Gene Therapy

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Somatic Gene Therapy;Gene Therapy of X linked Severe combined immunodeficiency by use of gammaretroviral vector : 

Somatic Gene Therapy;Gene Therapy of X linked Severe combined immunodeficiency by use of gammaretroviral vector V. Onur YILMAZ 130104002


X-SCID A recessive trait found in X chromosome No proper growht of T-cells and Natural Killer cells Disfunctional B-cells. Patients starts suffering from diseases with in 3 months of birth and die in first year of birth.


X-SCID There is a deficiency at gene coding region of γ chain at common cytokine receptors. γ chain


X-SCID If there is a donor for bone-marrow transplantation, over 90% survival rate. Donors should be matched as genotypically But What if there is not? Will they die? Somatic gene therapy!

Retroviral transduction basics : 

Retroviral transduction basics Virus producing cell lines In this research, PG113 cell lines are used. Provides gag and pol genes Retroviral vectors Vectors containing gene of interest Contains Env protein for packaging Self inactivation.

Slide 6: 

Map of pMFG retroviral vector MCS

MethodPatients : 

MethodPatients 4 children with ages of 10 months, 10 months, 3 months and 33 months are choosen. No matched bone-marrow donor Permission from family.

MethodsProcedures : 

MethodsProcedures mRNA isolation convert to cDNA from healty gene Clone into Transduction retroviral vector Bone Marrow Transplantation Control for Efficiency and further tests

Procedures : 

Procedures Cloning of γ chain cDNA with promoter pPGK into pMFG retroviral vector

Slide 10: 

PG113 Cells Transfection with pMFG TransfectedPG113 Cells starts producing viruses Coat empty 6well plates with retronectin Add Bone-Marrow Cells 34+ isolated from Patients. Work triplicate. Culture conditions: serum free X-VIVO 10 medium , 1% Human Serum Albumin, 300µg/L stem cell factor, 100µg/L thrombopoietin, 20µg/L IL-3 and 300µg/L Flt-3 ligand. Keep at 5% Carbondioxie incubators for 40 h Add virus supernatant for 3 times to BM cells after 40h, for 56h following Inject transduced BM cells to Patients after 96h of incubation.

Results : 

Results CD34+ BM cells from Patient 2 in culture condition expression of γ chain. Analyzed before transplantation.

Results : 

Results After transplantation, raise in T cells numbers along the weeks

Results : 

Results Number of Natural killer cells along the weeks

Results : 

Results T cell receptor variable region β chain repertoir after induction with mitogens Results gained after a year of transplantation.

Discussion : 

Discussion Recovery of patients Fully functional immune system after 1-2 years Medication was stopped after 1-2 years of transplantation at two patient Patients turned back to social life No chemotherapy before transplantation No major side effects, only imflammation during T cell emergence.(cytokine mediated) No insertional activation of proto-oncogenes. There is minor chance, have not been observed yet.

References : 

References Gene therapy of X-linked severe combined immunodeficiency by use of a pseudotyped gammaretroviral vectorThe Lancet, Volume 364, Issue 9452, Pages 2181-2187H.Gaspar, K.Parsley, S.Howe, D.King, K.Gilmour, J.Sinclair, G.Brouns, M.Schmidt, C.Von Kalle, T.Barington

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