51. Dual-regulated Lentiviral Vector for Gene Therapy of X-linked Chronic Granulomatosis
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Ferdinando Bombelli, Didier Trono, Andrea Finocchi, Luigi Naldini, Raisa Jofra Hernandez, Gigliola Di Matteo, Giada Farinelli, Paolo Rossi, Alessandro Aiuti, Valentina Capo, Bernhard Gentner, Ezio Giorda, Maria Chiriaco, Lucia Sergi Sergi, Maddalena Migliavacca, Samantha Scaramuzza, Erika Zonari, Manuel Grez, Anna Kajaste-Rudnitski, Chiriaco, M., Farinelli, G., Capo, V., Di Matteo, G., Zonari, E., Scaramuzza, S., Sergi Sergi, L., Migliavacca, M., Hernandez, R. J., Bombelli, F., Giorda, E., Kajaste Rudnitski, A., Trono, D., Grez, M., Rossi, P., Finocchi, A., Naldini, Luigi, Gentner, B., and Aiuti, Alessandro
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congenital, hereditary, and neonatal diseases and abnormalities ,Myeloid ,Transgene ,Genetic enhancement ,Genetic Vectors ,Antigens, CD34 ,Biology ,Granulomatous Disease, Chronic ,medicine.disease_cause ,Cell Line ,Viral vector ,Mice ,hemic and lymphatic diseases ,Drug Discovery ,microRNA ,Genetics ,medicine ,Animals ,Humans ,Myeloid Cells ,Molecular Biology ,Cells, Cultured ,Settore MED/38 - Pediatria Generale e Specialistica ,Pharmacology ,Membrane Glycoproteins ,Settore BIO/11 ,Lentivirus ,Hematopoietic Stem Cell Transplantation ,NADPH Oxidases ,Hematopoietic stem cell ,Genetic Therapy ,Hematopoietic Stem Cells ,Combined Modality Therapy ,Molecular biology ,3. Good health ,Disease Models, Animal ,MicroRNAs ,medicine.anatomical_structure ,Cell culture ,NADPH Oxidase 2 ,Cancer research ,Molecular Medicine ,Original Article ,Carcinogenesis - Abstract
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 gp91(phox) 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 gp91(phox) expressing LVs for CGD gene therapy. All vectors restored gp91(phox) expression and function in human X-CGD myeloid cell lines, primary monocytes, and differentiated myeloid cells. While unregulated LVs ectopically expressed gp91(phox) in CD34(+) cells, transcriptionally and posttranscriptionally regulated LVs substantially reduced this off-target expression. X-CGD mice transplanted with transduced HSC restored gp91(phox) 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.