Kidney pericytes and perivascular fibroblasts are the major progenitor population for myofibroblasts during kidney fibrosis and an important therapeutic target to slow the progression of fibrotic kidney diseases.1 There are currently no antifibrotic drugs approved for the treatment of CKD, which represents a major unmet medical need.
Recombinant adeno-associated virus (AAV) is a nonintegrating, nonenveloped, and replication-deficient parvovirus that has emerged as a powerful tool for gene delivery to mammalian cells. Advantages of AAV-directed gene transfer include stable transduction in nondividing cells, low immune response, and favorable safety profile in humans. Currently, AAV-based gene therapy trials are ongoing for a range of monogenic diseases, including neuromuscular disease, hemophilia, and inherited forms of blindness.2–6 In 2017, the US Food and Drug Administration approved the first gene therapy on the basis of an AAV vector for the treatment of a form of inherited retinal degeneration.7
Broader application of AAV gene therapy is limited by incomplete understanding of viral tropism. Recent efforts to engineer AAV vectors have shown that transduction efficiency can be improved and that tropism can be modified.8 Systemic injection of AAV achieves efficient transduction in several organs but not kidney, irrespective of capsid protein.9–15 Arterial injection of AAV2 transduces the proximal tubules of the kidney but at low efficiency.16–20 Either intravenous or in utero administration of AAV9 has been reported to transduce the proximal tubules, podocytes, and glomerular endothelium,21–24 and retrograde ureteral delivery results in transduction of tubular epithelium.25 Similarly, injection of AAV2 or -8 via the retrograde ureteral or pelvic approach transduces proximal tubule.26,27 Thus, various AAVs have been shown to transduce kidney epithelia, but efficiency is generally low; they may require arterial or ureteral administration, and no protocol to date has demonstrated transduction of kidney mesenchymal cells.
In this study, we systematically evaluated different AAV serotypes for their ability to deliver genetic material to kidney pericytes and fibroblasts, the myofibroblast progenitor populations.28 We report that the synthetic AAVAnc80 efficiently transduces kidney mesenchymal cells, including pericytes, fibroblasts, and mesangial cells. We validate use of Anc80 for gene transfer by knocking out Gli2 from kidney mesenchyme using Anc80-Cre, confirming an antifibrotic effect after chronic obstruction. We implicate the β-catenin pathway in myofibroblast expansion, because knockout of mesenchymal β-catenin expression using the same strategy was also antifibrotic. Finally, we show successful mesenchymal cell transduction in human kidney organoids, suggesting that Anc80 may be useful for targeting this lineage in humans.
AAV Preparation and Injection
Native AAV-2/2 and alternative serotypes AAV5, -6, -8, and -9 and Anc80 were generated with either the CMV or the synthetic CASI promoter. Transgenes were CMV.eGFP.WPRE, CASI.eGFP.WPRE, or CASI.Cre.WPRE. AAV production and titration were performed by the Gene Transfer Vector Core (http://vector.meei.harvard.edu/) at the Grousbeck Gene Therapy Center at the Schepens Eye Research Institute and Massachusetts Eye and Ear Infirmary as described previously.8,29 Briefly, all AAV preparations were made by plasmid cotransfection in human embryonic kidney 293 cells using polyethyleneimine. The packaging plasmid AAV Rep2-CapX (X indicating the serotype) was cotransfected with an AAV2 ITR-flanked transgene and adenohelper constructs. Three days after transfections, cells and media were harvested and underwent lysis and digestion with Benzonase. Viral vector was further purified from the lysate by tangential flow filtration and iodixanol gradient ultracentrifugation before reformulation in PBS.8,29 A final volume of 100 μl per mouse was injected via the retro-orbital venous plexus.
All mouse experiments were performed according to the animal experimental guidelines issued by the Animal Care and Use Committee at Washington University. Gli2-flox (JAX #007926), Rosa26tdTomato (JAX #007909), β-catenin–flox (JAX #004152), and C57BL/6J (JAX #000664) mice were purchased from Jackson Laboratories (Bar Harbor, ME). Anesthesia was achieved with continuous evaporated isoflurane (2%) using Anesthesia System RC2 (922100; VetEqip) during surgery. Buprenorphine (0.05 mg/kg), meloxicam (1 mg/kg), and lidocaine (1%) were given subcutaneously to achieve analgesia. Unilateral ureteral obstruction (UUO) surgery was as follows. After flank incision, the right kidney was exposed and freed from the perirenal fat tissue, and the ureter was tied off at the level of the lower pole using two 4.0 silk ties. Wounds were closed by staples. Mice were euthanized 10 days after UUO surgery. UUO surgeries in C57Bl6 wild-type mice were performed at 8 weeks of age using the same technique. For characterization of kidney transduction efficiency and dose, all experiments used three mice, except for the dose of 1012 genome copies (GCs) per mouse, which used n=2 due to cost. For the Gli2 experiments, both groups had n=6. For the β-catenin and lineage-tracing experiments, both groups had n=4.
Tissue Preparation and Histology
Mice were anesthetized with isofluorane (Baxter) and subsequently perfused via the left ventricle with 4°C PBS for 1 minute. For histologic analyses, tissue sections were fixed in 10% formaldehyde for 1 hour, paraffin embedded, cut with a rotating microtome at a thickness of 3 μm, and stained according to routine histologic protocols. For immunofluorescence studies, kidneys were fixed in 4% paraformaldehyde on ice for 1 hour and then incubated in 30% sucrose in PBS at 4°C overnight. OCT-embedded (Sakura Finetek) tissues were cryosectioned into 5-μm sections and mounted on superfrost slides (Fisher Scientific). Sections were washed in 1× PBS, blocked in 10% normal goat serum (Vector Labs), and incubated with primary antibodies (Supplemental Table 1). Secondary antibodies were FITC or Cy3 conjugated (Jackson ImmunoResearch). Sections were then stained with 4′,6-diamidino-2-phenylindole and mounted in Prolong Gold (Life Technologies). All images were obtained by confocal imaging (Nikon C2+).
Real Time PCR Experiments
Tissue was harvested and immediately snap frozen in liquid nitrogen. RNA was extracted according to the manufacturer’s instructions using the RNeasy Mini Kit (Qiagen), and 600 ng of total RNA was reverse transcribed with iScript (BioRad). During the RNA extraction, DNA was removed by a DNAse digestion step (Life Technologies). Quantitative PCRs were carried out with iQ-SYBR Green Supermix (BioRad) and the Applied Biosystems 7300 Teal-Time PCR System. Cycling conditions were 95°C for 3 minutes and then 40 cycles of 95°C for 15 seconds and 60°C for 1 minute followed by one cycle of 95°C for 10 seconds. Glyceraldehyde-3-phosphate dehydrogenase was used as a housekeeping gene. Data were analyzed using the 2−ΔΔct method. Primers are listed in Supplemental Table 2.
Protein was extracted from tissues or cells with RIPA or urea lysis buffer (urea: 8 M) with cOmplete Mini Protease Inhibitor Cocktail (Sigma-Aldrich). Proteins samples were separated in standard SDS polyacrylamide gel, transferred to polyvinylidene difluoride membranes, and incubated with primary antibodies with 5% skim milk or 3% BSA TBST buffer for 2 hours at room temperature or overnight at 4°C followed by the reaction with secondary antibody dissolved in 5% skim milk TBST buffer for 1 hour at room temperature. Goat anti-mouse (P0447), goat anti-rabbit (P0448), or rabbit anti-goat (P0449) Igs conjugated with HRP (DAKO) were used as secondary antibodies. For developing the signal, ECL Plus Western Blotting Substrate (Pierce Biotechnology) or SuperSignal West Femto Maximal Sensitivity Substrate (Pierce Biotechnology) was used according to the manufacturer’s instruction, and the signals were detected using the ChemDoc Detection System (BioRad).
Quantification of Fluorescent Reporter Proteins
Mesangial and cortical interstitial areas were focused to quantify the fraction of fluorescent reporter proteins. At least six different areas in cortex were taken, and percentage of fluorescent protein–positive area per PDGFRβ-positive area was calculated using Nikon NIS elements.
Five-week-old male mice were injected with a low dose of AAV (1010 GCs per mouse) followed by 3 weeks of incubation. UUO surgery was performed, and mice were euthanized 10 days after surgery; 15-μm-thick frozen sections were directly stained with 4′,6-diamidino-2-phenylindole and then analyzed using confocal microscopy. Coherent clusters of tdTomato-positive cells were counted for total cell number by DAPI counterstaining.
Data are presented as mean±SD. Comparison of two groups was performed using unpaired t tests. Paired t tests were used for comparison of repeated measures in the same group. Statistical analyses were performed using GraphPad Prism 5.0c (GraphPad Software Inc., San Diego, CA). A P value of <0.05 was considered significant.
In Vivo Kidney Transduction by AAV Variants
The capsid protein is a major determinant of cellular tropism, and we generated a panel of hybrid AAV vectors using the genome of the approved AAV serotype 2 and capsid proteins from AAV serotypes 5, 6, 8, and 9. We generated an additional AAV using the synthetic capsid protein Anc80, which has been engineered to reduce antigenic epitopes and has been shown to have unique transduction characteristics.8,30–32 All AAV serotypes contained the same CMV.eGFP.WPRE transgene construct. We reasoned that systemic delivery would be most practical for future clinical application, and therefore, all studies were conducted with intravenous administration; unless noted otherwise, we evaluated transduction 3 weeks after injection. Using a dose of 1011 GCs per mouse, we confirmed efficient transduction by AAV2/8 and AAV2/Anc80 of liver, with lower levels in heart, lung, and kidney (Figure 1A, Supplemental Figure 1). AAV2/8 and AAV2/Anc80 were the only AAV serotypes to exhibit kidney transduction, with Anc80 achieving three- to fivefold higher transduction than AAV8. GFP expression was primarily interstitial and enriched in the outer medulla.
We further characterized Anc80 cellular tropism in the kidney by immunofluorescence staining. GFP expression was limited to interstitial, glomerular, and juxtaglomerular regions. Interstitial GFP-positive cells uniformly expressed PDGFRβ, indicating a pericyte and fibroblast identity (Figure 1B). In the glomerulus, GFP-positive cells were WT1 negative and PDGFRβ positive, consistent with mesangium (Figure 1B). The only epithelial cells transduced were always found in a tubule adjacent to the glomerulus in cells that express the Na+:K+:2Cl– cotransporter Solute Carrier Family 12 Member 1, indicating the thick ascending limb. Intriguingly, only a subset of epithelia was transduced, however, and these were always cells adjacent to renin and nitric oxide synthase 1–expressing cells that themselves were not transduced (Figure 1C). These results indicate that AAV Anc80 efficiently transduces a subset of epithelia in the thick ascending limb adjacent to the juxtaglomerular apparatus (JGA).
We next asked whether the synthetic CASI promoter, consisting of a truncated CMV enhancer, chicken β-actin promoter, and UBC enhancer as well as splice donor and splice acceptor sequences, could increase sensitivity to detect transduction compared with the CMV promoter. Although overall kidney cell tropism was unchanged, we observed much better pericyte and fibroblast transduction with the CASI promoter compared with the CMV promoter (18% versus 4% of PDGFRβ-positive cells, respectively) (Figure 1, D and E). By contrast, mesangial cell transduction was somewhat reduced with the CASI promoter. Quantitation of transduction efficiencies between AAV2/2 and AAV2/Anc80 with either CMV or CASI promoters is shown in Figure 1, H and I. eGFP expression in JGA epithelia was detectable within 1 week of injection of AAV2/Anc80, whereas expression in mesangium and interstitium required 2 weeks for detection. Expression in mesangium increased from 2 to 4 weeks, whereas interstitial expression remained constant over this timeframe (Supplemental Figure 2).
Enhanced Sensitivity to Detect Gene Delivery by AAV-Cre
The fact that the CASI promoter increased apparent transduction efficiency compared with the CMV promoter suggested that we might lack adequate sensitivity to accurately measure viral transduction using a GFP reporter system. We reasoned that AAV encoding Cre recombinase in combination with a reporter mouse might provide superior sensitivity. We, therefore, generated AAV driving expression of Cre recombinase and injected 3×1011 GC per mouse into R26-tdTomato reporter mice. We observed very strong expression in all three kidney cell types (pericytes/fibroblasts, mesangial cells, and JGA), and overall transduction efficiency was far superior compared with AAV-GFP (Figure 1, F and G). Importantly, there was no transduction of peritubular endothelial cells in either the medulla (Figure 1F) or the cortex (Supplemental Figure 3). Anc80-Cre could transduce nearly 70% of interstitial pericytes and perivascular fibroblasts and nearly 90% of mesangium (Figure 1J). A dose-response study of AAV2/Anc80-Cre revealed increasing transduction through the highest dose tested (3×1011 GC per mouse) (Supplemental Figure 4).
We next investigated whether AAV causes adverse effects on uninjured kidney and other major organs. Somewhat surprisingly, our studies showed that AAV8 and the CASI.GFP.WPRE cassette (but not the CMV.GFP.WPRE cassette) led to liver toxicity at 1012 GC per mouse, including hepatocyte hypertrophy with nuclear swelling, monocellular infiltration along central veins, and bridging fibrosis (Figure 2A). It was unclear whether this toxicity was related to the vector or the expression of the transgene, but it was somewhat reflective of the previous findings on AAV8 by Wang et al.33 We also observed evidence of spontaneous myofibroblast transition in normal kidneys at the 1012 GC per mouse dose of the same AAV8 and cassette. This was evident by both upregulated expression of αSMA and immunofluorescence and increased mRNA encoding both αSMA and fibronectin (Figure 2, B–D). Of note, kidney histologic abnormalities were not detectable, and no histologic abnormalities were observed in the lungs or heart with any of the AAV serotypes tested (data not shown). It is unclear whether the kidney damage was the primary result of pericyte transduction by the highest dose of AAV8 or alternatively, a secondary effect of liver fibrosis; however, it is likely the latter, because transgene expression levels were very high in liver but lower in kidney. There was no toxicity observed with any of the other AAVs, including Anc80. In particular, we did not observe heart or lung inflammation or fibrosis 8 weeks after injection of Anc80-CASI-Cre dosed at 3×1011 GC per mouse (Supplemental Figure 5). Taken together, these results indicate that, in contrast to AAV8, Anc80 allows efficient gene transfer to kidney pericytes, mesangial cells, and JGA without toxicity.
Anc80 Transduces Myofibroblast Progenitors
Any gene therapy approach to treat kidney fibrosis requires transduction of myofibroblast progenitors. We, therefore, formally tested whether the pericytes and perivascular fibroblasts transduced by Anc80 are myofibroblast progenitors. We administered Anc80-CASI-Cre in doses ranging from 1010 to 3×1011 GCs per mouse to R26-tdTomato reporter mice and performed UUO. The lowest dose labeled about 5% of kidney pericytes, whereas 1×1011 GCs per mouse labeled 45% (Figure 3, A and B). In the UUO kidney, 15% of tdTomato-positive interstitial cells were αSMA-positive myofibroblasts at the low dose of AAV-Cre, and 60% were αSMA-positive myofibroblasts at the medium to midhigh dose. Injection of AAV-Cre at the low dose labeled isolated pericyte clones with tdTomato, and after UUO, these clones had expanded. We quantitated clonal expansion, measured an overall 4.7-fold increase in tdTomato-positive interstitial cells after UUO, and observed a right shift in the clone size distribution after UUO (Figure 3C). These results confirm that Anc80 transduces kidney myofibroblast progenitors that undergo proliferative expansion during fibrotic injury.
Pericyte-Specific Knockout of Gli2 by AAV-Cre Ameliorates Kidney Fibrosis
We have previously shown that Gli2 is required for myofibroblast proliferation during kidney fibrosis.34 We next aimed to evaluate the feasibility of gene editing using AAV-Cre. We generated R26tdTomato reporter mice that were either Gli2flox/flox homozygotes or Gli2flox/wt heterozygotes and injected 3×1011 GCs per mouse of AAV-Anc80-CASI-Cre 3 weeks before UUO surgery. On the basis of tdTomato expression, we achieved a transduction efficiency of 60% of cortical pericytes and perivascular fibroblasts and observed no significant histologic abnormalities in the contralateral, uninjured kidney (Figure 4, A and B). The Gli2flox/flox homozygotes had substantially reduced fibrosis compared with the Gli2flox/wt heterozygote controls by a variety of measures. These included reduced trichrome-positive collagen area and αSMA and Col1a1 expression by immunofluorescence (Figure 3, A and B) and reduced protein expression of Col1a1 by immunofluorescence (Figure 4, C and D). There were also reduced levels of total αSMA and fibronectin by Western blot (Figure 4, E–G). Additionally, the Gli2 knockout kidneys had reduced levels of mRNA encoding Gli1, Gli2, αSMA, Col3a1, Col1a1 and fibronectin (Figure 4, H–M). Injected mice from either group did not develop histologic abnormalities in contralateral kidneys, lung, heart, or liver (data not shown). These results indicate that Anc80-Cre can efficiently mediate recombination of floxed alleles other than R26 locus in kidney stroma.
Pericyte-Specific Gene Targeting of β-Catenin by AAV-Cre Ameliorates Kidney Fibrosis after UUO
Several lines of evidence have implicated canonical Wnt–β-catenin signaling in myofibroblast proliferation and differentiation during kidney fibrogenesis. This has never been tested directly, however, due to a lack of appropriate mouse models. We took advantage of Anc80-Cre tropism to test the hypothesis that canonical β-catenin signaling is necessary for renal fibrogenesis. We injected Anc80-Cre into β-cateninf/f versus β-cateninf/wt mice and subsequently induced fibrosis by UUO. Once again, 3×1011 GCs per mouse was injected 3 weeks before UUO surgery. After 10 days, mice with β-catenin ablation by Anc80-Cre had an even greater reduction in fibrosis than we observed with Gli2 knockout (Figure 5, A and B). We could detect substantially reduced levels of interstitial and total β-catenin protein as well as αSMA and fibronectin (Figure 5, C–E). Importantly, we also saw substantial reductions in a variety of fibrosis readouts as assessed by real time PCR (Figure 5, F–M). These included reductions in β-catenin targets Axin2 and Lef1, indicating successful ablation of canonical β-catenin signaling.
Analogous Tropism of Anc80 in Human Mesenchymal Cells
Mice may not predict the biology of AAV vectors in primates, and therefore, we next evaluated whether Anc80 is capable of transducing human mesenchymal cells. We first cultured adult human kidney fibroblasts and transduced them with Anc80.CASI.GFP. We observed GFP expression in PDGFRβ+ mesenchymal cells, suggesting a similar tropism of this virus in human as in mouse (Figure 6A). Because primary cells may lose differentiation marker expression in culture, we also asked whether Anc80-GFP could transduce the mesenchymal lineage in human kidney organoids generated from induced pluripotent stem cells. We observed that Anc80.CASI.GFP drove GFP expression exclusively in the interstitium of kidney organoids and that 26% of stromal marker Meis1-positive cells35 and 10% of PDGFRβ-positive cells were GFP positive (Figure 6, B–D). Although we cannot be certain of the identity of interstitial GFP-positive cells that are negative for MEIS1 and PDGFRβ, we speculate that these are mesenchymal lineage progenitors that have not yet fully differentiated, hence their lack of terminal differentiation markers, including MEIS1 and PDGFRβ. Collectively, these observations suggest that Anc80 AAV is capable of transducing the human kidney mesenchymal cell lineage.
We report three major findings. We have identified the first AAV serotype, namely Anc80, that efficiently transduces adult kidney mesenchymal cells and myofibroblast progenitors. We also show that Anc80-Cre mediates high-efficiency recombination of two separate floxed alleles, Gli2 and β-catenin, confirming important roles for these signaling pathways in mediating renal fibrosis and validating this approach in mice. Finally, we show that Anc80 also can also deliver genetic material to human mesenchymal cells in culture and in human kidney organoids, providing a proof of principle for the use of AAV-based vectors in gene therapy approaches to treat kidney fibrosis. The mechanistic basis for the enhanced transduction properties of the mesenchymal lineage in adult murine kidney is the subject of future studies, but it may relate to the rapid kinetics of expression after Anc80 infection that may enable transduction of less permissive cell targets.30,32
Our findings have applications for preclinical fibrosis models, because a major limitation of current investigations of stromal biology is the lack of high-efficiency Cre driver lines for the efficient manipulation of gene expression in adult kidney. Although FoxD1-Cre mediates recombination in nearly all kidney stroma, it is only active during nephrogenesis, and the FoxD1-CreERt2 driver is not expressed in adult kidney.36 We have used the Gli1-CreERt2 driver with success,34,37,38 but kidney expression is limited to outer medullary stroma in healthy adult kidney, with very limited expression in cortex. Thus, an important application of our findings is as a novel method for efficient, inducible gene knockout in adult kidney mesenchymal cells throughout cortex and medulla. Our approach carries the added benefit of not requiring breeding of a Cre driver allele onto the background of a floxed allele for a particular gene of interest.
These results confirm and extend important roles for Gli2 and canonical Wnt–β-catenin signaling in kidney fibrogenesis. We had already shown a role for Gli2 in myofibroblast differentiation, and here, we used this knowledge as a positive control to assess whether Anc80-Cre can delete genes in loci other than the R26 locus. Previously, tubular-specific β-catenin ablation was shown to have no antifibrotic effects on injured kidney,39 and recently, it was reported that tubule-derived Wnts are required for fibroblast activation and kidney fibrogenesis.40 However, whether interstitial stroma are the targets of tubule-derived Wnts has not been formally tested because of the lack of an inducible CreERt2 line with expression specific to kidney stroma. Our results, therefore, confirm that interstitial pericytes and fibroblasts do respond to Wnt ligands and that canonical β-catenin signaling is required for myofibroblast differentiation during fibrosis. This result is consistent with our prior demonstration that genetic stabilization of β-catenin in pericytes is sufficient to drive myofibroblast differentiation.41
Translation of these findings as a therapeutic approach to treat CKD could take several forms. AAV could be used, for example, to deliver siRNA targeting either Gli2 or β-catenin. Alternatively, it could deliver genes encoding inhibitors of these pathways, such as hedgehog-interacting protein or the secreted Wnt inhibitor Wnt inhibitory factor 1.42,43 Several hurdles, however, will need to be overcome to bring such a strategy into the clinic. Anc80 exhibits high-efficiency transduction of liver, muscle, retina, and other issues, leading to potential extrarenal toxicities from expression of hedgehog or Wnt pathway inhibitors. One potential solution is continued engineering of AAV tropism to achieve kidney specificity, and recent findings show the feasibility of such efforts.31 Alternatively, use of a kidney mesenchymal cell–specific promoter would limit extrarenal expression. Finally, although AAV supports gene expression for many months, the slowly progressive nature of CKD may present a need for repeated injections, raising the possibility of immunogenicity. Most mammals acquire humoral immunity against AAV capsids early in life, and as a consequence, they mount an immune response against conventional AAVs.8,44–47 Anc80 evokes a weaker immune response, because it is a synthetic virus, but repeated injections could lead to acquired immunity.8,30
In summary, this study evaluated five different conventional AAVs and a synthetic AAV for gene therapy applications in kidney myofibroblast progenitors. We identify Anc80 as a promising candidate for gene therapy applications targeting the kidney mesenchymal cell lineage.
L.H.V. is an inventor on several technologies licensed to pharmaceutical and biotechnology companies, including Anc80 technology, which was licensed to Astellas, Vivet Therapeutics, Lonza Houston, and Selecta Biosciences. L.H.V. receives research funding from and consults for Lonza Houston, Solid, and Selecta Biosciences. L.H.V. is a cofounder and equity holder of GenSight Biologics and Akouos. L.H.V. is also a member of the Scientific Advisory Board of GenSight Biologics, Akouos, and NightStarX. For all other authors, no competing financial interests exist.
The antirenin antibody was a gift from Ariel R. Gomez.
This work was supported by Giving/Grousbeck (L.H.V.) and National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases grants DK103740 (to B.D.H.), DK107374 (to B.D.H.), DK076169 (to B.D.H.), and DK115255 (to B.D.H.).
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