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Lentivirus-mediated gene transfer of uroporphyrinogen III synthase fully corrects the porphyric phenotype in human cells

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Abstract

Congenital erythropoietic porphyria (CEP) is an inherited disease due to a deficiency in the uroporphyrinogen III synthase, the fourth enzyme of the heme biosynthesis pathway. It is characterized by accumulation of uroporphyrin I in the bone marrow, peripheral blood and other organs. The prognosis of CEP is poor, with death often occurring early in adult life. For severe transfusion-dependent cases, when allogeneic cell transplantation cannot be performed, the autografting of genetically modified primitive/stem cells may be the only alternative. In vitro gene transfer experiments have documented the feasibility of gene therapy via hematopoietic cells to treat this disease. In the present study lentiviral transduction of porphyric cell lines and primary CD34+ cells with the therapeutic human uroporphyrinogen III synthase (UROS) cDNA resulted in both enzymatic and metabolic correction, as demonstrated by the increase in UROS activity and the suppression of porphyrin accumulation in transduced cells. Very high gene transfer efficiency (up to 90%) was achieved in both cell lines and CD34+ cells without any selection. Expression of the transgene remained stable over long-term liquid culture. Furthermore, gene expression was maintained during in vitro erythroid differentiation of CD34+ cells. Therefore the use of lentiviral vectors is promising for the future treatment of CEP patients by gene therapy.

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Abbreviations

ALA :

δ-Aminolevulinate

BMT :

Bone marrow transplant

CEP :

Congenital erythropoietic porphyria

CFC :

Colony-forming cell

EGFP :

Enhanced green fluorescent protein

Epo :

Erythropoietin

FL :

FLT3 ligand

HIV :

Human immunodeficiency virus

HSPC :

Hematopoietic stem/progenitor cell

IL :

Interleukin

LB :

Lymphoblastoid

LBCEP :

Lymphoblastoid cells from patients with congenital erythropoietic porphyria

LBN :

Lymphoblastoid cells from normal individuals

LTC-IC :

Long-term culture initiating cell

LTCM :

Long-term culture medium

LTR :

Long terminal repeat

MOI :

Multiplicity of infection

mPB :

Cytokine-mobilized peripheral blood

RM+CK :

RM-B00 medium + cytokines

SCF :

Stem cell factor

SCID :

Severe combined immunodeficiency

TEEW :

TRIP-ΔU3-EF1α-EGFP-WPRE

TEU :

TRIP-ΔU3-EF1α-UROS

TEUW :

TRIP-ΔU3-EF1α-UROS-WPRE

TPO :

Thrombopoietin

UROS :

Uroporphyrinogen III synthase

WPRE :

Woodchuck hepatitis virus posttranscriptional regulatory element

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Acknowledgements

We thank P. Charneau, A. Dubart-Kupperschmidt, and F. Pflumio for donating the TRIPΔU3-EF1α vector, J. Reiffers and B. Dazey for the normal CD34+ mPB cells, and M. Morey for the porphyric cells. This work was supported by grants from Association Française contre les Myopathies and Conseil Régional d'Aquitaine.

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Correspondence to H. de Verneuil.

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Géronimi, F., Richard, E., Lamrissi-Garcia, I. et al. Lentivirus-mediated gene transfer of uroporphyrinogen III synthase fully corrects the porphyric phenotype in human cells. J Mol Med 81, 310–320 (2003). https://doi.org/10.1007/s00109-003-0438-7

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