gene therapy for sickle cell disease

WIDYA SETYANINGTYAS1106014892GENETIKA TERAPAN

Stem Cell Therapy for Sickle Cell Disease: Transplantation and Gene Therapy Mark C. Walters 2005

OUTLINE

Sickle Cell Disease

Hematopoietic Stem Cell Transplantation (HSCT)

Allogeneic Stem Cell Transplantation

Autologous Stem Cell Transplantation (Gene Transfer)

Conclusion

Sickle Cell Disease (SCD)Sickle cell disease (SCD) is a genetic

disorder (autosomal recessive) that affect the red blood cells.

Red blood cells curve into a sickle shape due to hemoglobin S.

Sickle cell occurs when a person has gene for sickle hemoglobin (HbS).

In normal person they has gene for normal hemoglobin (HbA).

Sickle Cell Disease (SCD)

Structure of Red Blood Cells (RBC)

• Red blood cells are small and biconcave in shape as they lack a nucleus.

• Red blood cells contain several hundres hemoglobin molecule to carry the oxygen.

Structure Hemoglobino Hemoglobin are globular proteins . Each hemoglobin consists of four polypeptide and heme group.

o  Four polypeptida, two α-globin chains and two β-globin chains .

o Heme groups contain positively-charged iron (Fe2+) molecules which can reversibly bind to oxygen 

What are the causes of SCD ?

The molecular basis for SCD is mutation in β-globin gene in chromosome 11, A to T transversion in the 6th codon.

This simple transversion changes a polar glutamic acid to a non-polar valine in the β globin chain.

Non polar valine make formation of HbS polymers/agregation that cause a multitude of changes in RBC.

What Are The Causes of SCD ?

GAG

Glutamic acid

GTG

Valine

What Are The Causes of SCD ?

WHAT IS HEMATOPOIETIC STEM CELL TRANSPLANTATION (HSCT) ?

Hematopoietic stem cell transplantation (HSCT) is the transplantation of multipotent hematopoietic stem cells (HSC).

HSC usually derived from bone marrow, peripheral blood, or umbilical cord blood.

Hematopoietic Stem Cells (HSC) from Bone Marrow

HEMATOPOIETIC STEM CELL TRANSPLANTATION (HSCT)

Allogeneic stem cell

transplantation

•Transplantation using allogeneic stem cells from a normal donor (HLA-matches sibling).

Autologousstem cell

transplantation

•Transplantation using autologous stem cells which have been corrected by transfer of a normal gene (viral vectors).

Allogeneic Stem Cell Transplantation

Allogeneic stem cell transplantation involves two people: the (healthy) donor and the (patient) recipient.

Donors must have a HLA/Human Leukosit Antigen) type that matches the recipient.

Mismatches HLA type (i.e. HLA-A, HLA-B, or HLA-C) increase the risk of graft rejection.

Allogeneic Stem Cell Transplantation

Two Types Allogeneic Stem Cell Transplantation

Myeloablative allogeneictransplant

Non-myeloablativeallogeneic transplant

Myeloablative Allogeneic

Transplant

Myeloablative allogeneic transplant : uses very high doses of chemotherapy or radiation therapy before transplantation.

Before transplantation: use busulfan (BU), cyclophosphamide (CY), After transplantation : use cyclosporine (CSP) for immunosuppression..

The purpose of which is to help eradicate the patient's bone marrow cells and to suppress immune reactions.

Result After Myeloablative

Allogeneic Transplant Before transplantation, patient SCD had an abnormal hemoglobin levels < 12-16 g/dL. But, after ± 6 months after transplantation, patient SCD had normal hemoglobin levels. About 90-95% transplantation successfull, only 5-10% that had graft rejection.Patient SCD who had a stroke before transplantation had no further strokes after transplantation.

Non-myeloablativeAllogeneic

transplant

A non-myeloablative allogeneic transplant : is a newer treatment approach using lower doses of chemotherapy and radiation.Allows to have less intensive chemotherapy before transplantation, which are too low to eradicate all the bone marrow cells of a recipient.

Result After Non-myeloablative

Allogeneic Transplant

35% patient experienced graft rejection. GVHD (graft-versus host disease) were also more frequent (about 20%). This approach has been accompanied by a higher rate of graft rejection and overall a lower probability of cure.

Myeloablative VS Non-myeloablative

Myeloablative

oUses high doses of chemotherapy or radiation.oMore toxic and can have serious side effects ex: loss of hair, infertility.oGraft will not be rejected.

Non-myeloablativ

eoUses a less doses of chemotherapy or radiation.

o Less toxicity

oHigh risk of graft rejection (GVHD).

Autologous Stem Cell Transplantation

Transplantation using autologous stem cells which have been corrected by transfer of a normal gene.

Normal gene insertion of an anti-sickling globin gene (βAS3)

Insertion of an anti-sickling globin gene (βAS3) use lentiviral vectors.

Lentiviral Vectors

• .Lentiviral vector : type of virus that can infect both dividing and nondividing cells because their virus “shell” can get through the intact membrane of the nucleus of the target cells.Lentiviral can be used to provide highly effective gene therapy because can change the expression of their target cell's gene.

HIV is a very effective lentiviral vector

Structrure of HIV

Genetic Organization of HIV

HIV Lentiviral Vector Expresses Anti-Sickling Globin (βAS3)

To obtain a lentiviral gene therapy vector, anti-sickling globin gene (βAS3) insert into sequence.LTRs are necessary for integration anti-sickling globin gene (βAS3) into the chromosome cell target.

Mechanism of Autologous Stem Cell Transplantation

Hematopoietic stem cell from bone marrow patient are removed

Use HIV as vector

Normal gene/ anti-sickling globin gene (βAS3) is inserted into vector.

Patient with condition

chemotherapy Vector with normal gene inserted into stem cell from patient

Cell grown in medium culture

Stem cell with normal gene are

reintroduced to the body/patient

Inside the body, genetically altered cells produce normal hemoglobin.

Mechanism of Autologous Stem Cell Transplantation

Things That Must be Considered

1 •Optimizing the cells target

2 •Optimizing transduction of by vector selection and design

3 •Optimizing repopulating activity of target cells after successful transduction.

4 •Optimizing stable expression of globin gene expression.

Conclusion

• Hematopoietic stem cell transplantation (HSCT) have curative potential for SCD, and these continue to be actively investigated.

• Allogeneic stem cell transplantation has a track record of success if applied under optimal conditions.

• Autologous stem cell transplantation has the potential for a lower toxicity profile compared to Allogeneic transplantation.

References

• Mann R, Mulligan RC, Baltimore D. 1983. Construction of aretrovirus packaging mutant and its use to produce helper-freedefective retrovirus. Cell. Vol 33:153—159.•Mark C. Walters, Arthur W. Nienhuis, and Elliott Vichinsky. 2002. Novel therapeutic approaches in sickle cell disease. Hematology. Vol 23:10—34.•Paul S. Frenette and George F. Atweh .2007. Sickle cell disease: old discoveries, new concepts, and future promise. The Journal of Clinical

Investigation. Vol 117: 850--858.•Franklin.H. Eipsten. Pathogenesis and treatment for sickle cell disease. 1997. The New England Journal of Medicine. Vol 337: 762—769.