pros and cons of gene therapy in ß-thalassemia presentation… · •many ß-thalassemic patients...
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Pros and Cons of Gene Therapy in ß-Thalassemia
ß-Thalassemia: established and potential new ß-Thalassemia: established and potential new therapeutic approachestherapeutic approaches
•Many ß-thalassemic patients are treated with blood transfusion and iron chelation. However, the complexity of this disorder and new findings suggest that this conventional approach could be improved upon.
•Jak2 inhibitors and hepcidin agonists might soon be utilized to limit ineffective erythropoiesis, EMH, splenomegaly, limit iron absorption and improve the transfusional therapy.
•Gene transfer offers an alternative approach to bone marrow transplant since utilizing autologous hematopoietic stem cells avoids the limitations of finding a compatible donor and prevents GVHD.
•The only definitive cure is bone marrow transplantation. But this approach presents severe limitations such as finding a compatible bone marrow donor and avoiding GVHD.
Gene therapy of ß-thalasemiaGene therapy of ß-thalasemia
•Gene transfer using a viral vector (lentiviral vector)Gene transfer using a viral vector (lentiviral vector)
•Conditioning of the patient (complete or partial)Conditioning of the patient (complete or partial)
•Preclinical and clinical dataPreclinical and clinical data
•Potential pitfallsPotential pitfalls
•Poor chimerism (genetically modified vs. Poor chimerism (genetically modified vs. untransduced cells)untransduced cells)
•GenotoxicityGenotoxicity
•Potential solutionsPotential solutions
Hematopoietic Hematopoietic stem cellsstem cells
ReinfusionReinfusion TransductionTransduction
Vector carrying the Vector carrying the therapeutic genetherapeutic gene
Gene Therapy Schematic ApproachGene Therapy Schematic Approach
Therapeutic Levels of Hb in Mice Affected by Therapeutic Levels of Hb in Mice Affected by
ß-Thalassemia ß-Thalassemia
3´LTR3´LTR5’LTR5’LTR ß-globinß-globin LCRLCR
TNS9TNS9
WTWT
Transfusion Transfusion independent independent
Thal MajorThal Major Thal MajorThal Major
+ vector+ vector
Hb 13-15g/dLHb 13-15g/dL
Hb 2-4g/dLHb 2-4g/dL
Correction of Sickle Cell DiseaseCorrection of Sickle Cell Disease
Potential problemsPotential problems
GenotoxicityGenotoxicityThe possibility that the chromosomal random integration of the vector can lead to
insertional mutagenesis
Poor chimerism Poor chimerism (genetically modified vs. untransduced cells)(genetically modified vs. untransduced cells)
The challenge of obtaining therapeutic levels of genetically modified hematopoietic stem cells in beta-thalassemia patients
Chimerism, power of the vector and genotype of the Chimerism, power of the vector and genotype of the patientpatient
Bone marrow vs peripheral blood chimerism of genetically modified cellsBone marrow vs peripheral blood chimerism of genetically modified cells
It is expected that the selection will be more effective as the vector will be able to express high levels of the beta-globin gene at single copy, irrespective of the chromosomal site of integration
Selection?Selection?
Peripheral bloodPeripheral blood
Bone marrowBone marrow
Gene Therapy Clinical Trial for SCID-X1 Halted Due to Insertional Gene Therapy Clinical Trial for SCID-X1 Halted Due to Insertional Mutagenesis Mutagenesis
ATGATG
LMO2 gene LMO2 gene
+ Oncoretroviral Vector Encoding the T and NK cells + Oncoretroviral Vector Encoding the T and NK cells γγc-c-Cytokine Receptor SubunitCytokine Receptor Subunit
Potential solutions:Potential solutions:
InsulatorsInsulatorsSuicide geneSuicide gene
Targeted integrationTargeted integration
Ankyrin-ST9W Lentiviral Vector
ß-globin gene LCR
e+ ßp HS2 HS3 HS4AnkST9W
3’ SIN-LTR + Ankyrin sequenceSilent mutations
WPRECMV-5’ LTR
They might prevent “genome toxicity” or insertional mutagenesis
Insulators are elements thought to reduce expression interference and favor chromatin opening
A Pre-Clinical Approach
To establish a simple method to analyze, after gene transfer, a large number of thalassemic erythroid cells derived from patients with different mutations
Exapansio In vitro Exapansio In vitro (Phase 1)(Phase 1)
Transduction of human erythroid precursors isolated from peripheral blood
Ficoll separation
Erythroid Differentiation Erythroid Differentiation (Phase 2)(Phase 2)
1 Kb1 Kb
ee++ pp
SASA
RRERRE
SDSD
ß-globin geneß-globin gene
LTRLTR
Locus Control RegionLocus Control Region
Ankyrin-sinLTRAnkyrin-sinLTRHS2HS2 HS3HS3 HS4HS4
840 bp840 bp 1069 bp1069 bp1308 bp1308 bp
+/-
ConclusionsConclusions
•However, some concerns are still present regarding the possibility that these vectors might However, some concerns are still present regarding the possibility that these vectors might trigger the activity of oncogenes.trigger the activity of oncogenes.
•It is unclear what is the level of chimerism that need to be achieved.It is unclear what is the level of chimerism that need to be achieved.
•Additional preclinical studies using patient cells might be able to address these questions.Additional preclinical studies using patient cells might be able to address these questions.
•Gene transfer might offer an alternative approach to bone marrow transplant since utilizing Gene transfer might offer an alternative approach to bone marrow transplant since utilizing autologous hematopoietic stem cells avoids the limitations of finding a compatible donor and autologous hematopoietic stem cells avoids the limitations of finding a compatible donor and prevents GVHD.prevents GVHD.
•Preclinical studies in mice are extremely promising.Preclinical studies in mice are extremely promising.