High-level erythroid-specific gene expression in primary human and murine hematopoietic cells with self-inactivating lentiviral vectors

Use of oncoretroviral vectors in gene therapy for hemoglobinopathies has be en impeded by low titer vectors, genetic instability, and poor expression. Fifteen self-inactivating (SIN) lentiviral vectors using 4 erythroid promot ers in combination with 4 erythroid enhancers with or without the woodchuck hepatitis virus postregulatory element (WPRE) were generated using the enh anced green fluorescent protein as a reporter gene. Vectors with high eryth roid-specific expression in cell lines were tested in primary human CD34(+) cells and in vivo in the murine bone marrow (BM) transplantation model. Ve ctors containing the ankyrin-1 promoter showed high-level expression and st able proviral transmission. Two vectors containing the ankyrin-1 promoter a nd 2 erythroid enhancers (HS-40 plus GATA-1 or HS-40 plus 5-aminolevulinate synthase intron 8 [18] enhancers) and WPRE expressed at levels higher than the HS2/beta -promoter vector in bulk unilineage erythroid cultures and in dividual erythroid blast-forming units derived from human BM CD34+ cells. S ca1(+)/lineage(-) Ly5.1 mouse hematopoietic cells, transduced with these 2 ankyrin-1 promoter vectors, were injected into lethally irradiated Ly5.2 re cipients. Eleven weeks after transplantation, high-level expression was see n from both vectors in blood (63%-89% of red blood cells) and erythroid cel ls in BM (70%-86% engraftment), compared with negligible expression in myel oid and lymphoid lineages in blood, BM, spleen, and thymus (0%-4%). The 18/ HS-40-containing vector encoding a hybrid human beta/gamma -globin gene led to 43% to 113% human gamma -globin expression/copy of the mouse alpha -glo bin gene. Thus, modular use of erythroid-specific enhancers/promoters and W PRE in SIN-lentiviral vectors led to Identification of high-titer, stably t ransmitted vectors with high-level erythroid-specific expression for gene t herapy of red cell diseases. (C) 2001 by The American Society of Hematology .