Functional Reconstruction of Oxidase Activity in X-linked Chronic Granulomatous Disease by Retrovirus-Mediated Gene Transfer

patients affected by X-linked Chronic Granulomatous Disease (CGD). CGD is a rare inherited disease that induces a profound predisposition to severe, often fatal infections. Mutations in the gp9l-phox (ph phagocytes; ox oxidase) protein, the large subunit of cytochrome b558 , lead to an X-linked inherited form of CGD (X-CGD) that accounts for the majority of the cases. CGD is a very suitable disease for a gene therapy approach, since the molecular defect is functionally relevant only in cells of the myelomonocytic lineage, and bone marrow transplantation has been curative in some cases. Furthermore, it has been reported that carrier individuals with as little as 10% of functional phagocutes are perfectly healthy. These observations suggest that transfer of the genetically correct gene in hematopoietic precursors followed by autologous transplantation should be curative of the disease. The experimental work presented in this thesis has been divided into three main sections: i) Characterization of the genetic defect in the cells from three X-CGD patients. Three different point mutations have been detected in three Italian X-CGD patients. All the mutations lead to a shift in the mRNA reading frame predicting the consequent formation of a truncated protein. ii) Construction of retroviral vectors for gp91-phox gene transfer and development of a quantitative PCR procedure for retrovirus titering. Four different retroviral vectors were constructed, in which transcription of the gp91-phox cDNA was driven either by the Moloney murine leukemia virus long terminal repeat (LTR), or by internal promoters (including the herpes simplex virus-thymidine kinase (HSV-TK) and cytomegalovirus immedite early (CMV IE) promoters and an interferon-y inducible promoter). A method for rapid titering of retroviral vectors was developed based on competitive PCR quantification of proviral DNA molecules formed in target cells at few hours after infection. iii) Transduction of the gp91-phox cDNA in myeloid cell lines, lymphoblastoid cell lines derived from X-CGD patients and in hematopoietic progenitors. The results obtained upon infection showed that all the four vectors were able to functionally reconstitute oxidase activity in X-CGD cell lines and primary hematopoietic cells. The retroviral construct expressing the therapeutic gene directly from the viral LTR scored as the most effective in directing expression of the gp91-phox cDNA. In addition, the Moloney LTR promoter strongly affected the transcription driven by the internal promoters in myeloid cell lines and primary myeloid cells obtained upon differentiation of hematopoietic progenitors. The results achieved represent a first step in a gene therapy programme aiming at the correction of the genetic defect by ex viva gene transfer into the hematopoietic cells of the patients