Skip to main content
NCBI home page
Molecular Therapy logoLink to Molecular Therapy
. 2010 Apr 27;18(7):1302–1309. doi: 10.1038/mt.2010.71

Combined Paracrine and Endocrine AAV9 mediated Expression of Hepatocyte Growth Factor for the Treatment of Renal Fibrosis

Stephanie Schievenbusch 1, Ingo Strack 1, Melanie Scheffler 1, Roswitha Nischt 2, Oliver Coutelle 3,4, Marianna Hösel 3,4, Michael Hallek 3,4, Jochen WU Fries 1, Hans-Peter Dienes 1, Margarete Odenthal 1, Hildegard Büning 3,4
PMCID: PMC2911253  PMID: 20424598

Abstract

In chronic renal disease, tubulointerstitial fibrosis is a leading cause of renal failure. Here, we made use of one of the most promising gene therapy vector platforms, the adeno-associated viral (AAV) vector system, and the COL4A3-deficient mice, a genetic mouse model of renal tubulointerstitial fibrosis, to develop a novel bidirectional treatment strategy to prevent renal fibrosis. By comparing different AAV serotypes in reporter studies, we identified AAV9 as the most suitable delivery vector to simultaneously target liver parenchyma for endocrine and renal tubular epithelium for paracrine therapeutic expression of the antifibrogenic cytokine human hepatocyte growth factor (hHGF). We used transcriptional targeting to drive hHGF expression from the newly developed CMV-enhancer-Ksp-cadherin-promoter (CMV-Ksp) in renal and hepatic tissue following tail vein injection of rAAV9-CMV-Ksp-hHGF into COL4A3-deficient mice. The therapeutic efficiency of our approach was demonstrated by a remarkable attenuation of tubulointerstitial fibrosis and repression of fibrotic markers such as collagen1α1 (Col1A1), platelet-derived growth factor receptor-β (PDGFR-β), and α-smooth muscle actin (SMA). Taken together, our results show the great potential of rAAV9 as an intravenously applicable vector for the combined paracrine and endocrine expression of antifibrogenic factors in the treatment of renal failure caused by tubulointerstitial fibrosis.

Introduction

Terminal renal failure is a life-threatening disease controllable only by chronic dialysis or renal transplantation. Over the past decade, the prevalence and incidence of chronic renal failure have increased continuously to >150,000 new cases per year in the United States, predominantly driven by the rise in obesity, hypertension, and diabetes in the aging Western populations.1 As the first pathophysiological alteration in the course of chronic renal disease, dysfunction of glomerular filtration occurs causing hyperfiltration and proteinuria. As a consequence, the glomerular basement membrane thickens and inflammation is induced. This is followed by tubulointerstitial fibrosis, which in turn is associated with inflammatory cell infiltration, myofibroblastic precursor transdifferentiation and proliferation, and with extracellular matrix (ECM) accumulation in the tubulointerstitium.2,3

The main mediator triggering fibrogenic processes of chronic injuries is transforming growth factor (TGF)-β.2 TGF-β's fibrogenic activity is balanced by hepatocyte growth factor (HGF), a cytokine which possesses morphogenic, mitogenic, and motogenic functions.4 Under physiological conditions, liver, kidney, and spleen produce low amounts of HGF. Upon acute damage, HGF and its receptor c-met rapidly become upregulated and stimulate epithelial organ regeneration, thereby counteracting TGF-β-induced tissue fibrosis.5 This protective activity, however, is lost in chronic damage, mainly as a result of downregulated HGF expression.6 Thus, HGF has been used therapeutically as a recombinant protein to interfere with fibrogenic processes, especially in chronic renal disease.7 However, a major drawback of this approach is the rapid clearance of HGF in the circulation with a half-life of only 3–5 minutes.8 In order to achieve prolonged therapeutically relevant HGF levels, alternative strategies are required.

Recent advances in a variety of areas including vector design have put the once-daunting task of developing gene therapy treatments within grasp. A portfolio of gene delivery systems, divided into nonviral and viral vectors, is currently being evaluated in clinical trials (http://www.wiley.co.uk/genetherapy/clinical/). For the majority of potential kidney-related gene therapies, the ideal gene-transfer vehicle has to have little immunogenicity, provide for sustained transgene expression, and be applicable via the intravenous delivery route. When taking into consideration these requirements, vectors based on the nonpathogenic AAV emerge as promising candidates.

Recombinant adeno-associated viral vectors (rAAVs) have been applied in over 60 clinical trials mainly for the treatment of inherited diseases and cancer (http://www.wiley.co.uk/genetherapy/clinical/). With the exception of a transient, nonsymptomatic rise in liver enzymes in a clinical trial for hemophilia B, no vector-related adverse events were reported, owing to the excellent safety profile of this vector system.9 Furthermore, a clinical benefit was reported for the treatment of rare diseases like Leber's congenital amaurosis or lipoprotein lipase deficiency.10,11,12 In addition to the most commonly used serotype 2, alternative AAV serotypes and variants have been isolated from human or simian tissue, or from adenoviral stocks, and developed as gene-transfer vehicles.13 They differ in epitopes recognized by the immune systems, in receptors used for cell binding and entry, and in their intracellular processing, increasing the likelihood that a suitable AAV vector is available for any particular application. In addition, naturally occurring serotype capsids can be optimized for a specific application either by combining capsid proteins of different serotypes, by domain swapping or by combinatorial approaches.14

The most efficacious and least invasive delivery route for the treatment of renal diseases is intravenous application. All rAAV serotypes, when systemically administered, show a natural affinity for hepatic tissue. The liver holds about 30% of the total blood volume enabling an efficient systemic distribution of the recombinant factors following secretion by rAAV-transduced liver cells. After nonviral gene transfer, HGF, overexpressed in the liver, is efficiently delivered through the circulation to the site of damage proving an efficient and safe means of interfering with fibrogenic processes.15 Local HGF expression at the site of damage, however, was superior to systemic delivery of HGF in prolonging renal allograft survival.16 Therefore, we aimed to develop a combined treatment strategy using the liver as the primary and the kidney as the secondary gene-transfer target. To identify the most suitable serotype, we compared the commonly used rAAV2 with two less well-characterized, but highly efficient serotypes for hepatic transduction.17,18,19 Specifically, we employed rAAV8 and rAAV9 in the COL4A3-knockout mouse model of renal failure. This genetic mouse model,20 in contrast to experimentally induced fibrosis,7,21 reflects the natural course of human chronic renal disease with all its pathophysiological alterations including proteinuria and tubulointerstitial fibrosis. In reporter studies as well as in our therapeutic approach, rAAV9 proved to be superior compared with other serotypes for the purpose of hepatic and renal gene transfer. High levels of HGF transgene expression mediated by systemically applied rAAV9 resulted in dramatic attenuation of renal fibrosis revealing for the first time the therapeutic potential of rAAV for combined paracrine and endocrine HGF expression in chronic renal disease.

Results

AAV9 is superior to AAV8 and AAV2 in renal and hepatic transduction in COL4A3-knockout mice

In order to identify an AAV serotype that is suitable for hepatic and renal transgene expression following intravenous administration, we directly compared rAAV2, rAAV8, and rAAV9. All three serotypes contained the green fluorescent protein (GFP) reporter gene controlled by the ubiquitously active cytomegalovirus (CMV) promoter. The expression cassette was flanked by serotype 2 packaging signals (ITR), thus, rAAV8 and rAAV9 were packaged as pseudotypes.22 Equivalent numbers of vector genomes (5 × 1011 vector genomes) were administered by a single intravenous injection into the tail vein of 6-week-old male COL4A3-knockout mice (n = 6). Animals were killed 2 weeks after injection, and the kidneys and liver were harvested. Quantitative PCR (qPCR) was performed to compare the relative amounts of vector genomes and their transcriptional activity in these organs. Tissue sections were stained with anti-GFP antibodies to visualize the extent of GFP expression and its distribution across renal and hepatic tissue.

As expected, all three serotypes showed a preference for liver tissue with rAAV8 and rAAV9 being clearly superior in terms of transduction efficiency (Figure 1a) and transgene expression (Figure 1c) compared to rAAV2. Immunohistochemistry confirmed these results and revealed that GFP-expressing hepatocytes are scattered over the liver lobule (Figure 2).

Figure 1.

Figure 1

Open in a new tab

(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.

Figure 2.

Figure 2

Open in a new tab

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.

In the kidney, our second target organ, rAAV8 and rAAV9, again clearly outperformed rAAV2 with regard to renal transduction efficiency (Figure 1b) and transcriptional activity (Figure 1d). Compared to liver, however, renal transduction efficiency and transgene expression were low (Figure 1c). In renal sections of rAAV2- and rAAV8-treated animals, no (AAV2) or negligible (AAV8) amounts of GFP were detectable by immunostaining, whereas epithelial cells of the proximal and distal tubules, predominantly of the inner part of the kidney, stained positive for GFP when rAAV9 was used for in vivo gene transfer (Figure 2).

CMV-enhancer-Ksp-cadherin promoter restricts transgene expression

A 1,342-base-pair fragment within the 5′-region of the Ksp-cadherin promoter controls expression of the cell adhesion molecule Ksp-cadherin that is reported to be exclusively expressed in renal tubular epithelial cells.23 We combined this promoter sequence with the CMV-enhancer, a strategy shown to result in enhanced transcriptional activity,24 and we characterized the specificity of this enhancer–promoter construct (CMV-Ksp) in vivo using GFP as a reporter gene and rAAV9 as the delivery vector.

To this end, we administered 5 × 1011 genomic particles of rAAV9-CMV-GFP and 2.5 × 1011 genomic particles of rAAV9-CMV-Ksp-GFP, respectively, by tail vein injection into 6-week-old male COL4A3-knockout mice (n = 6). Animals were killed 2 weeks after injection, and spleens, lungs, hearts, kidneys, and livers were assayed for transgene expression. As depicted in Figure 3, the CMV promoter directed GFP expression to the liver, kidney, lung, spleen, and heart. In contrast, transgene expression for the CMV-enhancer-Ksp-cadherin promoter construct was tightly restricted. No GFP expression was detectable in the lung, heart, or spleen, whereas the CMV-Ksp promoter construct mediated strong transgene expression in the two target tissues, kidney and liver (Figure 3).

Figure 3.

Figure 3

Open in a new tab

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.

Tubulointerstitial fibrosis is significantly reduced following systemic administration of rAAV9-CMV-Ksp-hHGF

We investigated whether human HGF (hHGF) expressed from systemically applied rAAV vectors attenuates the development of tubulointerstitial fibrosis. We used rAAV8 and rAAV9, which possess the ability to transduce liver and kidney (Figure 1), as gene-transfer vectors and the CMV-enhancer-Ksp-cadherin promoter to restrict hHGF expression to the target tissues.

We administered intravenously 5 × 1011 genomic particles of rAAV9-CMV-Ksp-hHGF (n = 8) and of rAAV8-CMV-Ksp-hHGF (n = 8), respectively, or empty AAV8 capsids (mock; n = 6) into 4-week old male COL4A3-knockout mice. COL4A3-knockout mice at this age are symptom-free because histological signs of fibrosis are not detectable before 5–6 weeks of age.20 Mice were killed 5.5 weeks after vector application, and blood and kidneys were collected.

Kidneys of control mice showed a reduction in size of ~40% and a rough granular surface, both clear signs of renal injury. By contrast, the kidneys of rAAV8-CMV-Ksp-hHGF- and rAAV9-CMV-Ksp-hHGF-treated mice were obviously less affected (data not shown).

The mean level of hHGF expression in sera of rAAV-treated mice was significantly higher than in untreated controls particularly for rAAV9-treated animals with 340 pg/ml serum hHGF versus 100 pg/ml in rAAV8-treated animals (Figure 4).

Figure 4.

Figure 4

Open in a new tab

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.

Next, we examined the effect of hHGF overexpression on three markers of fibrosis, collagen 1α1 (Col1A1), α-smooth muscle actin (SMA), and platelet-derived growth factor receptor-β (PDGFR-β), in the kidneys by qPCR.

Col1A1 is the main matrix protein accumulating during fibrosis, and a known target of HGF regulation.25 As shown in Figure 5a, in rAAV-CMV-Ksp-hHGF-treated mice, Col1A1 expression was significantly reduced to 40% (rAAV8) and 50% (rAAV9), respectively, compared with control mice. Further, a significant inverse correlation between serum hHGF level and Col1A1 expression was detectable (R2 = 0.409), consistent with the antifibrogenic activity of hHGF overexpression (Figure 5b).

Figure 5.

Figure 5

Open in a new tab

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 < 0.01. COL1A1, collagen 1α1; hHGF, human hepatocyte growth factor; PDGFR-β, platelet-derived growth factor receptor-β SMA, smooth muscle actin.

An early event of tubulointerstitial fibrosis is the peritubular accumulation of myofibroblasts, which express SMA, thereby contributing to the abnormal matrix production seen in chronic renal disease.3 Indeed, SMA expression may be an indicator of fibrosis initiation and progression.2,3 Similar to the reduction seen for Col1A1 expression, rAAV-CMV-Ksp-hHGF-treated animals showed a reduced expression of SMA (Figure 5c). Again, the reduction was more pronounced in animals that had received rAAV9-CMV-Ksp-hHGF (20% reduction in rAAV8- and 47% in rAAV9-treated mice compared to control mice). Again, SMA and hHGF expression correlated well (R2 = 0.923) (Figure 5d).

The PDGFR-β is involved in fibrogenesis, presumably by driving proliferation of interstitial myofibroblasts.26 Expression of PDGFR-β was reduced in animals treated with rAAV-CMV-Ksp-hHGF (Figure 5e) compared to untreated controls and most obvious in rAAV9-treated animals (~45%). However, a significant reduction was also seen in animals that had received rAAV8 (23%). The reduction in PDGFR-β expression correlated with elevated hHGF serum level (R2 = 0.233) (Figure 5f).

In line with these findings, a remarkable attenuation in ECM accumulation was noticeable in renal sections of rAAV-CMV-Ksp-hHGF-treated mice (Figure 6). Five out of six of the control mice that had received empty capsids exhibited a fibrosis of stages 4 and 5 (Figure 6a), especially in the medullary region of the kidney (Figure 6b). In contrast, six out of eight rAAV8-CMV-Ksp-hHGF-treated animals showed a fibrosis of stage 3, whereas the remaining two animals were classified with stage 4 fibrosis (Figure 6a). Even more pronounced, four out of eight animals of the rAAV9-CMV-Ksp-hHGF cohort had stage 2 fibrosis, whereas three showed stage 3 and only one animal exhibited ECM accumulation and fibrotic alterations of stage 4 (Figure 6a). Again, the therapeutic effect could be correlated with hHGF expression in all animals (R2 = 0.355) (data not shown).

Figure 6.

Figure 6

Open in a new tab

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).

Discussion

In this study, we show the great potential of rAAV vectors applied intravenously as a delivery system for antifibrogenic factors such as HGF for the treatment of renal failure caused by chronic fibrogenic renal disease. Of the three serotypes compared in this study, rAAV9 was the most efficacious serotype for combined kidney- and liver-directed gene transfer. We showed that expression of hHGF following intravenous application of rAAV9-CMV-Ksp-hHGF attenuated the course of chronic renal disease (Figures 4–6). Thus, our data clearly demonstrate the efficacy of viral vector–mediated combined paracrine and endocrine delivery of antifibrogenic factors (Figure 7).

Figure 7.

Figure 7

Open in a new tab

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.

Similar to rAAV9-CMV-Ksp-hHGF-mediated gene transfer, systemic delivery of rAAV8-CMV-Ksp-hHGF resulted in a significant decrease in Col1A1, SMA, and PDGFR-β expression, and a reduction of tubulointerstitial fibrosis as indicated by diminished ECM deposition (Figures 5 and 6). However, the therapeutic effect was less pronounced than with rAAV9-CMV-Ksp-hHGF (Figure 6). In agreement with this, rAAV9 outperformed rAAV8 in our reporter gene study in terms of transgene expression in renal and hepatic tissue.

AAV serotypes show distinct variations in the amino acid composition of their capsids that are responsible for their serotype-specific serological recognition profile and for the use of distinct cell attachment and internalization receptors. Furthermore, serotypes may also differ in the stability of their capsids. Less stringent interactions may facilitate viral uncoating, a limiting step in viral infectivity and vector transduction. Therefore, the differences in expression efficiency per intracellular vector genome copy observed for different serotypes could be a result of distinct entry routes leading to differences in the intracellular processing of incoming AAV particles and/or in the efficiency of viral uncoating. With respect to rAAV8 and rAAV9, the serotypes of our study, different entry routes have not been described because both serotypes employ the same cellular receptor, namely the laminin receptor,27 leaving the differences in viral uncoating the most likely option.

Low efficiency of uncoating of AAV2 has been cited for the low transgene expression efficiency of rAAV2 compared to rAAV8 in mouse hepatocytes.28 In addition to impaired rAAV2-mediated gene expression, significantly smaller numbers of vector genomes were detected in hepatic tissue compared to rAAV8 and rAAV9 (Figure 1) precluding the use of rAAV2 for endocrine expression of hHGF in our proof-of-concept study. Similarly, only negligible numbers of vector genomes were detectable in renal tissue by qPCR, and no reporter gene expression could be traced by immunohistochemistry (Figures 1 and 2). In contrast to tail vein injection of rAAV2 vectors, the local delivery in rats via the renal artery,29 or by intraparenchymal injection in mice,29,30 did result in transgene expression in renal tubular epithelial cells, the same cell type in which reporter gene expression was detectable following systemic delivery of rAAV9 (Figures 2 and 3). Because all of our experiments were performed in COL4A3-knockout mice, we cannot formally exclude animal model–specific barriers for renal transduction following systemic application of rAAV2. However, it seems more likely that rAAV2 does not accumulate in sufficient quantity in renal tissue, presumably due to nonspecific retention of the vector in the liver and spleen as previously reported by us and others.31,32,33

The transduction efficiency of rAAV8 and rAAV9 in the liver and kidney clearly exceeded that of rAAV2 in the reporter gene assay (Figure 1), and both serotypes also performed well when applied therapeutically (Figures 4–6). However, previous studies have also shown that intravenous injection of rAAV8 into adult mice produced strong transgene expression not only in the liver, but also ectopically in the heart and to a lesser degree in skeletal muscle when ubiquitously active promoters were used to drive transgene expression.34 In agreement with our observations (Figure 3), strong muscle and liver tropism for rAAV9 has been reported previously.18,19 Here, our goal was to use rAAV as a platform for kidney- and liver-directed gene expression; however, targeted transgene expression presents a formidable challenge. One possibility involves cell surface or receptor targeting (reviewed in refs. 14,35). This technique retargets viral vector tropism by insertion of peptides into exposed surface positions of the viral capsid allowing targeted receptor binding while simultaneously interfering with natural viral receptor interactions. Alternatively, viral vector tropism can be modified nongenetically, for example, by using bispecific antibodies to mediate target cell binding. Here, we used a different methodology, namely transcriptional targeting, employing the renal tubular epithelial–specific Ksp-cadherin promoter23 in combination with the CMV enhancer, that is known to increase expression of tissue-specific promoters.24 This enhancer–promoter construct drives efficient transgene expression in kidney and liver, while avoiding expression from nontarget organs like lung, heart, and spleen (Figure 3). Although we cannot currently explain why the tubular epithelial cell–specific Ksp-cadherin promoter also drives expression in hepatocytes when combined with the CMV enhancer, this new enhancer–promoter construct is a valuable tool for improving renal- and liver-related gene therapy.

Target cell–restricted transgene expression is believed to assist in reducing recognition by the host immune system. In this regard, adoption of the CMV-enhancer-Ksp promoter, that, in contrast to the ubiquitously active CMV promoter, avoids transgene expression in the spleen (Figure 3), a secondary lymphoid organ, should be beneficial. In agreement with this, we did not observe signs of inflammation in rAAV-CMV-Ksp-hHGF-treated mice, although they efficiently expressed human HGF, a nonself-antigen (data not shown). In addition to the transgene, delivery vectors themselves may be recognized by the host immune system. Viral vectors like their natural counterparts possess pathogen-associated molecular patterns that are recognized by pattern recognition receptors of the innate immune system. The innate immune system, once activated, induces adaptive immune responses interfering with sustained transgene expression and vector readministration. Interestingly, although AAV should display pathogen-associated molecular patterns, systemically applied rAAV8 and rAAV9 seem not to induce proinflammatory immune responses, which are a hallmark of the innate immune system (ref. 19 and (M. Hösel and H. Büning, unpublished results) for rAAV8). The mechanisms responsible for the low-immunogenicity phenotype attributed to AAV and AAV-derived vectors in numerous in vivo studies are still unclear. However, at least for the intravenous application route, it is likely that rAAV's inability to productively transduce nonparenchymal cells of the liver (Figure 2 and ref. 19) could be one of the contributing factors.

Although various organs participate in the pathophysiological production and secretion of HGF,36,37,38,39 its natural activity is restricted to sites of tissue injury.39 This restriction is caused by HGF's dependency on activation by coagulation-associated proteases,39 which in turn are activated only at the sites of tissue injury.40 In the present study, we took advantage of the naive liver tropism of the AAV system to facilitate high levels of endocrine HGF expression and delivery of this inactive form of HGF via the circulation to the site of tissue damage and activation (Figure 7). Because local overexpression was shown to result in superior therapeutic efficiency compared to systemic muscle-mediated HGF delivery using nonviral gene delivery,16 we aimed to combine paracrine (renal) and endocrine (hepatic) expression of HGF (Figure 7).

Even though activation of HGF is tightly regulated, highly elevated and prolonged HGF expression could potentially increase the risk of malignant transformation.41 To minimize this potential risk, HGF expression should be restricted to the target organs. Indeed, using the CMV-enhancer-KSP-promoter, expression was observed in the liver and the kidney but not in nontarget tissues such as spleen, lung, and heart (Figure 3). Although beyond the scope of this proof-of-concept study, the inclusion of additional regulatory elements need to be considered to improve the safety profile further prior to human application.

In agreement with previous studies, where HGF was administered as a recombinant protein or overexpressed following nonviral vector gene transfer into kidney, muscle, or hepatic tissue,7,16,21,42,43,44 our study using rAAV-mediated HGF expression shows a beneficial effect on the course of chronic renal disease (Figures 4–6). Additional benefits are associated with our therapeutic approach: dosing intervals are significantly prolonged due to improved stability in comparison with nonviral gene therapy approaches21,43,44 and second, cost is reduced compared to strategies relying on recombinant HGF protein that is rapidly cleared (half-life of 3–5 minutes).8 In conclusion, this proof-of-concept study shows that intravenous administration of rAAV, especially rAAV9, is a highly effective strategy for the treatment of chronic kidney diseases by mediating simultaneous paracrine and endocrine expression of antifibrogenic factors.

Materials and Methods

Plasmids. The construction of pscAAV/EGFP was described in ref. 45. EGFP reporter gene expression was driven by CMV promoter. pAAV-CMV-Ksp-EGFP was cloned by replacing the MLC260-β-globin IgG promoter in pdsCMV-MLC0.26-EGFP (kindly provided by Oliver Müller, University of Heidelberg, Heidelberg, Germany) with the Ksp-cadherin promoter. The Ksp-cadherin promoter was PCR-amplified from mouse genomic DNA as described in ref. 23 using primers flanked by PpUMI and BamHI restriction sites for cloning. pCMV-Ksp-hHGF was cloned by PCR amplification of the Ksp-cadherin promoter23 using primers containing XhoI and HindIII recognition sequences. The PCR fragment was inserted into the pGL3 vector (Promega, Mannheim, Germany) resulting in pGL3-Ksp-Luc. Next, the CMV-enhancer of pEGFP-C1 (Clontech, Heidelberg, Germany) was cloned upstream of the Ksp-cadherin promoter into the SmaI site giving pGL3-CMV-Ksp-Luc. The luciferase gene was replaced by the hHGF open-reading frame including a poly(A) site—both isolated from pBlast49-hHGF (InvivoGen, San Diego, CA)—by HindIII and SalI digestion. The resulting pGL3-CMV-Ksp-hHGF was used for isolating the hHGF expression cassette for cloning into pSUB201 (ref. 46) replacing the rep and cap genes of AAV2. This plasmid—pSUB-CMV-Ksp-hHGF—served as the vector plasmid for packaging rAAV vectors encoding for hHGF.

Preparation of rAAV vectors. All rAAV vectors were produced by triple plasmid transfection as described previously47,48 with a self-complementary (experiments in Figures 1–3) or single-stranded (experiment in Figures 3–6) vector genome conformation. Briefly, subconfluent HEK293 cells were transfected with a vector plasmid (pscAAV/EGFP,45 pGFP,45 pAAV-CMV-Ksp-EGFP, or pSUB-CMV-Ksp-hHGF), pXX6 (ref. 47, kindly provided by Jude Samulski, University of North Carolina, Chapel Hill), and an AAV helper plasmid [pRC,49 pXR8 (ref. 17, kindly provided by Jim Wilson, University of Pennsylvania), or pXR9 (ref. 18, kindly provided by Jim Wilson, University of Pennsylvania)]. The empty capsid preparation used as control was produced using pXR8 and pXX6. Cells were harvested 48 hours after transfection, and lysed by three freeze-and-thaw cycles. Following Benzonase treatment, cell lysates were purified by iodixanol gradient ultracentrifugation as described previously.50 For all experiments, the genomic titer was determined by absolute quantification assays using qPCR.45

Animal procedures. COL4A3-knockout 129/Sv mice, kindly provided by Oliver Gross,20 had free access to regular chow and water. Only heterozygous COL4A3-knockout 129/Sv mice were crossbred. Genotyping of pubs was performed as previously described.20

Self-complementary rAAV2-CMV-GFP, rAAV8-CMV-GFP, rAAV9-CMV-GFP, rAAV9-Ksp-CMV-GFP or single-stranded rAAV9-CMV-GFP were administered via tail vein injection into 6-week-old COL4A3-knockout mice. Injection of 0.9% NaCl was used as a control. Animals were killed 2 weeks after injection, and organs were harvested for further analyses.

For the gene therapeutic approach, 4-week-old COL4A3-knockout mice were transduced with 5 × 1011 particles of single-stranded rAAV8-CMV-Ksp-hHGF or rAAV9-CMV-Ksp-hHGF. Control animals were injected with an equal volume of an empty AAV8 capsid preparation. Mice were killed 5.5 weeks after transduction. Blood was collected, and organs were harvested for further analyses.

All experiments were conducted in accordance with National Health and Medical Research Committee Guidelines for Animal Experimentation.

Immunohistochemical analyses of GFP expression. Three micrometer sections of formalin-fixed and paraffin-embedded tissues were treated with the avidin–biotin blocking kit (Vector Laboratories, Peterborough, UK) according to manufacturer's instructions to block endogenous biotin followed by overnight incubation with rabbit anti-GFP antibody (1:200; Abcam, Cambridge, UK). GFP antibodies were detected via avidin–biotin complex formation using the alkaline phosphatase–coupled Vectastain ABC-AP kit (Vector Laboratories) and detection by a chromogen reaction using Fast Red (Dako, Hamburg, Germany) as substrate. Sections were then counterstained with hematoxylin.

Evaluation of transduction efficiency by qPCR. To determine transduction efficiency in vivo, total genomic DNA was isolated by a standard phenol–chloroform extraction, followed by qPCR using GFP-specific primers (Supplementary Table S1) and normalization to the reference gene murine C-reactive protein (Supplementary Table S1). The assay was performed twice in triplicates.

Evaluation of transcriptional activity by qPCR. Total RNA was isolated using the NucleoSpin RNA II kit (Macherey-Nagel, Düren, Germany) according to the manufacturer's instructions. One microgram of total RNA was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (ABI, Darmstadt, Germany). Ten nanograms of cDNA were applied to qPCR analyses. The reference gene hypoxanthine phosphoribosyltransferase (HPRT) was used for normalization. All reactions were performed in triplicates and at least two independent PCR runs were performed.

Measurement of hHGF serum levels. Quantitative measurement of hHGF serum levels was performed using the Quantikine Human HGF Immunoassay (R&D Systems, Wiesbaden-Nordenstadt, Germany) according to the manufacturer's guidelines.

Classification of the fibrosis stage in transversal renal sections. Fibrosis of the renal specimens was independently assessed by two pathologists (M.O., H.-P.D.) using Gomori-stained transversal renal sections. Interstitial fibrosis was predominantly visible by ECM deposition, and the severity of fibrosis was graded as follows: stage 1, normal; stage 2, ECM accumulation in <5% of the section area; stage 3, ECM accumulation in 5 to <10% of the section area; stage 4, ECM accumulation in 10 to <20% of the section area; stage 5, ECM accumulation in >20% of the section area.

Statistical analyses. Statistical analyses were performed using SPSS software (SPSS, Chicago, IL). Expression profiles of Col1A1, SMA, and PDGFR-β were presented as box plots. Data with normal distribution (Kolmogorov–Smirnov test) were subjected to analysis of variance using Dunnett's post hoc comparison to test for differences in means of variables. Post hoc multiple comparisons were performed by Tukey test. A value of P < 0.05 was considered as statistically significant.

SUPPLEMENTARY MATERIAL Table S1. Primers used to amplify the indicated DNA sequences for qPCR analyses and cloning.

Acknowledgments

We gratefully acknowledge the technical assistance of Katharina Wendland, Ali Manav, and Hannah Janicki. We thank Jude Samulski (University of North Carolina, Chapel Hill, NC) and Jim Wilson (University of Pennsylvania, Philadelphia, PA) for kindly providing the adenoviral helper plasmid, and rAAV8 and rAAV9 helper plasmids, respectively. Furthermore, we thank Oliver Müller (University of Heidelberg, Heidelberg, Germany) for providing pdsCMV-MLC0.26-EGFP. This work was supported by the Center for Molecular Medicine Cologne (ZMMK) and the German Research Foundation (SBF 670) to H.B. and M. Ha. and the Medical Faculty of the University Cologne to M.O. and H.-P.D.

Supplementary Material

Table S1.

Primers used to amplify the indicated DNA sequences for qPCR analyses and cloning.

Click here for additional data file. (38KB, doc)

REFERENCES

  1. Collins AJ, Foley R, Herzog C, Chavers B, Gilbertson D, Ishani A, et al. Excerpts from the United States Renal Data System 2007 annual data report. Am J Kidney Dis. 2008;51 1 suppl. 1:S1–320. doi: 10.1053/j.ajkd.2007.11.001. [DOI] [PubMed] [Google Scholar]
  2. Wolf G., and, Ziyadeh FN. Cellular and molecular mechanisms of proteinuria in diabetic nephropathy. Nephron Physiol. 2007;106:p26–p31. doi: 10.1159/000101797. [DOI] [PubMed] [Google Scholar]
  3. Eddy AA. Molecular basis of renal fibrosis. Pediatr Nephrol. 2000;15:290–301. doi: 10.1007/s004670000461. [DOI] [PubMed] [Google Scholar]
  4. Inoue T, Okada H, Kobayashi T, Watanabe Y, Kanno Y, Kopp JB, et al. Hepatocyte growth factor counteracts transforming growth factor-beta1, through attenuation of connective tissue growth factor induction, and prevents renal fibrogenesis in 5/6 nephrectomized mice. FASEB J. 2003;17:268–270. doi: 10.1096/fj.02-0442fje. [DOI] [PubMed] [Google Scholar]
  5. Matsumoto K, Mizuno S., and, Nakamura T. Hepatocyte growth factor in renal regeneration, renal disease and potential therapeutics. Curr Opin Nephrol Hypertens. 2000;9:395–402. doi: 10.1097/00041552-200007000-00011. [DOI] [PubMed] [Google Scholar]
  6. Ramadori G, Neubauer K, Odenthal M, Nakamura T, Knittel T, Schwögler S, et al. The gene of hepatocyte growth factor is expressed in fat-storing cells of rat liver and is downregulated during cell growth and by transforming growth factor-beta. Biochem Biophys Res Commun. 1992;183:739–742. doi: 10.1016/0006-291x(92)90545-v. [DOI] [PubMed] [Google Scholar]
  7. Mizuno S, Matsumoto K., and, Nakamura T. Hepatocyte growth factor suppresses interstitial fibrosis in a mouse model of obstructive nephropathy. Kidney Int. 2001;59:1304–1314. doi: 10.1046/j.1523-1755.2001.0590041304.x. [DOI] [PubMed] [Google Scholar]
  8. Liu KX, Kato Y, Narukawa M, Kim DC, Hanano M, Higuchi O, et al. Importance of the liver in plasma clearance of hepatocyte growth factors in rats. Am J Physiol. 1992;263 5 Pt 1:G642–G649. doi: 10.1152/ajpgi.1992.263.5.G642. [DOI] [PubMed] [Google Scholar]
  9. Manno CS, Pierce GF, Arruda VR, Glader B, Ragni M, Rasko JJ, et al. Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response. Nat Med. 2006;12:342–347. doi: 10.1038/nm1358. [DOI] [PubMed] [Google Scholar]
  10. Bainbridge JW, Smith AJ, Barker SS, Robbie S, Henderson R, Balaggan K, et al. Effect of gene therapy on visual function in Leber's congenital amaurosis. N Engl J Med. 2008;358:2231–2239. doi: 10.1056/NEJMoa0802268. [DOI] [PubMed] [Google Scholar]
  11. Maguire AM, Simonelli F, Pierce EA, Pugh EN, Jr, Mingozzi F, Bennicelli J, et al. Safety and efficacy of gene transfer for Leber's congenital amaurosis. N Engl J Med. 2008;358:2240–2248. doi: 10.1056/NEJMoa0802315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Stroes ES, Nierman MC, Meulenberg JJ, Franssen R, Twisk J, Henny CP, et al. Intramuscular administration of AAV1-lipoprotein lipase S447X lowers triglycerides in lipoprotein lipase-deficient patients. Arterioscler Thromb Vasc Biol. 2008;28:2303–2304. doi: 10.1161/ATVBAHA.108.175620. [DOI] [PubMed] [Google Scholar]
  13. Wu Z, Asokan A., and, Samulski RJ. Adeno-associated virus serotypes: vector toolkit for human gene therapy. Mol Ther. 2006;14:316–327. doi: 10.1016/j.ymthe.2006.05.009. [DOI] [PubMed] [Google Scholar]
  14. Büning H, Perabo L, Coutelle O, Quadt-Humme S., and, Hallek M. Recent developments in adeno-associated virus vector technology. J Gene Med. 2008;10:717–733. doi: 10.1002/jgm.1205. [DOI] [PubMed] [Google Scholar]
  15. Xia JL, Dai C, Michalopoulos GK., and, Liu Y. Hepatocyte growth factor attenuates liver fibrosis induced by bile duct ligation. Am J Pathol. 2006;168:1500–1512. doi: 10.2353/ajpath.2006.050747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Franquesa M, Alperovich G, Herrero-Fresneda I, Lloberas N, Bolaños N, Fillat C, et al. Direct electrotransfer of hHGF gene into kidney ameliorates ischemic acute renal failure. Gene Ther. 2005;12:1551–1558. doi: 10.1038/sj.gt.3302569. [DOI] [PubMed] [Google Scholar]
  17. Gao GP, Alvira MR, Wang L, Calcedo R, Johnston J., and, Wilson JM. Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy. Proc Natl Acad Sci USA. 2002;99:11854–11859. doi: 10.1073/pnas.182412299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gao G, Vandenberghe LH, Alvira MR, Lu Y, Calcedo R, Zhou X, et al. Clades of Adeno-associated viruses are widely disseminated in human tissues. J Virol. 2004;78:6381–6388. doi: 10.1128/JVI.78.12.6381-6388.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Vandendriessche T, Thorrez L, Acosta-Sanchez A, Petrus I, Wang L, Ma L, et al. Efficacy and safety of adeno-associated viral vectors based on serotype 8 and 9 vs. lentiviral vectors for hemophilia B gene therapy. J Thromb Haemost. 2007;5:16–24. doi: 10.1111/j.1538-7836.2006.02220.x. [DOI] [PubMed] [Google Scholar]
  20. Gross O, Beirowski B, Koepke ML, Kuck J, Reiner M, Addicks K, et al. Preemptive ramipril therapy delays renal failure and reduces renal fibrosis in COL4A3-knockout mice with Alport syndrome. Kidney Int. 2003;63:438–446. doi: 10.1046/j.1523-1755.2003.00779.x. [DOI] [PubMed] [Google Scholar]
  21. Yang J, Dai C., and, Liu Y. Systemic administration of naked plasmid encoding hepatocyte growth factor ameliorates chronic renal fibrosis in mice. Gene Ther. 2001;8:1470–1479. doi: 10.1038/sj.gt.3301545. [DOI] [PubMed] [Google Scholar]
  22. Rabinowitz JE, Rolling F, Li C, Conrath H, Xiao W, Xiao X, et al. Cross-packaging of a single adeno-associated virus (AAV) type 2 vector genome into multiple AAV serotypes enables transduction with broad specificity. J Virol. 2002;76:791–801. doi: 10.1128/JVI.76.2.791-801.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Shao X, Johnson JE, Richardson JA, Hiesberger T., and, Igarashi P. A minimal Ksp-cadherin promoter linked to a green fluorescent protein reporter gene exhibits tissue-specific expression in the developing kidney and genitourinary tract. J Am Soc Nephrol. 2002;13:1824–1836. doi: 10.1097/01.asn.0000016443.50138.cd. [DOI] [PubMed] [Google Scholar]
  24. Gruh I, Wunderlich S, Winkler M, Schwanke K, Heinke J, Blömer U, et al. Human CMV immediate-early enhancer: a useful tool to enhance cell-type-specific expression from lentiviral vectors. J Gene Med. 2008;10:21–32. doi: 10.1002/jgm.1122. [DOI] [PubMed] [Google Scholar]
  25. Schievenbusch S, Strack I, Scheffler M, Wennhold K, Maurer J, Nischt R, et al. Profiling of anti-fibrotic signaling by hepatocyte growth factor in renal fibroblasts. Biochem Biophys Res Commun. 2009;385:55–61. doi: 10.1016/j.bbrc.2009.05.010. [DOI] [PubMed] [Google Scholar]
  26. Tang WW, Ulich TR, Lacey DL, Hill DC, Qi M, Kaufman SA, et al. Platelet-derived growth factor-BB induces renal tubulointerstitial myofibroblast formation and tubulointerstitial fibrosis. Am J Pathol. 1996;148:1169–1180. [PMC free article] [PubMed] [Google Scholar]
  27. Akache B, Grimm D, Pandey K, Yant SR, Xu H., and, Kay MA. The 37/67-kilodalton laminin receptor is a receptor for adeno-associated virus serotypes 8, 2, 3, and 9. J Virol. 2006;80:9831–9836. doi: 10.1128/JVI.00878-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Thomas CE, Storm TA, Huang Z., and, Kay MA. Rapid uncoating of vector genomes is the key to efficient liver transduction with pseudotyped adeno-associated virus vectors. J Virol. 2004;78:3110–3122. doi: 10.1128/JVI.78.6.3110-3122.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Takeda S, Takahashi M, Mizukami H, Kobayashi E, Takeuchi K, Hakamata Y, et al. Successful gene transfer using adeno-associated virus vectors into the kidney: comparison among adeno-associated virus serotype 1-5 vectors in vitro and in vivo. Nephron Exp Nephrol. 2004;96:e119–e126. doi: 10.1159/000077378. [DOI] [PubMed] [Google Scholar]
  30. Lipkowitz MS, Hanss B, Tulchin N, Wilson PD, Langer JC, Ross MD, et al. Transduction of renal cells in vitro and in vivo by adeno-associated virus gene therapy vectors. J Am Soc Nephrol. 1999;10:1908–1915. doi: 10.1681/ASN.V1091908. [DOI] [PubMed] [Google Scholar]
  31. Kern A, Schmidt K, Leder C, Müller OJ, Wobus CE, Bettinger K, et al. Identification of a heparin-binding motif on adeno-associated virus type 2 capsids. J Virol. 2003;77:11072–11081. doi: 10.1128/JVI.77.20.11072-11081.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. White SJ, Nicklin SA, Büning H, Brosnan MJ, Leike K, Papadakis ED, et al. Targeted gene delivery to vascular tissue in vivo by tropism-modified adeno-associated virus vectors. Circulation. 2004;109:513–519. doi: 10.1161/01.CIR.0000109697.68832.5D. [DOI] [PubMed] [Google Scholar]
  33. Perabo L, Goldnau D, White K, Endell J, Boucas J, Humme S, et al. Heparan sulfate proteoglycan binding properties of adeno-associated virus retargeting mutants and consequences for their in vivo tropism. J Virol. 2006;80:7265–7269. doi: 10.1128/JVI.00076-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wang Z, Zhu T, Qiao C, Zhou L, Wang B, Zhang J, et al. Adeno-associated virus serotype 8 efficiently delivers genes to muscle and heart. Nat Biotechnol. 2005;23:321–328. doi: 10.1038/nbt1073. [DOI] [PubMed] [Google Scholar]
  35. Vandenberghe LH, Wilson JM., and, Gao G. Tailoring the AAV vector capsid for gene therapy. Gene Ther. 2009;16:311–319. doi: 10.1038/gt.2008.170. [DOI] [PubMed] [Google Scholar]
  36. Liu Y, Tolbert EM, Lin L, Thursby MA, Sun AM, Nakamura T, et al. Up-regulation of hepatocyte growth factor receptor: an amplification and targeting mechanism for hepatocyte growth factor action in acute renal failure. Kidney Int. 1999;55:442–453. doi: 10.1046/j.1523-1755.1999.00267.x. [DOI] [PubMed] [Google Scholar]
  37. Yanagita K, Nagaike M, Ishibashi H, Niho Y, Matsumoto K., and, Nakamura T. Lung may have an endocrine function producing hepatocyte growth factor in response to injury of distal organs. Biochem Biophys Res Commun. 1992;182:802–809. doi: 10.1016/0006-291x(92)91803-x. [DOI] [PubMed] [Google Scholar]
  38. Kono S, Nagaike M, Matsumoto K., and, Nakamura T. Marked induction of hepatocyte growth factor mRNA in intact kidney and spleen in response to injury of distant organs. Biochem Biophys Res Commun. 1992;186:991–998. doi: 10.1016/0006-291x(92)90844-b. [DOI] [PubMed] [Google Scholar]
  39. Miyazawa K, Shimomura T, Naka D., and, Kitamura N. Proteolytic activation of hepatocyte growth factor in response to tissue injury. J Biol Chem. 1994;269:8966–8970. [PubMed] [Google Scholar]
  40. Miyazawa K, Shimomura T., and, Kitamura N. Activation of hepatocyte growth factor in the injured tissues is mediated by hepatocyte growth factor activator. J Biol Chem. 1996;271:3615–3618. doi: 10.1074/jbc.271.7.3615. [DOI] [PubMed] [Google Scholar]
  41. Takayama H, LaRochelle WJ, Sharp R, Otsuka T, Kriebel P, Anver M, et al. Diverse tumorigenesis associated with aberrant development in mice overexpressing hepatocyte growth factor/scatter factor. Proc Natl Acad Sci USA. 1997;94:701–706. doi: 10.1073/pnas.94.2.701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Yazawa K, Isaka Y, Takahara S, Imai E, Ichimaru N, Shi Y, et al. Direct transfer of hepatocyte growth factor gene into kidney suppresses cyclosporin A nephrotoxicity in rats. Nephrol Dial Transplant. 2004;19:812–816. doi: 10.1093/ndt/gfh064. [DOI] [PubMed] [Google Scholar]
  43. Yang J., and, Liu Y. Blockage of tubular epithelial to myofibroblast transition by hepatocyte growth factor prevents renal interstitial fibrosis. J Am Soc Nephrol. 2002;13:96–107. doi: 10.1681/ASN.V13196. [DOI] [PubMed] [Google Scholar]
  44. Yang J., and, Liu Y. Delayed administration of hepatocyte growth factor reduces renal fibrosis in obstructive nephropathy. Am J Physiol Renal Physiol. 2003;284:F349–F357. doi: 10.1152/ajprenal.00154.2002. [DOI] [PubMed] [Google Scholar]
  45. Hacker UT, Wingenfeld L, Kofler DM, Schuhmann NK, Lutz S, Herold T, et al. Adeno-associated virus serotypes 1 to 5 mediated tumor cell directed gene transfer and improvement of transduction efficiency. J Gene Med. 2005;7:1429–1438. doi: 10.1002/jgm.782. [DOI] [PubMed] [Google Scholar]
  46. Samulski RJ, Chang LS., and, Shenk T. Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression. J Virol. 1989;63:3822–3828. doi: 10.1128/jvi.63.9.3822-3828.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Xiao X, Li J., and, Samulski RJ. Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus. J Virol. 1998;72:2224–2232. doi: 10.1128/jvi.72.3.2224-2232.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Shi W, Arnold GS., and, Bartlett JS. Insertional mutagenesis of the adeno-associated virus type 2 (AAV2) capsid gene and generation of AAV2 vectors targeted to alternative cell-surface receptors. Hum Gene Ther. 2001;12:1697–1711. doi: 10.1089/104303401750476212. [DOI] [PubMed] [Google Scholar]
  49. Girod A, Ried M, Wobus C, Lahm H, Leike K, Kleinschmidt J, et al. Genetic capsid modifications allow efficient re-targeting of adeno-associated virus type 2. Nat Med. 1999;5:1438. doi: 10.1038/71021. [DOI] [PubMed] [Google Scholar]
  50. Zolotukhin S, Byrne BJ, Mason E, Zolotukhin I, Potter M, Chesnut K, et al. Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Ther. 1999;6:973–985. doi: 10.1038/sj.gt.3300938. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Table S1.

Primers used to amplify the indicated DNA sequences for qPCR analyses and cloning.

Click here for additional data file. (38KB, doc)

Articles from Molecular Therapy are provided here courtesy of The American Society of Gene & Cell Therapy

RESOURCES