Pros and Cons of Gene Therapy in -Thalassemia -Thalassemia: - - PowerPoint PPT Presentation

Pros and Cons of Gene Therapy in ß-Thalassemia

ß-Thalassemia: established and potential new ß-Thalassemia: established and potential new therapeutic approaches therapeutic 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-thalasemia Gene 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 data

Preclinical and clinical data

• Potential pitfalls

Potential pitfalls

• Poor chimerism (genetically modified vs.

Poor chimerism (genetically modified vs. untransduced cells) untransduced cells)

• Genotoxicity

Genotoxicity

• Potential solutions

Potential solutions

Hematopoietic Hematopoietic stem cells stem cells Reinfusion Reinfusion Transduction Transduction Vector carrying the Vector carrying the therapeutic gene therapeutic gene

Gene Therapy Schematic Approach Gene Therapy Schematic Approach

Therapeutic Levels of Hb in Mice Affected by Therapeutic Levels of Hb in Mice Affected by ß-Thalassemia ß-Thalassemia

3´LTR 3´LTR 5’LTR 5’LTR ß-globin ß-globin LCR LCR

TNS9 TNS9

WT WT Transfusion Transfusion independent independent Thal Major Thal Major Thal Major Thal Major + vector + vector Hb 13-15g/dL Hb 13-15g/dL Hb 2-4g/dL Hb 2-4g/dL

Correction of Sickle Cell Disease Correction of Sickle Cell Disease

Potential problems Potential problems

Genotoxicity Genotoxicity

The 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 patient patient

Bone marrow vs peripheral blood chimerism of genetically modified cells Bone 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 blood Peripheral blood Bone marrow Bone 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

ATG ATG

LMO2 gene LMO2 gene + Oncoretroviral Vector Encoding the T and NK cells + Oncoretroviral Vector Encoding the T and NK cells γ γc- c- Cytokine Receptor Subunit Cytokine Receptor Subunit

Potential solutions: Potential solutions: Insulators Insulators Suicide gene Suicide gene Targeted integration Targeted integration

Ankyrin-ST9W Lentiviral Vector

ß-globin gene LCR e+ ßp HS2 HS3 HS4

AnkST9W 3’ SIN-LTR + Ankyrin sequence Silent mutations WPRE CMV-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 Kb 1 Kb e e+

+

p p SA SA RRE RRE SD SD

ß-globin gene ß-globin gene

LTR LTR

Locus Control Region Locus Control Region

Ankyrin-sinLTR Ankyrin-sinLTR

HS2 HS2 HS3 HS3 HS4 HS4 840 bp 840 bp 1069 bp 1069 bp 1308 bp 1308 bp

+/-

Conclusions Conclusions

• 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.