2. • >350,000 children are born each year with a severe inherited Hb disorder
(WHO, June 2008 ; Weatherall DJ, Blood 2010)
Births with a
pathological Hb disorder
per 1,000 live births
Epidemiology of Hemoglobin disorders
b
αα
b
Hemoglobin
(Hb)
Red Blood Cells
(RBC)
3. β-hemoglobinopathies
b
αα
b
fetal genes adult genes
HbA
(adult)
β-thalassemia
reduction or absence of β-globin
>200 different mutations
HbA
b
αα
b
α
α
α
α
α
α
α
g g
αα
HbF
(fetal)
chr11
Hemoglobin
bg
Sickle-cell disease (SCD)
HbS
b
αα
b★★
production of a mutant β-globin
★ SCD Mutated residue
HbS
b
αα
b★★
HbS
b
αα
b★★
HbS
b
αα
b★
4. Reduced or absent synthesis of β-globin
chain (α-globin precipitates) Intramedullary death of red
blood cell (RBC) precursors Anemia
β-thalassemia
Erythroid development
6. • Red blood cell transfusions: not definitive, side effects
• Pharmacological treatments (pain-killers, hydroxyurea): not definitive, not effective in
all the patients, side effects
• Allogeneic hematopoietic stem cell (HSC) transplantation: definitive but limited by the
donor availability
Therapeutic approaches for β-hemoglobinopathies
HSC (“long-lasting”)
RBC
(half-life:
120 days)
7. Gene therapy and genome editing for β-hemoglobinopathies
HSCβ-globin expressing
lentiviral vectors
Transplantation of autologous, genetically corrected hematopoietic stem cells is an
alternative therapy for patients lacking a compatible donor
(Miccio et al., PNAS 2008; Cavazzana et al., Nature, 2010)
Genome editing tools
12. Conclusions
• Efficacy:
- Transfusion independence observed in b+-thalassemic patients.
- Higher levels of b globin production may be necessary to
correct the b0-thalassemic and SCD phenotype.
Modify the endogenous locus (genome editing)
• Safety:
- No adverse events have been reported so far.
- LV integrate into the genome and have the potential to
deregulate genes.
13. Genome editing-based approaches for β-
hemoglobinopathies
- Correct the b-globin gene mutations
★
★ SCD or b-thal mutation
Endogenous β-globin locus
fetal genes adult genes
14. Genome editing-based approaches for β-
hemoglobinopathies
- Increase fetal g-globin levels
★ SCD or b-thal mutation
Persistence of Fetal Hemoglobin benefit
thalassemic and SCD patients
★
Endogenous β-globin locus
fetal genes adult genes
g g
αα
18. Genome editing-based approaches for β-
hemoglobinopathies
- Increase fetal g-globin levels
★ SCD or b-thal mutation
★
Endogenous β-globin locus
fetal genes adult genes
19. Genome editing-based approaches for β-
hemoglobinopathies
- Increase fetal g-globin levels
★ SCD or b-thal mutation
★
Endogenous β-globin locus
fetal genes adult genes
g-globin repressors
20. Genome editing-based approaches for β-
hemoglobinopathies
- Increase fetal g-globin gene expression
★ SCD or b-thal mutation
★
Endogenous β-globin locus
fetal genes adult genes
g-globin repressors
g g
αα
21. non-homologous end-joining
targeted
disruption
*
Genome editing-based approaches for β-
hemoglobinopathies
- Increase fetal g-globin gene expression
Canver, Nature 2016
SCD cells
ZFN
CRISPR/Cas9
RBC
g repressor
g-repressors= BCL11A, LRF
genes or genomic regions
bound by HbF repressors
Increased g-globin
production
KO mutation of Fetal g-globin repressors Traxler, Nature Medicine 2016
22. Conclusions
• Efficacy:
- Correct the b-globin gene mutations (SCD): ideal strategy, but
poorly efficient in hematopoietic stem cells (HSC).
- Increase fetal g-globin gene expression: high and curative levels
of endogenous g-globin, not tested HSC
Increase/test genome editing efficiency in HSC
• Safety:
- Theoretically “targeted” genome editing.
- Safety studies to evaluated the off-target activity are mandatory
Improve/test the specificity of the genome editing tools
