NEWER THERAPIES IN THE MANAGEMENT OF

THALASSEMIA

PROF JANET POOLEDEPT OF PAEDIATRICS, HAEM/ONC, CMJAH,

FACULTY OF HEALTH SCIENCES,UNIVERSITY OF THE WITWATERSRAND,

JOHANNESBURG

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HAEMOGLOBINPATHIES

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Background• Thalassemias are the most common human

mongenic disorders related to deficiency in

production of the or globin chains.

• -Thalassemia Major/Cooley’s

Anaemia/Mediterranean Anaemia is the most

severe.

• Despite prenatal diagnosis in some countries,

> 50,000 children are born each year worldwide,

leading to the disease burden of this condition

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Short Stature, HSM

Maxillary Hyperplasia

BETA- THALASSAEMIA MAJOR•Present 3-6mths – 4 years

(β0/β+)

•Anaemia

•Jaundice

•Hepatosplenomegaly

•CCF

•Maxillary Hyperplasia & Frontal Bossing

•Frequent Infections

•Hypersplenism

•Poor Growth

•Death in 1st decadeUPTOSPAED2019

EFFECTS OF EXCESS α–GLOGIN FREE CHAINS

Excess free globin chains

Chain precipitates

Cell membrane damage

Red blood cells Bone marrow

Haemolysis Ineffective erythropoesis

ANAEMIA

Erythropoeitin

increased

Bone marrow

expansion

Skelatal changes

Hypermetabolic state

Extramedullary

haemopoeisis

Blood transfusion

Iron loading

Cardiac, Hepatic, Diabetic DEATHHSM

Iron absorption

increased

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SURVIVAL OF THALASSEMIA

MAJOR

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THE CONTINUED CHALLENGE

• Today β-Thalassemia patients

are living to 40-50 years but

patients are still dying

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Therapies for -Thalassemia

Supportive therapy▪ Transfusion

– Leukoreduction– Viral testing

▪ Iron overload– Deferoxamine (DFO)– Deferiprone– Deferasirox

▪ Endocrinopathies– Hormone replacement

▪ Osteoporosis– Osteoclast replacement– Vitamin D

Curative therapy▪ Hematopoietic stem cell transplantation

– Bone marrow– Cord blood– Unrelated donor– Non-myeloablative

▪ Experimental therapy– EPO– Fetal hemoglobin modifiers

(hydroxyurea, butyrate)– Antioxidants

Future therapy▪ Gene therapy

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Current and future therapies

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CONVENTIONAL THERAPIES

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Complications of iron overload

Non-transferrin-bound iron

circulates in the plasma

Excess iron promotes

the generation of free

hydroxyl radicals,

propagators of oxygen-

related tissue damage

Liver cirrhosis/

fibrosis/cancer

Insoluble iron complexes

are deposited in body

tissues and end-organ

toxicity occurs

Diabetes

mellitus

Growth

failure

Capacity of serum transferrin

to bind iron is exceeded

Iron overload

Cardiac

failureInfertility

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Dying RBC

Reticuloendothelial System

Free Iron

Liver

Heart

Endocrine organsCIRRHOSIS

ARRHYTHMIA HEART FAILURE

DIABETES

Delayed Puberty & InfertilityUPTOSPAED2019

SURVIVAL & COMPLICATIONS OF TM -

SICS• 977 Pts - 47% ♀

– If born > 1970 80% OS

– If born < 1970 60% OS

• Causes of death – 60% CCF– Probability of heart disease after age 10 – dec with

birth cohort

• Complcations (>1970)– Hypothyroid -11%

– Heart disease 13%

– Liver – HCV 85%, HBV 34%

– Diabetes 6%

– Hypogonadism – Male 55%, Female 50%

• If born 1985 -97 – most complication freeUPTOSPAED2019

CAUSES OF DEATH - ITALY

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NO. DEATHS OF TM PTS - UK

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NEWER THERAPIES1. Prenatal diagnosis – DNA based tests

– Shown to decrease Thal births in susceptible pop

2. HbF – switch– many studies with hydroxyurea, 5-Azacytidine,

Butyrate – works better in sickle cell disease because endpoint is not correction of anaemia (at best get 20-30% induction γ-globin gene)

3. Stem cell transplant– Lucarelli – good if MSD -95% survival

– MUD – 25% mortality – too high

4. Gene Therapy in intact mice – 2002

5. Gene Therapy in human blood progenitors -2004

6. 2008 – 1st patient successfully transplanted UPTOSPAED2019

Induction of HbF

• Largely ineffective

in Beta -

Thalassemia as

not sufficient to

become

transfusion

independant

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Allogeneic HSCT

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STEM CELL TRANSPLANTATION• MSD – Lucarelli

– good results, but only 30% TM have a MSD

• Adult >17yrs – 3 risk classes depending on chelation, hepatomegaly

& liver fibrosis

– More transplant related morbidity esp GvHD

– 67% EFS (vs 50% survival if no transplant)

• Sibling cord blood vs marrow– Need a specific program

• MUD – Transplant Related Morbidity & mortality too high for non-malignant disease – only 60% EFS

• Unrelated cord blood – high rate of rejection

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MSD Transplantation - Lucarelli

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Survival according to Class

• Defined

according to

the presence of

hepatomegaly

and portal

fibrosis

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Success depends on Chimerism

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Gene Therapy

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Fundamental Issues for Gene

therapy

• The source of haemopoietic stem/progenitor cells should

contain an adequate dose of early stem cells to ensure

long-term engraftment

• The choice of BM conditioning regimen should create

space for the transduced cells but reduce toxicity

• The dose of transduced HSPCs and the therapeutic level

of transgene expression which depends on the vector

output and average vector copy number

• The presence of a favourable BM microenvironment

enabling regeneration of complete haemopoiesis

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GENE THERAPY

• Lentivirus vector is derived from HIV-1 with SIN insulator at both ends

• Issues

– Stem cell transduction efficiency

– Stem cell recovery and engraftment

– Appropriate preparative regimen

– Would insulators improve safety?

– Do other regulatory elements have advantages

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GLOBIN GENE TRANSFER

• History

– 1960 – dream

– 1981 – prematurity – Cline experiment

– 1988 – hope & disillusion – Low expression in mice, low transfer, vector instability

– 2000 – efficacy• Lentiviral TNS9 vector

• Req – lineage restricted, erythroid specific, position independent, sustained over time

• Correction of anaemia, prevention of 20 organ damage, long term expression, peripheral differentiation, expression in human cells

– 2005 – safety – risk of insertional oncogenesis as in SCID trials

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2010• Cavazzna-Calvo reported first successful use of gene

therapy in a HbE/beta Thal patient

• The HSCs were tansduced ex-vivo with a lentivirus

vector carrying an extended -globin gene structure.

• Gradual stabilisation of the haemoglobin levels rendered

him transfusion independent for up to 6 years

• However transduced cell engraftment was associated

with clonal expansion of the erythroid cells with vector

insertion site at the HMGA2 locus – need to be followed

closely for possible adverse events

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MSKCC/NIH/UW TRIAL for Gene

Therapy

• >15yrs age

• No MSD

• Transduce normal globin gene using TNS9.3.55 lentivirus vector into CD34+ G-CSF mobilised cells from patient

• Non-myeloablative regimen

• Transplant transducted autologous stem cells

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Preliminary Results – NEJM 2018• 22 Patients with tranfusion dependant - Thalassemia

were enrolled in 2 Phase 1-2 studies

• CD34+ cells were mobilised and transduced with the

Lentiglobin BB305 vector

• The cells were re-infused after Busulphan myeloablative

conditioning

• All but 1 out of 13 non-0 0 genotype became

transfusion independent

• In 9 patients with a 0 0 genotype there was a 73%

reduction in transfusion requirements, and only 1 patient

became transfusion independent

• No clonal dominance related to the vector integration

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HAEMOPOIETIC & EMBRYONIC STEM

CELL CORRECTIONS IN MOUSE

MODELS

• Therapeutic vs reproductive cloning

– Somatic cell (fibroblast) nucleus is transferred

into an unfertilised enucleated egg

– Nuclear transfer embryo cultured in lab

– Blastocyst inner cell mass cultured to colonies

of Embryonic stem cells (ESC)

• Can be cultured indefinitely

• Mutations can be corrected

• Transplant cell back into host (no GvHD)

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Β – Thal Mouse

Derive EScells

Lentiviral Transduction

Human β-globin gene

transfer

Homologous recombination

Knock-in human β-globin

Genetic correction

of ES cells

Back into mouse

Both have been done & have been successful in correcting

anaemia in the Thal Mouse UPTOSPAED2019

• Globin gene transfer

– Adv

• all steps have been successful in animal models

• Clinical trials could be initiated soon

– Disadv

• Insertion of therapeutic genes at random sites

• Cells cannot be expanded before transplantation

• Therapeutic cloning

– Adv

• Site specific insertions can be expanded before

transplantation

– Disadv

• Not ever done for Haemoglobinopathy

• Ethical considerations for blastocyst == human being

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• Future

– Limit interaction between vector & neighbouring oncogenes – looking for ? A superior vector

– Efficient vector production

– Host conditioning – minimal conditioning vs low engraftment

– First need non-human primate studies

– Clinical transduction protocol of human CD34+ cells

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Β – Thal Mouse

Derive EScells

Lentiviral Transduction

Human β-globin gene

transfer

Homologous recombination

Knock-in human β-globin

Genetic correction

of ES cells

Back into mouse

Both have been done & have been successful in correcting

anaemia in the Thal Mouse UPTOSPAED2019

• Globin gene transfer

– Adv

• all steps have been successful in animal models

• Clinical trials could be initiated soon

– Disadv

• Insertion of therapeutic genes at random sites

• Cells cannot be expanded before transplantation

• Therapeutic cloning

– Adv

• Site specific insertions can be expanded before

transplantation

– Disadv

• Not ever done for Hbopathy

• Ethical considerations for blastocyst == human being

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Gene Editing Stem Cells

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Fetal Haemoglobin Revival

• Fetal Hb production only occurs in the fetus

• A gene called BCL11A represses this production

in adulthood

• By modifying the expression of this gene,

production of HbF could be derepressed

• Rationale behind the recent announced

CRISPR-based trial.

• Remains to be seen whether this approach is

able to cure Beta Thalassemia patients

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CONCLUSION

• Conventional therapy can polong life, but

as patients are living longer, morbidity

increases, and the cost is high

• The only cure at present is a HSCT with a

Matched Sibling donor at an early age

• Gene therapy is in its infancy, and at

present is out of reach for the majority of

Beta Thalassemia patients, most of whom

live in LMICUPTOSPAED2019