1. Volume 9, Issue 4, Pages 548-556 (April 2004)
Long-Term Lowering of Plasma Cholesterol Levels in LDL-Receptor-Deficient WHHL Rabbits by Gene Therapy 
Hanna M Kankkonen, Elisa Vähäkangas, Robert A Marr, Timo Pakkanen, Anniina Laurema, Pia Leppänen, Johanna Jalkanen, Inder M Verma, Seppo Ylä-Herttuala 
Molecular Therapy 
Volume 9, Issue 4, Pages (April 2004)
DOI: /j.ymthe
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

2. Fig. 1
Vector design showing the rabbit LDLR- and GFP-expressing provector constructs. The internal promoters driving the transgenes are indicated by arrows (CMV, human cytomegalovirus promoter, and LSP, liver-specific promoter). All vectors utilized the HIV-1 central polypurine tract (cppt) and the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), while the LSP-LDLR and LSP-GFP vectors also utilized the LE6 hepatic-specific enhancer. Long terminal repeat (LTR) sequences with self-inactivating (SIN) deletions (∇) are shown on the 3′ ends. Ψ, packaging signal.
Molecular Therapy 2004 9, DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

3. Fig. 2
Analysis of liver cell specificity of the lentiviral vectors. GFP expression (A–D) from (A, B) CMV-GFP- and (C, D) LSP-GFP-transduced (m.o.i. 5) HepG2 cells (left) and WHHL rabbit skin fibroblasts (right) was detected using fluorescence microscopy 3 weeks after the gene transfer. Binding and uptake of fluorescent DiI-LDL (10 μg/ml, 4 h) to (E, F) CMV-LDLR- and (G, H) LSP-LDLR-transduced (m.o.i. 5) HepG2 cells (left) and WHHL rabbit skin fibroblasts (right) was used as a measure of LDLR expression. Cells were photographed using fluorescence microscopy 4 weeks after the gene transfer. Original magnification (A–D) 20×, (E–H) 40×.
Molecular Therapy 2004 9, DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

4. Fig. 3
Analysis of LDLR functionality in vitro. (A) 125I-LDL degradation analysis (10 μg/ml, 12 h) in HepG2 cells transduced with LSP-LDLR or CMV-LDLR (m.o.i. 5). GFP-transduced cells were used as controls. The data were collected from four independent experiments performed 1–4 weeks after the gene transfer. (B) Analysis of the uptake of fluorescent DiI-LDL (10 μg/ml, 12 h) into the lentivirus-transduced HepG2 cells. Percentage of the DiI-LDL-positive cells in LSP-LDLR- or CMV-LDLR-transduced (m.o.i. 5) cells was compared to that of DiI-LDL-positive GFP-transduced control cells. *P < 0.05; ***P < (one-way ANOVA with Tukey's posttest).
Molecular Therapy 2004 9, DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

5. Fig. 4
Transgene expression after in vivo injection of lentiviral vectors (1 × 109 IU) into the portal vein of WHHL rabbits. Representative photomicrographs of a liver biopsy sample (A) 2 weeks after CMV-GFP transduction and (B) 4 weeks after LSP-GFP transduction. Paraffin-embedded sections were stained with anti-GFP antibody (dilution 1:100). GFP-positive hepatocytes are indicated with arrows. Bar, 200 μm. (C) Analysis of gene transfer efficiency after LSP-GFP and CMV-GFP transduction into WHHL rabbits. **P < 0.01; ***P < (one-way ANOVA with Tukey's posttest) in comparison to controls. (D) RT-PCR analysis of HepG2 and WHHL rabbit skin fibroblast cells transduced in vitro with LSP-LDLR or CMV-LDLR (m.o.i. 5) and representative WHHL rabbit liver biopsy samples 4 weeks–1 year after the LSP-LDLR gene transfer (n = 13) into the portal vein. Nested-RT-PCR fragments. Lanes: (1) molecular weight (MW) marker; (2) HepG2/LSP-LDLR RNA (0.5 μg); (3) HepG2/CMV-LDLR RNA (0.5 μg); (4) WHHL rabbit fibroblast/LSP-LDLR RNA (0.5 μg); (5) WHHL rabbit fibroblast/CMV-LDLR RNA (0.5 μg); (6) liver RNA (2 μg) 4 weeks after the LSP-LDLR gene transfer; (7) liver RNA (2 μg) 1 year after the LSP-LDLR gene transfer; (8) positive control, LSP-LDLR expression plasmid; (9) RT− control, reverse transcription step omitted from the reaction; (10) no RNA control, RNA omitted from the reaction. (E) PCR (DNA) and nested RT-PCR (RNA) fragments from a representative set of rabbit biodistribution tissue samples 4 weeks after the LSP-LDLR (n = 13) gene transfer. Lanes: (1) MW marker; (2) liver DNA (2 μg); (3) spleen DNA (3 μg); (4) spleen RNA (2 μg); (5) lung DNA (3 μg); (6) lung RNA (2 μg); (7) muscle DNA (3 μg); (8) testis DNA (3 μg); (9) positive control, LSP-LDLR expression plasmid; (10) negative (−) control, polymerase enzyme omitted from the reaction.
Molecular Therapy 2004 9, DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

6. Fig. 5
Effect of direct in vivo lentiviral vector-mediated LDLR gene transfer on plasma lipids in WHHL rabbits. Changes in plasma (A) cholesterol and (B) triglyceride levels after LSP-LDLR (n = 13) and CMV-GFP (n = 2) or LSP-GFP (n = 9) control vector-mediated gene transfer to the portal vein of WHHL rabbits. Data are expressed as percentage (mean ± SEM) change in comparison to the pretreatment values in each treated animal. Two-way ANOVA for multiple comparisons was used for statistical analyses.
Molecular Therapy 2004 9, DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

7. Fig. 6
Immunohistological characterization of liver samples transduced with LSP-LDLR and LSP-GFP lentiviral vectors 4 weeks after the gene transfer. (A, D) Immunostaining for macrophages using RAM-11 antibody (1:1000 dilution). (B, E) Immunostaining for T cells using MCA-805 antibody (1:1000 dilution). (C, F) Hematoxylin–eosin stain. Bar, 200 μm.
Molecular Therapy 2004 9, DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions