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.