Lentiviral Mediated Gene Therapy For X-Linked Agammaglobulinemia

X-linked agammaglobulinemia(XLA)is a primary immunodeficiency disease caused by a mutation in the BTK gene.BTK plays a vital role in pro-B and mature B cell antigen receptor(BCR)signaling transduction.XLA patients suffer from developmental defects of the B cells and do not produce mature B cells and immunoglobulins.Therefore,XLA patients are susceptible to micorbial infections.At present,intravenous injection of immunoglobulin is the main treatment for XLA,which has disadvantages such as high cost,shortage of blood products and lifelong maintenance.There is an urgent need for more effective treatments for XLA.Gene therapy is a new strategy for genetic diseases,which is considered an innovative way to cure XLA.Hematopoietic stem cell-based ex vivo gene therapy strategies have been successfully applied in the clinical treatment of a variety of genetic diseases,but this treatment approach may increase the risk of infection complications and toxicity for patients due to the strong chemotherapy conditioning.In addition,ex vivo gene therapy approaches require complex facilities,as well as highly technical and professional handling of the gene-modified cells.Given these obstacles,we have focused on in vivo gene therapy research for XLA.The lentivirus vector as an integration genetic vector has been engineered to carry a B-cell specific promoter desigh,either B29 or B29CD19,to drive the expression of BTK protein in B lineage of cells.We transferred a therapeutic BTK gene using lentivectors in vivo into the xid mice,which had been pre-treated with short-term immunosuppression and AMD3100,a hematopoietic stem cell mobilizing agent,to enhance long term transgene persistence.In vitro,the lentivirus carrying the BTK gene and the B-specific promoters B29 and B29CD19 have high packing efficiency,showed tissue specificity in B cells,and could normally initiate the expression of BTK protein.In vivo,the proportion of B cells(B220/CD45R~+)in peripheral blood lymphocytes of B29 group mice increased by17.34±7.614%.The proportion of B cells(B220/CD45R~+)in peripheral blood lymphocytes of B29CD19 group mice was increased by 17.17±3.721%;and the serum immunoglobulin lgM and lgG3 in two B-specific promoter group mice showed significant improvement(p﹤0.01)after receiving in vivo gene therapy of LV-/B29/B29CD19-BTK.In order to further evaluate the effectiveness of in vivo gene therapy,the hematopoietic stem cells from the treated mice were transplanted into na?ve xid mice which had been treated with lethal irradiation.In four months after transplantation,we detected 20%-30%restored B cells in the peripheral blood of the xid mice;and the serum immunoglobulin lg M and lg G3 of the xid mice showed significant improvement(p﹤0.01).All of the treated mice survived without tumor formation during 6 mouths of observation.In summary,these preliminary results demonstrate the feasibility and effectiveness of this novel in vivo gene therapy strategy.