1. Volume 18, Issue 7, Pages 1302-1309 (July 2010)
Combined Paracrine and Endocrine AAV9 mediated Expression of Hepatocyte Growth Factor for the Treatment of Renal Fibrosis 
Stephanie Schievenbusch, Ingo Strack, Melanie Scheffler, Roswitha Nischt, Oliver Coutelle, Marianna Hösel, Michael Hallek, Jochen WU Fries, Hans-Peter Dienes, Margarete Odenthal, Hildegard Büning 
Molecular Therapy 
Volume 18, Issue 7, Pages (July 2010)
DOI: /mt
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
(a,b) Transduction efficiency and (c,d) transcriptional activity of rAAV2-CMV-GFP, rAAV8-CMV-GFP, and rAAV9-CMV-GFP in liver and kidney. 5 × 1011 particles were intravenously injected into 6-week-old male COL4A3-knockout mice (n = 6). Mice were killed 2 weeks after injection, and liver and kidneys were harvested. Total DNA (for transduction efficiency) and total RNA (for expression analyses) were extracted from liver and kidney, and subjected to qPCR analyses. Vector genomes were normalized to mCRP (a,b), whereas HPRT was used to normalize transgene expression (c,d). *Statistical significance (P < 0.05). Control animals received 0.9% NaCl solution. RU, relative units.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
Anti-GFP immunohistology of renal and hepatic tissue in rAAV2-CMV-GFP-, rAAV8-CMV-GFP-, and rAAV9-CMV-GFP-treated animals. Liver and kidney samples of rAAV2-CMV-GFP-, rAAV8-CMV-GFP-, and rAAV9-CMV-GFP-treated animals (see Figure 1) were formalin-fixed upon organ harvesting. Formalin-fixed and paraffin-embedded tissue sections were stained for GFP using a rabbit anti-GFP antibody (Abcam, Cambridge, UK) and counterstained with hematoxylin. GFP, green fluorescent protein.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
Comparison of rAAV9-CMV-GFP and rAAV9-CMV-Ksp-GFP. 5 × 1011 genomic particles of single-stranded rAAV9-CMV-GFP or 2.5 × 1011 genomic particles of self-complementary rAAV9-CMV-Ksp-GFP were administered by intravenous injection into 6-week-old male COL4A3-knockout mice (n = 6). Mice were killed 2 weeks after injection, and formalin-fixed and paraffin-embedded tissue sections of spleen, lung, heart, kidneys, and liver were stained for GFP using a rabbit anti-GFP antibody (Abcam, Cambridge, UK) and counterstained with hematoxylin. GFP, green fluorescent protein.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
hHGF serum level of COL4A3-knockout mice following intravenous injection of rAAV-CMV-Ksp-hHGF. 5 × 1011 vector particles of rAAV8-CMV-Ksp-hHGF (rAAV8, n = 8) and rAAV9-CMV-Ksp-hHGF (rAAV9, n = 8) were injected into 4-week-old male COL4A3-knockout mice. Control animals were treated with an equal volume of a preparation of empty AAV8 capsids (mock, n = 6). Mice were killed 5.5 weeks after injection. Blood was collected, and kidneys and liver were harvested. hHGF serum levels were assayed by enzyme-linked immunosorbent assay. Values are means of duplicates. *Significant differences in expression levels (P < 0.05). hHGF, human hepatocyte growth factor.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
Effect of hHGF overexpression on transcriptional activity of genes involved in fibrogenic processes. Total RNA was isolated from kidneys of rAAV-CMV-Ksp-hHGF-treated animals (see Figure 4) and assayed by qPCR for expression of (a) COL1A1, (c) SMA, and (e) PDGFR-β. Values normalized to the reference HPRT mRNA are shown as relative units (RU). Expression values were correlated to hHGF serum levels (Figure 4), and results are presented in b for COL1A1, in d for SMA, and in f for PDGFR-β. Correlation was considered as significant with *P < 0.05 and **P < COL1A1, collagen 1α1; hHGF, human hepatocyte growth factor; PDGFR-β, platelet-derived growth factor receptor-β; SMA, smooth muscle actin.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

7. Figure 6
Histological evaluation of human hepatocyte growth factor (hHGF) overexpression following intravenous rAAV-CMV-Ksp-hHGF injection in COL4A3-knockout mice. (a) Staging of extracellular matrix deposition in renal sections of COL4A3-knockout mice following rAAV8- and rAAV9-mediated hHGF expression based on the Gomori staining of renal tissue. (b) Representative examples of Gomori staining of transversal renal sections at different stages of fibrosis. Control mice were injected with an equal amount of empty AAV8 capsids. **Statistical significance (P < 0.01).
Molecular Therapy  , DOI: ( /mt )
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

8. Figure 7
Simultaneous paracrine and endocrine human hepatocyte growth factor (hHGF) delivery for the treatment of chronic renal disease by rAAV via intravenous route of administration. Intravenous application of rAAV9-CMV-Ksp-hHGF results in a simultaneous paracrine and endocrine production of the antifibrogenic factor hHGF by renal and hepatic tissue, respectively. At the kidney, the site of tissue damage in chronic renal disease, hHGF is expressed by the renal tubular epithelial cells and can counteract the fibrogenic activity of TGF-β. In addition, hHGF expressed by hepatocytes and secreted in its inactive form (pHGF) is delivered via the circulation to the kidney, where it is activated and supports the paracrine hHGF effects. In our proof-of-concept study, this combined paracrine and endocrine approach was highly efficient in ameliorating the course of chronic renal disease implicating it as potential treatment strategy. aHGF, activated HGF; pHGF, proHGF.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions