Gene Therapy Of Chronic Granulomatous Disease(CGD) Based On Lentiviral Vector

Chronic granulomatous disease（CGD）is a congenital immunodeficiency caused by functional deficiency of NADPH oxidase in phagocytes.So far,the antibacterial and azole antifungal medicine are effective in the treatment of CGD,however,the widely use of these medicine prone to induce bacterial resistance.Currently,hematopoietic stem cell transplantation（HSCT）is the only curable strategy for CGD.The difficulty in finding a matched donor,graft-versus-host disease（GVHD）,low efficiency of transplantation,and the high risk of death are known limitations to HSCT.Gene therapy（GT）is an advanced therapeutic strategy for CGD.Thus besides HSCT,GT has become the most promising treatment for CGD.Insertional mutagenesis and transgene silencing may be caused by inappropriate vector use.In addition,short survival time of gene-corrected HSCs,the tedious transplantation process,and high cost are all concerning issues associated with gene therapy in CGD.The selection and design of vector,the use of tissue-specific promoter,and the mode of vector administration are crucial factors to the safety,effectiveness,sustainability and economy of gene therapy.In this study,an improved high-safety lentivirus vector（LV）system together with three type of promoters:universal promoter EF1α,myeloid-specific promoters miR223and CD68,were used to drive the expression of either a green fluorescent protein（GFP）reporter gene or the CYBB gene（cytochrome B-245 beta chain,or p91-phox）.The safety,tissue specificity and persistence of the vector and promoter were investigated using HSCT in X-CGD mice(X-CGD;B6.129S-Cybb^tm1Din/J).Also,for the purpose to reduce the cost and operation difficulty of gene therapy,the feasibility of direct injection of lentiviral vector into the tail vein was explored in the X-CGD mice.The results of this study are summarized below:1.The effectiveness and tissue specificity of the three promoters were investigated in vitro in C57 mouse HSCs and in various human cell lines.The results showed that the EF1αpromoter facilitated the expression of GFP in all types of cells.The miR223 and CD68 promoters displayed tissue specificity,while the miR223 showed higher specificity than the CD68 promoter.Furthermore,in vivo experiments in X-CGD mice showed consistent results as with the in vitro experiments.Based on the X-CGD mouse pneumonia model,it was found that all three promoters could restore the antibacterial function of NADPH oxidase under high transgenic conditions,while in the low transgenic conditions,the EF1αpromoter showed the best antibacterial activity,and the miR223promoter worked better than the CD68 promoter.Any toxicity was not observed in the X-CGD mice after LV-mediated gene therapy in 66 weeks.2.This study further developed an“in vivo”intravenous（iv）LV gene transfer strategy to overcome the high cost,complicated process and high risk associated with the traditional HSCT gene therapy strategy.Mice were pretreated with immunosuppressive preconditioning through either a nonmyeloablative radiotherapy（6Gy）approach or chemotherapy（busulfan+cyclophosphamide+dexamethasone）to avoid inactivation of the iv injected LV by the mouse immune system.The results showed that the“in vivo”approach could restore the production of gp91-phox and reactive oxygen species（ROS）in peripheral blood phagocytes of the X-CGD mice.To increase gene transfer efficiency,a second injection of LV was given,which illustrated increased expression of gp91-phox and ROS,and did not detect the formation of anti-LV-P24 antibody.However,the vector copy number（VCN）of LV in peripheral blood gradually decreased after 12 weeks.To increase HSC gene transfer,the CGD mice were treated with AMD3100 to mobilize HSCs before LV injection.Bone marrow HSCs from the LV treated CGD mice were retransplanted into new CGD mice 12 weeks after gene therapy.The results showed that gp91-phox positive HSCs were successfully retransplanted into recipient mice,suggesting that chemotherapy conditioning and AMD3100 mobilization could substantially improve the efficiency of LV gene delivery into HSCs.This improved iv LV gene delivery strategy could reduce both the risk and the cost of CGD gene therapy with great potential in future translational applications.3.An advanced LV design（4711-LV）with reduced immunogenicity was designed to avoid the use of harmful chemotherapy pretreatment to CGD patients.The feasibility of 4711-LV“in vivo”regimen was explored without immunosuppression.After 4 weeks treatment via iv LV gene delivery,the expression of gp91-phox and ROS was restored in peripheral blood phagocytes in the CGD mice.in order to further improve the gene delivery efficiency of the 4711-LV in vivo,a variety of small molecule drugs（rapamycin,cyclosporine A,cyclosporine H,prostaglandin E2,2-valproic acid and diphenyl phosphate）were tested using the X-CGD mice model to investigate their effective on the gene delivery efficiency.Through flow cytometry,some small molecules such as rapamycin,cyclosporine A,prostaglandin E2,2-propylpropionic acid and diphenyl phosphate could effectively improve the gene delivery efficiency of 4711-LV into HSCs,and rapamycin has the most potential.In conclusion,both miR223 and CD68 promoters displayed myeloid-specificity,while miR223 is superior to CD68 promoter in specificity.The pneumonia model indicated that under low-transgenic condition,EF1αexhibited the best antibacterial activity.Tail vein injection experiments indicated that radiotherapy or chemotherapy conditioning is essential for the iv injection of the VSVG-pseutyped LV.In addition,AMD3100 mobilization could improve the LV gene delivery into HSCs.In the absence of immunosuppression,the newly developed 4711-LV could efficiently deliver genes into peripheral blood cells in the X-CGD mice.Small molecules such as rapamycin could effectively improve the efficiency of in vivo LV gene delivery into HSCs.