascorbate deficiency

Serum ferritin

No quantitative correlation to iron burdenSerum transferrin saturation

Expensive; not widely available; reliable up to LIC of15 mg/g dry wt.

MRI R2

Expensive; not widely available; require a skilledradiologist; validated on the heart; less validated onthe liver

MRI T2*

NTBI, LPI - methods commercially availableLIP – research tool at present

NTBI/ LPI/ LIP

Still not widely availableSerum hepcidin

MONITORING IRON OVERLOAD BY MRI

An R2 image of an iron-overloaded human liver superimposed on a T-2 weighted image.

Bright areas represent high iron concentration; dark areas represent low iron concentration.

Clark PR, et al. Magn Reson Med. 2003;49:572-575. Image courtesy of T. St. Pierre

Hepcidin, the iron hormone regulator, acts

on Ferroportin, the iron exporter

Hepcidin

Enterocyte

FPN

Absorption

P

Fe2+

Hepatocyte

FPN

Fe2+

P

Release

Macrophage

Fe2+

P

FPN

Recycling

The ß-Thalassemia Mouse Models Show Massive Iron Overload in Liver

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.82.0

BM +/+; N: 7

BM Th3/+; N: 4

BM Th3/Th3; N: 7

+/+ Th3/Th3+/Th3

+/+ versus Th3/Th3: P<0.01

Iron content by atomic absorption

Fe

g/g

dry

weight

+/+

Th3/Th3

Gomori Staining

0.38

0.95

2.45

0.7

0.38

0.9

0.00

0.50

1.00

1.50

2.00

2.50

3.00

Hepcidin IREG NGAL HFE TfR1 TfR2

C57B1/6 wild-tipe

C57B1/6 Hbbth3/+

Expression of iron regulatory genes in the liver of a

mouse model of -thalassemia

NATURE MEDICINE, 2007

Hypoxia

Erythropoietin

Globin chain

imbalance

?Other

GDF15

Hepcidin production

Duodenum

Iron absorption

Ineffective

erythropoiesis

Model for iron regulation in thalassemia patients

The hypercoagulable state in thalassemia, Blood 2002

Prevalence of Thromboembolic Events

Among 8860 Patients with Thalassemia Major and Intermedia in the Mediterranean

Area and Iran

Total: 1.65% (53% females)

Thal. Major - 61/6670 - 0.9%

Thal. Intermedia – 85/2190 - 4% *

* 95% splenectomized

Chart 1: Percentage of Thrombotic Events in

TM & TI

66

9.4

39

12

19

8

12

47.6

28

23

8

11

0

30

0 10 20 30 40 50 60 70

Venous

Stroke

DVT

PE

Portal Vein

STP

Others

Typ

e

Percentage

85 TI patients 61 TM patients

Study Number of patients Age (years) Prevalence of SCI (95% CI)

Manfre et al. 1999 16 Mean:29 37,5% (18.4-61.7)

Taher et al. 2010 30 Range: 9-48

Mean:32.1

60.0% (42.2-75.5)

Karimi et al. 2010 30 Mean: 24.3 26.7% (14.2-44.6)

Teli et al. 2012 24 Range: 18-34

Mean:12

0%

Karimi e t al. 2012 95 * ** Range: 23±8

Mean: 23

15.8%

Karimi et al. 2015 40 (1) (2) Range: 12-45

Mean: 27±7

37.5%

• 59 Splenectomized ** 46 Regularly transfused

(1) 21 Splenectomized (2) 40 Regularly transfused

Incidence of silent cerebral infarction (SCI) in

195 patients with β-TI obtained by MRI

Coronal FLAIR thin section through parietal

& occipital lobes and cerebellum

demonstrates high intensity lesions as

marked by arrows.

Axial FLAIR superior thin section

demonstrates high intensity lesions in

the frontal & parietal lobes as marked by

arrows.

3T MR Imaging of the Brain of a Multi-

Transfused β-TM Patient

1

2

3

Treatment:PREVENTION

Prenatal Diagnosis in Pregnancy

Country of origin?

Well known cases in the family

of both siblings!!

MCV<80fl

% Fetal Hb

Hb A2 (α2δ2) > 3.5 % β Thal.

Hb A2 (α2δ2) < 3.5% α Thal. (or β Thal. +IDA

Family history

Complete blood count

Hb electrophoresis

Placental Aspiration, Chorion Villi Sampling

Treatment of iron overload -a question of balance

Too muchiron

Too muchchelator

• Uncoordinated iron

• Free radical

generation

• Organ damage

• Organ failure

• Cardiac death

• Uncoordinated chelator

• Inhibition of

metalloenzymes

• Neurotoxicity

• Growth failure

• Bone Marrow toxicity

Deferoxamine

Deferiprone

Hexadentate

Bidentate

DeferasiroxTridentate

STRUCTURE OF CHELATOR-IRON COMPLEXES

Iron chelators that enter cells by different pathways remove iron from different pathways remove iron from different subcellular

compartments: deferasirox and deferiprone target cytosolic ferritin iron, andesferrioxamine mesylate targets lysosomal ferritin

iron increased by stimulated autophagy, and damaged ferritin (hemosiderin) iron

66

Hours

2 μM

28242016128 32

Deferiprone (L1)

75 mg/kg/day

Each colour represents LPI values of individual patients starting at 8AM and followed for the next 24 hours

Effects of monotherapy and

combined therapy on LPI

Hours

mg/kg/day

DFO 40

028242016128 32

Deferiprone (L1)

75 mg/kg/day

DFO 40 mg/kg/day

28242016128

DFO 40 mg/kg/day

Hours

32

Reproduced from Cabantchik Z, et al. Best Pract Res Clin Hematol 2005;18:277–287

LPI

LPI

LPI

2 μM

2 μM

POTENTIAL ROLE OF ANTIOXIDANTS

TO AMELIORATE CLINICAL AND

LABORATORY PARAMETERS RESULTING

FROM OXIDATIVE STRESS

Summary: Vit E supplement

• Vitamin E supplement 200-300 mg/day:– Normalize serum vitamin E

– Decrease activity of anti-oxidant enz GPx

– Decrease lipid peroxidation

– Increase red cell survival (small series)

– No change in Hb level or transfusion requirement

• Vitamin E supplement 600 mg/day

– Normalize MDA level at 3 months

– Vit E in LDL remains low at 6 months

– No change in Hb level

24 -thalassemia/HbE patients

(11 splenectomized)

receiving curcumin 500 mg/d for 6 months

age 16 - 48 years

no blood transfusion at least 3 months

before donating their blood for this study

Hb level 4.7 – 9.5 g/l

Treatment of curcumin for 6 months

in -thalassemia/Hb E

MDA 30.73%

SOD 15.30%

GSH-Px 18.91%

GSH 19.48%

NTBI

No significant change in Hb and ferritin

Result: Red cell survival Red cell survival after 3 months of curcumin therapy

0

5

10

15

20

25

30

Pre-treatment 3-month post treatment

Red c

ell

half

life (

days

) Series1

Series2

Series3

Series4

Series5

Series6

Series7

Series8

0

1 0 0

2 0 0

3 0 0

4 0 0

5 0 0

6 0 0

R O S G S H R O S G S H

MF

C

FP P -

FP P +R B C P la te le ts

T h e e ffec ts o f F erm en ted P ap a ya P rep ara tio n (F P P )

In b e ta -T h a lassem ic P a tien ts

Mean Hb levels on various treatment dose of Darbepoetin

in 5 patients with β-TI and 4 with E-βo thalassemia (Singer et al, BJH 154:271; 2011)

Rivella S; Haematologica 2015

Hemoglobin switching in Humans

• Hereditary persistence of fetal hemoglobin (HPFH) and

pharmacological induction of HbF by hydroxyurea may

improve sickle cell anemia phenotype

Weatherall DJ, Clegg JB.

Hereditary persistence of fetal haemoglobin.

Br J Haematol. 1975 Feb;29(2):191-8.

McGann PT, Ware RE.

Hydroxyurea for sickle cell anemia: what have we learned and what questions still remain?

Curr Opin Hematol. 2011 May;18(3):158-65.

Increased synthesis of fetal

hemoglobin can alleviate sickle cell

anemia phenotype

The effect of Hydroxyurea on transfused

and untransfused Thalassemia patients:

(a review of 1469 patients out of 500 articles)

Results:

A modest significant increase in Hemoglobin

levels (p<0001) was found in the 2 groups of

patients

M. Kosaryan et al. Hemoglobin 2014;38(4): 262

HbF reactivation is mediated by forcing the

looping of the LCR on the g-globin promoter

LMO2

E2A

Promoter of the

g-globin gene

LCR

Promoter of the

-globin gene

ZF

LCR

Production of HbF in adult cells

ZF Zinc-finger, binding the g-globin

promoter, fused to LDB1

Deng et al. Cell, 2012 and Deng et al. Cell, 2014

Bone marrow transplantation (BMT) in Thalassemia Major (TM)

Results from 2000 patients

(18 reports)

Overall

Survival

66%-95%!

Thal. Free

Survival

55%-88%

Hematopoietic Stem Cell Transplantation (HSCT) in TM

Indications Transfusion Dependency

• Time of transplantation With HLA-identical sibling : as soon as possible

• Stem cell source from MSD Bone marrow, cord blood

• HSCT in adult TM patient If sufficient chelation was performed, within controlled clinical trials only

• HSCT from HLA-mismatched Within controlled clinical trials only

family members

• HSCT from phenotypically In TM-experienced HSCT-Centers only

Identical family members

• HSCT from unrelated donors Only from allelic matched donors; in patients without iron-related tissue damage

• HSCT with unrelated cord blood Within controlled clinical trials only, in expert Centers for CBT

New Developments in the Strategy of HSCT

Preimplantation Genetic Diagnosis

To Select an Embryo as a Stem Cell Donor

Average cost of medical treatment of

Thalassemia and SCD compared to BMT/year

17.000 $ 10.000 $ 1900 $

ThalassemiaSCD BMT

Gene Therapy Schematic Approach

Rivella S; Haematologica 2015

Gene Therapy Achieved Trough Viral Gene Transfer

To avoid

• Little knowledge of the biology and gene transfer in

human cells

To avoid

• Expression of the therapeutic gene in the wrong cells

Gene Therapy of ß-Thalassemia: Requirements

By J.No

The correct approach…

• To test the vector in human cells

• To prepare a safe and efficient clinical protocol

Allogeneic Bone Marrow

Transplant versus Gene Therapy

• No need to find a compatible BM donor)

• No rejection

• No GVHS

Sotatercept: Developed to Treat

Osteoporosis

• Developed to Treat Osteoporosis by Targeting Activin IIA

Signalling

• Sotatercept binds activin IIAand thereby prevents its

prosurvival effects on osteoclasts.

Another analogue Luspatercept, targets

Activin IIB. (This is the drug we will have soon)

• Unexpectedly, patients with osteoporosis who were given

Sotatercept had a 12% rise in Hb! And the same happened in normal controls.

93

Activin, a Member of TGFSuperfamily, has Type I and II receptors

GDF11 is an activin receptor IIA ligand94

95

Activin Receptor-II Trap Ligands improve ineffective erythropoiesis by targeting GDF11

Iancu-Rubin C et al, Exp Hematol. 2013 Feb;41(2):155-166

Red cells

ROS & GDF11

FAS/FASL Decreased apoptosisof erythroid precursors

SMAD 2/3 Decreased erythroid cell differentiation

FAS/FASL Increased apoptosis of erythroid precursors

SMAD 2/3 Increased erythroid cell differentiation

ACE-011 or ACE-536

Trap ligandACE-011 and ACE-536 improve ineffective

erythropoiesis and bone metabolism in NTDT mice

More TDT patients achieved a reduction in transfusion burden of ≥ 20% in the sotatercept 0.3, 0.5, and 0.75 mg/kg cohorts compared with the0.1 mg/kg cohort

a Transfusion burden evaluated up to the last known efficacy record, adjusted to 168 days; b

Change in transfusion burden (units/168 days) from baseline. Interim data as of 07 February 2014.

Reduction in transfusion burden in TDT patients

0%

50%

33%

0%0% 0%

67%

33%

Change in transfusion burdenb

0.3

mg/kg

(n = 3)

0.5

mg/kg

(n = 2)

0.75

mg/kg

(n = 3)a

0.3

mg/kg

(n = 3)

0.5

mg/kg

(n = 2)

0.75

mg/kg

(n = 3)a

Pati

en

ts (

%)

0.1

mg/kg

(n = 2)

0.1

mg/kg

(n = 2)

Progenitor erythroid cells

Red cell

Normal Erythropoiesis

Cooley’s Anemia

Ineffective Erythropoiesis

Apoptosis/Hemolysis

Erythroblast

Progenitor erythroid cells

Normal Erythropoiesis

Red cell

Cooley’s Anemia

Apoptosis

Jak2: a Gene That Controls Red Cell Production

: pJak2

Red cell

Cooley’s Anemia

Potential effect of Jak2 inhibitors on Ineffective Erythropoiesis

Jak2 Inhibitor

Ineffective Erythropoiesis

: pJak2

A Jak2 Inhibitor Decreases the Spleen Size in Thalassemic Mice

th3/th3

Placebo + TX

Jak2 inhib./TG101209 + TX

th3/th3

Jak2 inhib./ TG101209

+ TX

Placebo + TX

Hb g/dL

6.8

7.2

Day 0

10.2

7.4

Day 18

Luca Melchiori, Ella Guy

Possible Therapy: JAK-2 inhibitor?

103

Hypothesis: Increased levels of Hepcidin in thalassemia

intermedia are beneficial to prevent iron overload and

ameliorate erythropoiesis

Increased iron absorption

Hepcidin

Anemia

Hepcidin

Decreased iron absorption &

hemichrome formation

Amelioration of organ iron content

& erythropoiesis

Blood, 2012

Minihepcidins (MH): background

• Minihepcidins are short peptide mimetics (9 retro-inverso AA) of hepcidin

(25 AA)

N

HN NH2

HH

NH2HN

N

H

HOOCHN

HN

N

OH

H

H

H

O

O

O

O

NN

N

O

NO

H

NN

NN

H

O

O

H

NN

SH

N H

O

HO

CONH2

ONH

M004• MH are effective in reducing iron overload in

animal models of HFE- and HAMP-related

hemochromatosis

• Derived from the N-terminal amino acid sequence and modified for in vivo

activity

Casu C. et al . Blood 14, 128:265-276

http://content.nejm.org/content/vol353/issue11/images/large/09f1.jpeg

V. Nathan Subramaniam.Blood 2016;128:153

Pocket-Sized Iron Regulators: one size fits all?

BCL11A: a repressor of fetal hemoglobin and

phenotypic modifier for hemoglobinopathies

Treatment of hemoglobinopathies: new therapies and

emerging challenges

• Very heterogeneous disorders: several known and unknown modifiers

• “Dynamic” disorders: young vs. old patients

Novel findings and technologies might provide a way to “tailor” therapies

for different clinical needs and groups of patients

Hemoglobinopathies:

background

Thalassemias; abnormal hemoglobin variants

• Variable levels of Hb synthesis

• Abnormal globin chains

• Chronic transfusionsiron overload

• Increased iron absorptioniron overload

• Abnormal bone metabolism, thrombosis, etc

Hemoglobins:Embryonic: z2e2 2e2 z2g 2

Fetal: 2g2

Adult: 22 2d2

Hemoglobin switching in Humans-1

GgeLCR dAg

5 Kb

12zHS-40%

glo

bin

syn

thesis

0 3 6 9 3 6

20

40

60

80

100

0

Birth

e

d

g

Months

z

Peterson et al, PNAS 92, 5655-5695; 1995. Trimborn et al, Gen. Dev. 13, 112-124; 1999.

Bone Marrow Macrophages provide iron,

regulatory signals and phagocytize nucleii of

maturing RBC: “Nurse cells”

Liposomal Clodronate Eliminates Macrophages, Improves Thalassemic Mice

•20-40 hrs after Clodronate injection, Hb increased and

spleen decreased as much as 32%, persisted for 2

months of chronic Rx

•Increased numbers of differentiated RBC and reduction

of number of cycling RBC in spleen

•Increased hepcidin production and decrease serum

iron

•Not due to lack of iron (iron loaded mice: same effect)

•Conclusion: Macrophages impair erythroid development in beta thalassemia

Hemoglobin (g/dl)

Gene Transfer Raises Hemoglobin to Therapeutic Levels in Mice Affected by ß-Thalassemia Major

0

5

10

15

20

ß-Thal.Major

Normalß-Thal.Major

+ Vector

Transfusion Independent

Reinfusion

Gene Transfer Schematic Approach

Hematopoietic stem cells

Transduction

Vector carrying the therapeutic

gene

Meta Analysis on the effects of

HYDROXYUREAin 1469 patients with

β-TM and NTDT15-30mg/kg/day, for 6-180 mean (37)

Showed:

Modest significant increase in Hb levels (p<0.0001)

M. Kosaryan et al. Hemoglobin 2014; 38(4):262-271