Dual-regulated Lentiviral Vector for Gene Therapy of X-linked Chronic Granulomatosis
Authors: Maria Chiriaco, Giada Farinelli, Valentina Capo, Erika Zonari, Samantha Scaramuzza, Gigliola Di Matteo, Lucia Sergi Sergi, Maddalena Migliavacca, Raisa Jofra Hernandez, Ferdinando Bombelli, Ezio Giorda, Anna Kajaste-Rudnitski, Didier Trono, Manuel Grez, Paolo Rossi, Andrea Finocchi, Luigi Naldini, Bernhard Gentner, Alessandro Aiuti
Journal: Molecular Therapy (2014)

Regulated transgene expression may improve the safety and efficacy of hematopoietic stem cell (HSC) gene therapy. Clinical trials for X-linked chronic granulomatous disease (X-CGD) employing gammaretroviral vectors were limited by insertional oncogenesis or lack of persistent engraftment. Our novel strategy, based on regulated lentiviral vectors (LV), targets gp91phox expression to the differentiated myeloid compartment while sparing HSC, to reduce the risk of genotoxicity and potential perturbation of reactive oxygen species levels. Targeting was obtained by a myeloid-specific promoter (MSP) and posttranscriptional, microRNA-mediated regulation. We optimized both components in human bone marrow (BM) HSC and their differentiated progeny in vitro and in a xenotransplantation model, and generated therapeutic gp91phox expressing LVs for CGD gene therapy. All vectors restored gp91phox expression and function in human X-CGD myeloid cell lines, primary monocytes, and differentiated myeloid cells. While unregulated LVs ectopically expressed gp91phox in CD34+ cells, transcriptionally and posttranscriptionally regulated LVs substantially reduced this off-target expression. X-CGD mice transplanted with transduced HSC restored gp91phox expression, and MSP-driven vectors maintained regulation during BM development. Combining transcriptional (SP146.gp91-driven) and posttranscriptional (miR-126-restricted) targeting, we achieved high levels of myeloid-specific transgene expression, entirely sparing the CD34+ HSC compartment. This dual-targeted LV construct represents a promising candidate for further clinical development. Regulated transgene expression may improve the safety and efficacy of hematopoietic stem cell (HSC) gene therapy. Clinical trials for X-linked chronic granulomatous disease (X-CGD) employing gammaretroviral vectors were limited by insertional oncogenesis or lack of persistent engraftment. Our novel strategy, based on regulated lentiviral vectors (LV), targets gp91phox expression to the differentiated myeloid compartment while sparing HSC, to reduce the risk of genotoxicity and potential perturbation of reactive oxygen species levels. Targeting was obtained by a myeloid-specific promoter (MSP) and posttranscriptional, microRNA-mediated regulation. We optimized both components in human bone marrow (BM) HSC and their differentiated progeny in vitro and in a xenotransplantation model, and generated therapeutic gp91phox expressing LVs for CGD gene therapy. All vectors restored gp91phox expression and function in human X-CGD myeloid cell lines, primary monocytes, and differentiated myeloid cells. While unregulated LVs ectopically expressed gp91phox in CD34+ cells, transcriptionally and posttranscriptionally regulated LVs substantially reduced this off-target expression. X-CGD mice transplanted with transduced HSC restored gp91phox expression, and MSP-driven vectors maintained regulation during BM development. Combining transcriptional (SP146.gp91-driven) and posttranscriptional (miR-126-restricted) targeting, we achieved high levels of myeloid-specific transgene expression, entirely sparing the CD34+ HSC compartment. This dual-targeted LV construct represents a promising candidate for further clinical development.