1. Volume 13, Issue 1, Pages 88-97 (January 2006)
The inhibitory effects of anticoagulation on in vivo gene transfer by adeno-associated viral or adenoviral vectors 
Joerg Schuettrumpf, Jianxiang Zou, Yi Zhang, Alexander Schlachterman, Yi-Lin Liu, Shyrie Edmonson, Weidong Xiao, Valder R. Arruda 
Molecular Therapy 
Volume 13, Issue 1, Pages (January 2006)
DOI: /j.ymthe
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

2. FIG. 1
Inhibition of AAV-2 transduction by anticoagulants in HepG2 cells. Prior to cell transduction, AAV vector encoding human F.IX at a multiplicity of infection of 10,000 was incubated for 15 min at room temperature with serial dilutions of UFH (140 U/mg), LMWH (100 U/mg), hirudin, TAP, mTAP, and saline as control. F.IX levels were determined in the conditioned medium 48 h posttransduction. The inhibition rate was determined in triplicate experiments by comparing with F.IX levels of controls that average 150–200 ng/ml/48 h. Data are shown as mean values ± SEM.
Molecular Therapy  , 88-97DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

3. FIG. 2
Circulating F.IX levels in plasma samples of C57BL/6 mice injected with anticoagulant drugs as a function of time after tail vein delivery of AAV-2- F.IX. (A, B, and D) Mice were injected with AAV-2-F.IX at doses of 4 × 1012 vg/kg within 5–10 min post-injection of UFH at doses of 0.1 (n = 2), 1 (n = 8), or 10 mg/kg (n = 3); LMWH at 1 mg/kg (n = 7); hirudin at doses of 0.25 (n = 4) or mg/kg (n = 2); or TAP at 1 mg/kg or mTAP at 1 mg/kg (n = 4 for each group) and compared with saline-injected mice (n = 12). (C) Mice received AAV-2-F.IX at doses of 1 × 1013 vg/kg following UFH (n = 2) or hirudin (n = 2) at the doses indicated or saline (n = 2). Data are shown as mean values ± SEM. P values were calculated for drug-treated groups compared to saline-control animals with ANOVA for repeated measurements with Dunnett's multiple comparison adjustment: *P < 0.05 or **P < 0.005.
Molecular Therapy  , 88-97DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

4. FIG. 3
Immunofluorescence staining of murine liver 16 weeks following injection of AAV-2 vectors at doses of 4 × 1012 vg/kg in the presence of anticoagulant drugs. Immunofluorescent staining for human F.IX in liver sections of saline- or anticoagulant-injected C57BL/6 mice is presented. Representative sections from the injected muscle are shown. Excitation of fluorescence tags revealed both periportal and parenchymal signals in saline- or LMWH- (1 mg/kg) treated mice, but markedly reduced periportal signals in mice injected with UFH, TAP, or mTAP (each 1 mg/kg) or hirudin (0.25 mg/kg). Original magnification ×200. Values shown represent means ± SEM. P values were calculated using Student's t test comparing averages of sections for total positive hepatocytes of drug- with saline-treated animals and by χ2 test with Yate's continuity correction comparing number of positively staining perivascular and parenchymal cells between saline- and drug-injected mice: *P < 0.05, **P < 0.005.
Molecular Therapy  , 88-97DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

5. FIG. 4
Effects of anticoagulant drugs on AAV expression using vectors of distinct designs and different murine models. Animals received UFH (1 mg/kg), hirudin (0.25 mg/kg), TAP (1 mg/kg), or saline via the tail vein. (A) C57BL/6 mice (n = 4 per group) were injected with AAV-2 encoding A1AT under the control of albumin promoter at doses of 4 × 1012 vg/kg. (B) BALB/c mice (n = 4 per group) received AAV-2-F.IX at doses of 4 × 1012 vg/kg. (C) C57BL/6 mice (n = 4 per group) were injected with AAV-5-F.IX at doses of 1.2 × 1012 vg/kg. (D) C57BL/6 mice received AAV-8-F.IX at doses of 1 × 1011 vg/kg (n = 4 per group). Pvalues for comparisons between drug-treated mice and saline controls were calculated by ANOVA for repeated measurements with Dunnett's adjustment: *P < 0.001.
Molecular Therapy  , 88-97DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

6. FIG. 5
Effects of anticoagulant drugs on adenoviral vector-mediated gene expression. C57BL/6 mice (n = 4–6 mice per group) were tail vein injected with UFH (1 mg/kg), hirudin (0.25 mg/kg), TAP (1 mg/kg), or saline. Vector was administered at doses indicated within 10 min post-drug injection. Student's t test was used for comparison between saline and each anticoagulant drug group.
Molecular Therapy  , 88-97DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

7. FIG. 6
AAV-2-mediated gene transfer in genetic models for F.IX deficiency or factor V Leiden. (A) Hemophilia B mice with F.IX activity <1% due to large gene deletion (n = 4) or mice transgenic for F.IX-R180T (n = 4) were compared with mice transgenic for F.IX wild type (n = 6). Animals received AAV-2 encoding A1AT as transgene. (B) Female mice homozygous for FVL (+/+) or FV wild type (−/−) were injected via the tail vein with AAV-2-F.IX at a dose of 1 × 1012 vg/kg (n = 4/group). Student's t test was used for group comparisons. The P values obtained after week 6 are represented as *P < 0.05.
Molecular Therapy  , 88-97DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions