title: High-level erythroid-specific gene expression in primary human and murine hematopoietic cells with self-inactivating lentiviral vectors.
abstract: Use of oncoretroviral vectors in gene therapy for hemoglobinopathies has been impeded by low titer vectors, genetic instability, and poor expression. Fifteen self- inactivating (SIN) lentiviral vectors using 4 erythroid promoters in combination with 4 erythroid enhancers with or without the woodchuck hepatitis virus postregulatory element (WPRE) were generated using the enhanced green fluorescent protein as a reporter gene. Vectors with high erythroid-specific expression in cell lines were tested in primary human CD34(+) cells and in vivo in the murine bone marrow (BM) transplantation model. Vectors containing the ankyrin-1 promoter showed high-level expression and stable proviral transmission. Two vectors containing the ankyrin-1 promoter and 2 erythroid enhancers (HS-40 plus GATA-1 or HS-40 plus 5-aminolevulinate synthase intron 8 [I8] enhancers) and WPRE expressed at levels higher than the HS2/beta-promoter vector in bulk unilineage erythroid cultures and individual erythroid blast-forming units derived from human BM CD34(+) cells. Sca1(+)/lineage(-) Ly5.1 mouse hematopoietic cells, transduced with these 2 ankyrin-1 promoter vectors, were injected into lethally irradiated Ly5.2 recipients. Eleven weeks after transplantation, high-level expression was seen from both vectors in blood (63%-89% of red blood cells) and erythroid cells in BM (70%-86% engraftment), compared with negligible expression in myeloid and lymphoid lineages in blood, BM, spleen, and thymus (0%-4%). The I8/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-globin gene. Thus, modular use of erythroid-specific enhancers/promoters and WPRE in SIN-lentiviral vectors led to identification of high-titer, stably transmitted vectors with high-level erythroid-specific expression for gene therapy of red cell diseases.
authors.affiliation: Children's Hospital Los Angeles, University of Southern California School of Medicine, Los Angeles 90027, USA.
authors.forename: F
authors.initials: F
authors.lastname: Moreau-Gaudry
authors.forename: P
authors.initials: P
authors.lastname: Xia
authors.forename: G
authors.initials: G
authors.lastname: Jiang
authors.forename: N P
authors.initials: NP
authors.lastname: Perelman
authors.forename: G
authors.initials: G
authors.lastname: Bauer
authors.forename: J
authors.initials: J
authors.lastname: Ellis
authors.forename: K H
authors.initials: KH
authors.lastname: Surinya
authors.forename: F
authors.initials: F
authors.lastname: Mavilio
authors.forename: C K
authors.initials: CK
authors.lastname: Shen
authors.forename: P
authors.initials: P
authors.lastname: Malik
mesh_terms: D000818:Animals; D018952:Antigens, CD34; D001854:Bone Marrow Cells; D016026:Bone Marrow Transplantation; D004742:Enhancer Elements, Genetic; D015672:Erythroid Precursor Cells / Q000378:metabolism*; D015870:Gene Expression; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors / Q000378:metabolism* / Q000592:standards; D049452:Green Fluorescent Proteins; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D017919:Hepatitis B Virus, Woodchuck / Q000235:genetics; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D023421:Models, Animal; D011401:Promoter Regions, Genetic; D012323:RNA Processing, Post-Transcriptional / Q000235:genetics; D014161:Transduction, Genetic / Q000379:methods* / Q000592:standards; D005719:gamma-Globulins / Q000235:genetics / Q000378:metabolism
chemical_list: D018952:Antigens, CD34; D008164:Luminescent Proteins; D005719:gamma-Globulins; D049452:Green Fluorescent Proteins