1. Volume 12, Issue 5, Pages 860-866 (November 2005)
Long-term correction of hyperbilirubinemia in the Gunn Rat by repeated intravenous delivery of naked plasmid DNA into muscle 
Zhen Jia, István Dankó 
Molecular Therapy 
Volume 12, Issue 5, Pages (November 2005)
DOI: /j.ymthe
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

2. FIG. 1
HPLC analysis of bilirubin glucuronides in bile. Bile chromatogram from (A) an untreated Gunn rat, (B) a normal Wistar rat, (C) a Gunn rat expressing hUGT1A1 in muscle without immunosuppression, or (D) with FK506 and dexamethasone treatment on day 7 after the fifth intravenous injection. Results are representative of three animals in each experimental group. The eluate was monitored at 436 nm. The initial peak (*) represents solvent injection. Bilirubin diglucuronides are eluted between 8 and 10 min (B, C, D, peaks 1), monoglucuronides between 15 and 17 min (B, C, D, double peaks 2, two isomers), unconjugated bilirubin at 30–32 min (A, C, D, peaks 3). The minor diconjugate peak following peak 1 at approximately 10–11 min (B, C, D) represents bilirubin conjugates with other sugars [42].
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

3. FIG. 2
Effects of hUGT1A1 expression in Gunn rat muscle on serum bilirubin levels. pDNA was injected via (A) femoral artery or (B) great saphenous vein, with (hUGT1A1 + FK506) or without (hUGT1A1) immunosuppression. Arrows above graphs indicate time points of injections. Values are expressed as means ± SD of four animals. Controls (LacZ) were injected with pCI-LacZ. Dashed line indicates average serum bilirubin measured in normal Wistar rats. *P < 0.05, **P < 0.01 compared to lacZ-injected animals at the same time point. P values comparing serum bilirubin in animals injected intravenously with pcDNA3hUGT1A1 with and without immunosuppression were <0.01 at all time points except on days 0, 1, and 3 (P > 0.05).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

4. FIG. 3
Expression analysis of hUGT1A1 in Gunn rat muscle. Vein: gene delivery into great saphenous vein. Vein + FK: same as Vein, with immunosuppression. Artery: gene delivery into femoral artery without immunosuppression. (A) Western blot of Gunn rat muscle probed with polyclonal mouse anti-human UGT1A1 antibody at 1 and 4 weeks after gene delivery. Actin: β-actin internal control. (B) PCR of total DNA from Gunn rat muscle with primers amplifying a 438-bp segment of hUGT1A1. LacZ: animals injected with pCI-LacZ. hUGT1A1: animals injected with pcDNA3hUGT1A1. Plasmid control: amplification from pcDNA3hUGT1A1. (C) Semiquantitative RT-PCR of total muscle RNA after gene transfer into Gunn rat muscle. Experimental conditions same as in (B). GAPDH: internal control.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

5. FIG. 4
Development of antibodies against hUGT1A1. (A) Western blot of human liver probed with sera from three different animals, 4 weeks after intravenous injection of pcDNA3hUGT1A1 or pCI-LacZ. Each lane represents a single animal. (B) Western blots of human liver probed with sera from the same animal obtained at various time points after intravenous delivery of pcDNA3hUGT1A1. Top: sera from an animal without immunosuppression. Bottom: sera from an animal that received FK506/dexamethasone. Two animals tested exhibited similar patterns; the animal with more pronounced antibody response is shown.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

6. FIG. 5
Cellular immune response to hUGT1A1 expressed in muscle. (A) Immunohistochemical detection of CD4- and CD8-positive cells in Gunn rat muscles harvested 4 weeks after the last intravenous injection of pcDNA3hUGT1A1. Positive cells are stained black and are localized around the endomysium. (A1) Animals without immunosuppression. (A2) Animals treated with FK506/dexamethasone. Original magnification: 200×. (B) Quantitative analysis of lymphocytic infiltrate in muscle. Data are presented as average numbers ± SD of lymphocytes counted per 100 myofibers. **P < 0.01 compared to untreated animals; *P < 0.05 compared to treated animals without FK506/dexamethasone.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions