Gene Therapy of a Mouse Model of Protoporphyria with a Self-Inactivating Erythroid-Specific Lentiviral Vector without Preselection

Abstract

Successful treatment of blood disorders by gene therapy has several complications, one of which is the frequent lack of selective advantage of genetically corrected cells. Erythropoietic protoporphyria (EPP), caused by a ferrochelatase deficiency, is a good model of hematological genetic disorders with a lack of spontaneous in vivo selection. This disease is characterized by accumulation of protoporphyrin in red blood cells, bone marrow, and other organs, resulting in severe skin photosensitivity. Here we develop a self-inactivating lentiviral vector containing human ferrochelatase cDNA driven by the human ankyrin-1/beta-globin HS-40 chimeric erythroid promoter/enhancer. We collected bone marrow cells from EPP male donor mice for lentiviral transduction and injected them into lethally irradiated female EPP recipient mice. We observed a high transduction efficiency of hematopoietic stem cells resulting in effective gene therapy of primary and secondary recipient EPP mice without any selectable system. Skin photosensitivity was corrected for all secondary engrafted mice and was associated with specific ferrochelatase expression in the erythroid lineage. An erythroid-specific expression was sufficient to reverse most of the clinical and biological manifestations of the disease. This improvement in the efficiency of gene transfer with lentiviruses may contribute to the development of successful clinical protocols for erythropoietic diseases.

Overexpression and role of the ATPase and putative DNA helicase RuvB-like 2 in human hepatocellular carcinoma

“…Vesicular stomatitis virus G protein (VSV-G) pseudotyped lentivectors were produced by triple-transient transfection of 293T cells. 22 Titers were determined by the transduction of 293T cells through the serial dilution of the lentiviral supernatant and were analyzed for EGFP expression 5 days later. Finally, HuH7 cells were transduced with lentiviral vectors at a multiplicity of infection of 20 and/or 10 in Dulbecco/Vogt modified Eagle's minimal essential medium (DMEM)/10% fetal bovine serum (FBS) medium with 8 g/ml protamine sulfate (Sigma, St. Louis, MO).…”

Lentiviral vectors: optimization of packaging, transduction and gene expression

“…The addition of the γ -globin intron sequences to the ankyrin-1 promoter has further augmented vector expression [118]. Moreover, the enhancement for LV-mediated erythroid gene expression, driven by another chimeric promoter/enhancer element (ankyrin-1/α-globin HS40), has also been reported in a model of erythropoietic protoporphyria where the ferrochelatase (FECH) cDNA was used in gene repair experiments [119].…”

Microglia used as vehicles for both inducible thymidine kinase gene therapy and MRI contrast agents for glioma therapy

“…30 The construct with the TK-GFP gene under the control of the HSP70 promoter was isolated after bglII and SalI digestions. The insertion in the lentivirus PCS ferroch 31,32 was realized after BamHI and XhoI digestions. The transduction was performed with the microglial cell line grown in Dulbecco's modified Eagle's medium (DMEM)-fetal calf serum (FCS) medium: 5 Â 10 5 cells in suspension were incubated with 2 ml of viral supernatant collected at 24 h added of 4 mg/ml polybrenne (Gibco BRL, Carlsbad, CA, USA) corresponding to a MOI 15.…”