Infectious delivery of a 135-kb LDLR genomic locus leads to regulated complementation of low-density lipoprotein receptor deficiency in human cells  Richard Wade-Martins, Yoshinaga Saeki, E Antonio Chiocca  Molecular Therapy  Volume 7, Issue 5, Pages 604-612 (May 2003) DOI: 10.1016/S1525-0016(03)00060-1 Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 1 Conversion of the LDLR BAC into infectious vectors. (A) Sequence analysis of a 135-kb BAC insert located the LDLR locus and confirmed the presence of all 18 exons (marked as vertical lines) and the native promoter region, including the sterol response elements (SREs). The BAC contains the 45-kb LDLR transcribed region flanked by approximately 45 kb of 5′ and 45 kb of 3′ sequence. (B) The two retrofitting constructs used to convert the BAC into infectious vectors. pHG contains the HSV-1 amplicon elements oris and pac. In addition pEHHG contains the latent origin of replication (oriP) and the EBNA-1 gene, both episomal retention elements from EBV. Molecular Therapy 2003 7, 604-612DOI: (10.1016/S1525-0016(03)00060-1) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 2 pHSV-LDLR and pHSV/EBV-LDLR efficiently deliver an intact, functional 135-kb insert by infection. (A) Plasmid rescue analysis of infected MRC-5V2 human fibroblast cells 48 h postinfection shows intact delivery and recovery of the 149- and 156-kb constructs in E. coli. (B) Infectious delivery of pHSV-LDLR and pHSV/EBV-LDLR to the ldlr-deficient CHO ldl−/−a7 cell line results in LDLR expression assayed by uptake of fluorescently labeled DiI-LDL. Molecular Therapy 2003 7, 604-612DOI: (10.1016/S1525-0016(03)00060-1) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 3 pHSV/EBV-LDLR restores specific LDLR function to wild-type levels in CHO ldl−/−a7 cells. (A) GFP reporter gene analysis shows very efficient pHSV/EBV-LDLR infection of CHO ldl−/−a7 cells. Control infections of pEHHG were performed at the same m.o.i. (B) Quantitation of specific LDLR activity by measuring LDL binding and uptake shows LDLR function to be restored to wild-type levels in CHO ldl−/−a7 cells 72 h postinfection. Nonspecific binding was determined in the presence of a 50-fold excess of unlabeled LDL and subtracted from total binding to give specific binding. Means are from three independent experiments, each performed in duplicate; error bars are +1 SEM. Molecular Therapy 2003 7, 604-612DOI: (10.1016/S1525-0016(03)00060-1) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 4 pHSV/EBV-LDLR is retained episomally and expressed long term in rodent cells. (A) Plasmid rescue recovers only intact pHSV/EBV-LDLR from two clonal CHO ldl−/−a7 lines. (B) The episomal pHSV/EBV-LDLR is maintained at a copy number of ∼10–17/cell. (C) pHSV/EBV-LDLR has restored the ability of the cells to take up DiI-LDL. (D) Quantitative analysis shows that the clonal cell lines have specific LDLR expression levels broadly similar to those of wild-type CHO cells. Nonspecific binding was determined in the presence of a 50-fold excess of unlabeled LDL and subtracted from total binding to give specific binding. Means are from three independent experiments, each performed in duplicate; error bars are +1 SEM. All the data shown for (A) to (D) were collected after 3 months of continuous culture. Molecular Therapy 2003 7, 604-612DOI: (10.1016/S1525-0016(03)00060-1) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 5 Infectious delivery and expression of the 135-kb LDLR genomic DNA locus in human FH patient primary fibroblasts. (A) Primary fibroblast cell lines FH-486 and FH-488 show negligible specific binding or uptake of LDL, compared to wild-type primary human fibroblasts (MRC-9). Nonspecific binding and internalization were determined in the presence of a 50-fold excess of unlabeled LDL. (B) Infection of FH primary fibroblasts by pHSV-LDLR and pHSV/EBV-LDLR is highly efficient, as assayed by GFP reporter gene expression. (C) Specific LDLR expression in FH fibroblasts following pHSV-LDLR and pHSV/EBV-LDLR infection. Means are from three independent experiments, each performed in duplicate; error bars are +1 SEM. Molecular Therapy 2003 7, 604-612DOI: (10.1016/S1525-0016(03)00060-1) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 6 Specific LDLR expression from the infectious genomic DNA vectors is repressed by elevated sterol levels. (A) Patient primary fibroblast FH-488 cells were infected with iBAC-LDLR vectors and LDL binding and uptake were assayed after incubation in the absence or presence of sterols (12 μg/ml cholesterol and 0.6 μg/ml 25-hydroxycholesterol). Nonspecific binding was determined in the presence of a 50-fold excess of unlabeled LDL and subtracted from total binding to give specific binding. Means are from three independent experiments, each performed in duplicate; error bars are +1 SEM. *P < 0.05 and **P < 0.01 by t test. (B) The presence of sterols did not alter the infectivity of the amplicons nor reporter gene (GFP) expression. Molecular Therapy 2003 7, 604-612DOI: (10.1016/S1525-0016(03)00060-1) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions