Original Article

Gene Therapy (2017) 24, 308–313; doi:10.1038/gt.2017.21; published online 13 April 2017

AAV9-mediated engineering of autotransplanted kidney of non-human primates

S Tomasoni1, P Trionfini1, N Azzollini1, L Zentilin2, M Giacca2,3, S Aiello1, L Longaretti1, E Cozzi4,5, N Baldan6, G Remuzzi1,7,8 and A Benigni1

  1. 1IRCCS – Istituto di Ricerche Farmacologiche Mario Negri, Bergamo, Italy
  2. 2Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy
  3. 3Department of Medical, Surgical and Health Sciences, University of Trieste, Trieste, Italy
  4. 4Department of Cardiac, Thoracic and Vascular Sciences, Transplant Immunology Unit, Padua University Hospital, Padova, Italy
  5. 5Consortium for Research in Organ Transplantation (CORIT), Padua, Italy
  6. 6Department of Surgical, Oncological and Gastroenterological Sciences, Padua University Hospital, Padova, Italy
  7. 7Unit of Nephrology and Dialysis, Azienda Socio-Sanitaria Territoriale (ASST) Papa Giovanni XXIII, Bergamo, Italy
  8. 8Department of Biomedical and Clinical Sciences, University of Milan, Milan, Italy

Correspondence: Professor G Remuzzi, IRCCS - Istituto di Ricerche Farmacologiche Mario Negri, Centro Anna Maria Astori, Science and Technology Park Kilometro Rosso, Via Stezzano, 87, Bergamo 24126, Italy. E-mail: giuseppe.remuzzi@marionegri.it

Received 6 December 2016; Revised 21 February 2017; Accepted 16 March 2017
Accepted article preview online 27 March 2017; Advance online publication 13 April 2017

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Abstract

Ex vivo gene transfer to the graft before transplantation is an attractive option for circumventing systemic side effects of chronic antirejection therapy. Gene delivery of the immunomodulatory protein cytotoxic T-lymphocyte-associated protein 4–immunoglobulin (CTLA4-Ig) prevented chronic kidney rejection in a rat model of allotransplantation without the need for systemic immunosuppression. Here we generated adeno-associated virus type 2 (AAV2) and AAV9 vectors encoding for LEA29Y, an optimized version of CTLA4-Ig. Both LEA29Y vectors were equally efficient for reducing T-cell proliferation in vitro. Serotype 9 was chosen for in vivo experiments owing to a lower frequency of preformed antibodies against the AAV9 capsid in 16 non-human primate tested sera. AAV9-LEA29Y was able to transduce the kidney of non-human primates in an autotransplantation model. Expression of LEA29Y mRNA by renal cells translated into the production of the corresponding protein, which was confined to the graft but not detected in serum. Results in non-human primates represent a step forward in maintaining the portability of this strategy into clinics.

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Introduction

Suppression of the host immune system is still the main goal in organ transplantation to ensure long-term allograft survival. Preventing the rejection of major histocompatibility complex-mismatched organs requires continuous administration of immunosuppressive drugs to inhibit one or more key steps in the alloimmune response. In this context, CD4 T lymphocytes, key cells in coordinating the immune response, are the primary targets of the currently available drugs in clinical practice. In the past two decades, maintenance of immunosuppression has been accomplished using calcineurin-inhibitor-based steroid-containing regimens. The availability of newer immunosuppressive medications, showing comparable efficacy in terms of allograft and patient outcomes, enabled the minimization of calcineurin inhibitor as well as steroid-based regimens for selected groups of patients.1 The new agents have significantly improved short-term survival rates for solid organ allografts, including kidney allografts.2 Unfortunately, the long-term use of non-specific immunosuppressive drugs, eventually resulting in systemic and non-specific inhibition of the graft recipient’s immune system, increases the risk of serious side effects, including opportunistic infections and cancer.3, 4 Moreover, drug-specific side effects enhance the risk of cardiovascular and metabolic disease, as well as of nephrotoxicity,5 which negatively impact on allograft function and outcome limiting the benefits of organ transplantation for patients. Antirejection drugs significantly reduce patient's quality of life, often causing poor adherence to the treatment.6 This is clinically significant, because non-adherence to immunosuppressants is a major cause of graft loss in renal transplant patients.6 Moreover, these agents cannot prevent chronic allograft dysfunction, the primary cause of graft loss 1 year posttransplantation. The half-lives of the transplanted kidneys have only changed marginally in the past 20 years, notwithstanding the introduction of newer, more powerful immunosuppressive agents in the past decade.7 Accordingly, the estimated half-life of kidney transplants from deceased donors has increased from 9.1 years in 1991 to 10.9 years in 2010.8 Still, concerted efforts must be made to design novel strategies to prevent acute graft rejection and chronic graft dysfunction, allowing the reduction of the systemic adverse effects of lifelong immunosuppressive agents on the one hand and promoting a state of donor-specific tolerance in the host on the other.

An alternative approach to systemic immunosuppression is to promote local intragraft immunosuppression by engineering the kidney before transplantation. In this context, the graft is a particularly suitable candidate for gene therapy for a few reasons. Owing to its accessibility, the graft can easily be engineered after retrieval. Moreover, local transduction of the graft could potentially circumvent the unwanted adverse effects of systemic immunosuppression and target additional mechanisms promoting graft protection.

Allograft rejection is the consequence of complete T-cell activation, a process that is triggered by engagement of T-cell receptor interacting with antigenic peptides presented on the major histocompatibility complex (in humans, human leukocyte antigens) of antigen-presenting cells and through activation of a second costimulatory pathway.9 In the absence of costimulatory signal, T cells become anergic and undergo apoptosis. Among the several costimulatory pathways, which can either upregulate or downregulate T-cell activation, one of the best characterized is that between CD28/cytotoxic T-lymphocyte-associated protein 4 (CTLA4) on T cells and CD80 (B7-1) and CD86 (B7-2) on antigen-presenting cells.10 CD28 signals promote effector T-cell differentiation and increase the production of antibodies by B cells and the proliferation of activated T cells.11, 12 T-cell activation induced by CD28 engagement is then turned off by the increase in the negative T-cell regulator CTLA4,11 which binds CD86 and CD80 with higher avidity than CD28. The receptor fusion protein CTLA4-Ig (Abatacept), containing the extracellular portion of CTLA4 and the Fc portion of human immunoglobulin (IgG1), has been developed to inhibit costimulation and T-cell activation by binding to CD86/CD80.13, 14 More recently, a mutagenesis and screening strategy was used to obtain the second-generation CTLA4-Ig, Belatacept (LEA29Y). The latter binds CD80 and CD86 2–4-fold better, respectively, than Abatacept and is 10-fold more potent at inhibiting T-cell activation in vitro than Abatacept.15

We demonstrated previously that the systemic administration of the fusion protein could be circumvented by the transfer of the CTLA4-Ig gene into the graft using an adenoviral vector that assured the persistent and highly efficient expression of the fusion protein in the organ.16 Indeed, the injection of an adenovirus encoding CTLA4-Ig into donor grafts during the cold-preservation period—allowing transgene expression for up to 2 weeks—was capable of preventing acute rejection by prolonging graft survival and inducing unresponsiveness toward donor antigens in fully incompatible rats without additional systemic immunosuppression. More importantly, local CTLA4-Ig gene delivery to the donor kidney was subsequently applied successfully to prevent chronic graft rejection in rats by using the non-pathogenic, single-stranded DNA adeno-associated virus (AAV), capable of inducing high-level transgene expression, for a more prolonged period than adenoviruses do, without eliciting any inflammatory or immune response.17 Long-term and sustained CTLA4-Ig gene expression in the graft was achieved by AAV vector, which prevented progressive proteinuria—a biomarker of chronic graft injury—and protected the transplanted kidneys from renal structural damage in a fully major histocompatibility complex-mismatched rat strain combination. Moreover, in recipient draining lymph nodes as well as in the graft for up to 120 days after transplantation, we found a population of anergic T cells with regulatory activity, which were eventually responsible for the induction of immune tolerance. These results suggested that genetic engineering of the donor graft by AAV-mediated CTLA4-Ig gene transfer was a valuable strategy for preventing the development of chronic graft rejection, circumventing the side effects of systemic immunosuppression.

Before moving to the clinical practice, a study in large animals is mandatory. The aim of the present study is to evaluate whether AAV9-CTLA4-Ig, in the formulation of Belatacept (LEA29Y), can transduce the kidneys of non-human primates in a kidney autotransplantation model.

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Results

Generation of AAV9-LEA29Y vector and evaluation of its functionality

After sequence optimization for codons, which are mostly expressed in non-human primates without altering the final protein sequence, we synthesized the LEA29Y cDNA and cloned it into pZac2.1 plasmid containing the AAV vector backbone (Figure 1a). LEA29Y protein expression and secretion were then evaluated through in vitro transfection experiments. To this end, HEK293 cells were transfected with pZac-LEA29Y by lipofectamine. The medium was collected 24 and 48h after transfection and analyzed for the presence of the LEA29Y protein by western blot using an antibody specific for soluble CTLA4. As shown in Figure 1b, western blot revealed that the recombinant protein was secreted into the medium as soon as 24h after the transfection and increased at 48h. Moreover, using an enzyme-linked immunosorbent assay (ELISA) kit specific for soluble human CTLA4, we measured the amount of the released LEA29Y protein in the culture medium that reached a concentration >6.4ngμl−1. In order to evaluate the functional activity of the secreted LEA29Y, immunosuppressive effect of the conditioned medium collected 48h after transfection was tested on mixed lymphocyte reaction experiments. Dendritic cells isolated from the bone marrow of Brown Norway rats were used as stimulators of T cells isolated from Lewis rats. The addition of the LEA29Y containing medium from transfected cells inhibited T-cell proliferation, indicating immunosuppressive activity by LEA29Y (Figure 1c).

Figure 1.
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Analysis of pZac-LEA29Y plasmid and evaluation of its functionality in vitro. (a) Restriction analysis of pZac-LEA29Y plasmid: 1, uncut plasmid; 2, plasmid digested with EcoRI and NotI; 3 and 4, plasmid linearized with HindIII and XhoI, respectively. (b) Western blot analysis of the supernatants from HEK293 transfected cells. (c) Mixed lymphocyte reaction experiments conducted in the presence or absence (CTR) of supernatant from HEK293 cells transfected with pZac-LEA29Y. Proliferation was measured by incorporation of 3H-Thymidine at days 2–5 and expressed as c.p.m. Results are mean±s.d. of three independent experiments. *P<0.005 versus CTR.

Full figure and legend (59K)

The pZac-LEA29Y was then packaged into AAV capsid serotype 2 and 9. Both vectors have been tested in vitro for their ability to infect HT1080 cells, a cell line highly permissive to AAV-mediated transduction,18 and to release the recombinant protein LEA29Y in the culture medium. Western blot analysis showed that both vectors were able to produce and release the recombinant protein in the culture medium, which was functionally active in reducing the proliferation of T lymphocytes in in vitro mixed lymphocyte reaction experiments (Figure 2).

Figure 2.
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Immunosuppressive effect of medium from AAV2-LEA29Y- and AAV9-LEA29Y-infected cells. Proliferation of T cells to allogeneic dendritic cells was evaluated in the presence of the medium from HT1080 cells (CTR) or AAV2-LEA29Y- and AAV9-LEA29Y-infected cells. Proliferation was measured by incorporation of 3H-Thymidine at day 5 and expressed as c.p.m. Results are mean±s.d. of three independent experiments. *P<0.05 versus CTR.

Full figure and legend (33K)

Neutralizing antibody assay in the serum of non-human primates

The presence of preformed antibodies against the AAV2 and AAV9 capsids was analyzed in the serum of non-human primates selected as the most suitable candidates for transplantation experiments. To this end, 16 sera were analyzed in vitro using a neutralizing antibody assay based on the infectivity inhibition of the AAV-luciferase vector by a test serum in HELA cells. To this end, AAV2-Luciferase (5 × 103 multiplicity of infection (MOI)) or AAV9-Luciferase (3 × 105 MOI) was preincubated with serial dilutions (from 1/2 to 1/64) of sera or with culture medium as control. The percentage of inhibition was calculated by quantifying the luciferase activity in cellular extracts 24h postinfection in relation to the control medium. The output of the measure was considered the dilution that was able to inhibit the cellular transduction by 50%. The results obtained showed a low neutralizing response against both serotypes. Notably, we detected the presence of preexisting neutralizing antibodies with a titer 1/2 to 1/16 against AAV2 capsid in 9 out of the 16 tested sera, while we detected a titer of 1/4 to 1/32 against AAV9 capsid in 5 out of the 16 non-human primates. These results are in line with those reported in literature, showing that the frequency of preformed antibodies against the AAV9 capsids, which could negatively affect the efficacy of gene transfer, is lower in non-human primates and in a healthy human population than that of preformed antibodies against the AAV2 capsids.19, 20

On the basis of this evidence and our results, we decided to proceed with serotype 9 for subsequent experiments.

Large-scale production of AAV9-LEA29Y for in vivo experiments

AAV9-LEA29Y vector was produced in bulk so that all non-human primate kidneys could be treated with the same vector preparation, which has been tested for its efficacy both in vitro and in vivo. In vitro, infected HEK293 cells showed LEA29Y mRNA expression, which was accompanied by the release of the recombinant protein in the cell supernatant (data not shown). For in vivo experiments, LEA29Y mRNA expression was evaluated on total RNA obtained from rat-engineered kidneys 21 days after AAV9-LEA29Y injection. As shown in Figure 3, reverse transcriptase-PCR (RT-PCR) analysis demonstrated efficient transduction of all treated kidneys.

Figure 3.
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RT-PCR analysis for LEA29Y on total RNA obtained from rat transplanted kidneys (n=3, lanes #1–#3) injected with AAV9-LEA29Y into the renal artery and killed 21 days after transplantation. RT− samples represent the reverse transcriptase–negative controls. C+ represents the positive control (plasmid DNA).

Full figure and legend (35K)

Once the efficacy of the AAV9-LEA29Y preparation had been proven in rodents, we moved to renal transplantation in non-human primates following the experimental design depicted in Figure 4a.

Figure 4.
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Experiments in non-human primates. (a) Experimental design of kidney autotransplantation experiments in non-human primate. (b) RT-PCR analysis for LEA29Y transcript on total RNA from renal tissue. Lanes (+) represent the treated kidney; lanes (−) represent the contralateral kidney; Naive represents total RNA extracted from a kidney sample taken from an untreated non-human primate. (c) RT-PCR analysis for LEA29Y transcript on total RNA from heart (H) and liver (L) tissues. C+: positive control (plasmid DNA).

Full figure and legend (80K)

After left uni-nephrectomy and a 1h cold preservation period subsequent to organ injection through the artery with AAV9-LEA29Y (1 × 1013 vector genomes (vg) per graft), the kidney was re-transplanted in the same animal (n=4), which was killed 60 days posttransplantation. At killing, both the untreated and AAV9-LEA29Y-treated kidneys were analyzed for transgene expression by RT-PCR and for protein production in the renal tissue homogenates using the ELISA kit specific for the soluble human CTLA4.

As shown in Figure 4b, AAV9-LEA29Y was able to infect the non-human primate kidneys, as revealed by the presence of the specific mRNA transcript in all grafts. A faint band was observed in the contralateral kidney of the first two animals (samples 4919F- and 8063D-), which could reflect a spill-over of the vector after transplantation. No transcript was detected in the renal biopsy sample taken from an untreated non-human primate (naive). Based on the muscle tropism of the AAV vectors, and because the liver is the main off-target organ after intravenous administration of gene delivery vectors, we evaluated heart and liver gene expression of LEA29Y to monitor possible leakage of the vector from the kidney after transplantation. As shown in Figure 4c, no transcript was observed in the hearts of treated non-human primates while three out of the four livers expressed the transgene.

We then measured the production of LEA29Y protein in the renal homogenates and in the serum using an ELISA kit specific to soluble CTLA4. CTLA4 was detected in all the four treated kidneys (44; 67; 130 and 60ngmg−1 proteins). In the contralateral kidneys, three animals displayed levels that were close to the detection limit of the assay while one of the four animals showed CTLA4 levels that were fairly detectable (3; 2; 96 and 10ngmg−1 proteins).

No soluble CTLA4 was detected in the sera of the four animals at any point of evaluation (5, 14, 21, 30, 45 and 60 days).

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Discussion

Here we demonstrated the feasibility of genetically engineering the kidney graft through local delivery of AAV vectors encoding the immunosuppressant protein LEA29Y in non-human primates. This innovative approach, in a mammalian species closer to humans than rodents, is a step forward for gene therapy targeting to the donor organ as a means for overcoming major unwanted effects of systemic immunosuppression.

The clinical need for the development of novel immunosuppressive drugs to overcome the non-immune toxicities of calcineurin inhibitors has prompted the search for new immunomodulatory drugs, including the CTLA4-Ig—Abatacept. This molecule, used clinically to treat psoriasis and rheumatoid arthritis, was effective in rodent models of renal transplantation but inadequate in non-human primate renal transplantation. Studies on codon-based mutagenesis enabled the identification of a more effective molecule called LEA29Y, or Belatacept, which has been designed to provide effective immunosuppression but reduced toxicity with respect to calcineurin inhibitors (cyclosporine, tacrolimus) not only in non-human primate models but also in humans. Phase 3 clinical trial data in kidney transplantation have demonstrated that Belatacept (administered intravenously) is non-inferior to cyclosporine in 1-year patient and allograft survival.21 Three-year data demonstrate improvements in the glomerular filtration rate in patients receiving Belatacept versus cyclosporine A-treated patients. Compared with those receiving cyclosporine A, Belatacept-treated patients seemed to experience improvements in cardio-metabolic parameters. However, systemic administration of Belatacept created a safety concern owing to an increased risk of posttransplant lymphoproliferative diseases, observed in Epstein–Barr virus sero-negative recipients or patients treated with lymphocyte-depleting agents.22 In the long term, the cumulative frequencies of serious adverse effects were similar across the treated groups.23 Thus targeting costimulatory signals with Belatacept is a more specific way of modulating alloimmune response than using conventional immunosuppressive agents, possibly limiting their toxicities. Still, the systemic administration of Belatacept is not free from the risk of posttransplant malignancies. These findings have triggered an intense search for alternative approaches to best use the great potential of costimulatory blockade with Belatacept in kidney transplantation, avoiding the major side effects observed after systemic administration of this biological agent.

In a rodent model of kidney transplantation, we demonstrated that a recombinant AAV2 vector effectively transduced the graft in syngeneic animals without inducing local host immune response. Thanks to the sustained long-term CTLA4-Ig gene expression in the graft, CTLA4-Ig prevented renal structural and functional damage associated with chronic rejection.17 Our current results in a non-human primate kidney autotransplantation model demonstrate, first, that AAV9 is a suitable gene delivery vector for engineering the graft before transplantation. Second, AAV9-mediated expression of LEA29Y translates into the local production of the immunosuppressive agent. A faint transduction of two out of the four contralateral kidneys, possibly due to leakage of the vector from the kidney after the circulation was restored, does not translate into any release of the LEA29Y in the systemic circulation. The latter finding is extremely important for the prevention of the undesirable side effects observed in patients after systemic Belatacept administration. AAVs are considered the vectors of choice for cardiac gene transfer and particularly serotype 9.24, 25 Here we did not observe any transgene expression in the heart of transplanted non-human primates, meaning that the leakage of vector was minimal and not enough to transduce the heart. On the other hand, the vector possibly released in the circulation after surgery was indeed sequestered into the livers of three out of the four non-human primates to different extents. This was not unexpected, as earlier evidence reported liver transduction after direct cardiac injection of AAV9 vector.25 In view of the future application of our approach in clinics, the transduced kidney could be connected to a pulsatile perfusion machine that, pumping a cold solution containing oxygen and nutrients through the kidney,26 could enhance the preservation of the graft, avoiding any possible spill-over of the vector.

Collectively, these findings create promising prospects for gene therapy for donor organs to move to the clinics, with important implications for the ongoing challenge of preventing chronic rejection while avoiding the need for systemic immunosuppression in clinical transplantation.

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Materials and methods

AAV vector construction

The cDNA for LEA29Y was synthesized from Genscript (Piscataway, NJ, USA) after codon optimization in Macaca fascicularis. The cDNA was cloned in the AAV vector backbone pZac2.1 (Gene Therapy Program, Penn Vector core, University of Pennsylvania, Pennsylvania, PA, USA) and packaged into AAV capsid serotypes 2 and 9. Infectious vector stocks were prepared by the ICGEB AAV Vector Unit (http://www.icgeb.org/avu-core-facility.html). Briefly, pZac2.1-LEA29Y was co-transfected together with the packaging/helper plasmids into HEK293 cells. Viral stocks were obtained by CsCl2 gradient centrifugation as previously described.27, 28 The physical titer of AAV preparations was determined by quantifying vector genomes (vg) packaged into viral particles, by real-time PCR using a TaqMan probe (TGGGAGGTCTATATAAGC) designed on common cytomegalovirus promoter sequence: values obtained were in the range of 9 × 1012–2 × 1013 vector genomesml−1.

In vitro studies

HEK293 cells (human embryonic kidney cells, ATCC, Manassas, VA, USA) were cultured in Minimum Essential Medium Eagle–Earle's Balanced Salt Solution (Lonza, Walkersville, MD, USA) supplemented with 10% fetal bovine serum, 0.075% Sodium bicarbonate, 1mm Sodium Pyruvate, 100μgml−1 streptomycin and 100IUml−1 penicillin. HEK293 were transfected with Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturers’ instructions.

HT1080 cells (human fibrosarcoma cell line, ATCC) were grown in Dulbecco’s modified Eagle’s medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) containing high glucose (4.5gl−1) supplemented with 10% fetal bovine serum, 2mm L-Glutamine, 1mm Sodium Pyruvate, 100μgml−1 streptomycin and 100IUml−1 penicillin. Cell cultures were maintained at 37°C in a humidified incubator. HT1080 cells were infected with 1 × 105 MOI of AAV vectors.

All cell lines were routinely tested for mycoplasma contamination.

Western blot and ELISA

LEA29Y expression was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis on a 10% acrylamide gel and visualized by western and immunoblot using the monoclonal anti-CTLA4 antibody (WH0001493M8, Sigma-Aldrich, St Louis, MO, USA).

LEA29Y was also quantified in the cell culture medium or in renal tissue homogenate by ELISA using the human sCD152/CTLA-4 kit (RBMS276R, Biovendor, Asheville, NC, USA). For tissue homogenates, quantification of LEA29Y was normalized for mg of total proteins. For protein extraction, frozen kidneys were lysed in 10 volumes of Ripa Buffer 1 × (20mm Tris-HCl pH 7.5, 150mm NaCl, 5mm EDTA pH 8.00, 0.5% Sodium Deoxycholate, 1% TritonX-100), supplemented with protease inhibitor cocktail (Sigma-Aldrich) by mechanical homogenyzation and centrifuged at 15000g for 10min at 4°C to remove detergent-insoluble material. Protein concentration was calculated by bicinchoninic acid assay (Bio-Rad, Segrate, Italy) according to the manufacturer's instructions.

Mixed lymphocyte reaction studies

Responder Lewis lymph node T cells (1 × 106) were cultured in triplicate with bone marrow-derived Brown Norway dendritic cells (1 × 104) used as stimulators in the presence of conditioned medium (diluted 1:10) collected from control cells or cells transfected with pZac-LEA29Y or AAV2 and AAV9-LEA29Y. Cultures were maintained in RPMI medium (Gibco) with 20% fetal bovine serum in 5% CO2 at 37°C for 5 days. T-cell proliferation was measured at 2–5 days by adding 1μCi 3H-Thymidine. In detail, during the last 18h, cells were pulsed with 1μCi per well 3H-Thymidine and the uptake of radioactivity was measured by liquid scintillation counting. Proliferation was expressed as c.p.m.

In vivo studies in rats

All procedures involving animals were performed in accordance with institutional guidelines in compliance with national (D.L.n.26, 4 March 2014) and international laws and policies (directive 2010/63/EU on the protection of animals used for scientific purposes).

Rats were maintained in a specific pathogen-free facility with a 12h dark/12h light cycle, in a constant temperature room and with free access to standard diet and water. Animal studies were approved by the Institutional Animal Care and Use Committees of IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy.

Male Lewis rats (10 weeks of age, Charles River Italia, Calco, Italia) were used as donors and recipients for syngeneic kidney transplantation as previously described.17 AAV viral vectors (4 × 1011 vg per kidney) were diluted in saline solution to a final volume of 500μl and administered ex vivo into kidney grafts via injection into the renal artery. Cold ischemia time was 30min before transplantation. Animals were killed at 21 days for LEA29Y mRNA expression (n=3).

Total RNA extraction and RT-PCR evaluation

Total RNA from transfected cells or frozen tissues (kidney, heart and liver) was extracted using Trizol reagent (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA) and treated with RNase-free DNase (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Three μg of total RNA were used for reverse transcription reaction with SuperScript II First-Strand Synthesis System (Life Technologies) following the manufacturer's instructions. No enzyme was added for reverse transcriptase–negative controls (RT−). The cDNA was submitted to PCR using the following primers: forward 5′-AGATTCTCAGGTGACCGAAGT-3′; and reverse 5′-GTGCTGTTGTACTGTTC-3′. Samples were then separated on a 1.5% agarose gel (LEA29Y transcript 507bp).

Neutralizing antibody assay in the serum of non-human primates

The presence of preformed antibodies against the AAV2 and AAV9 capsids was analyzed in the serum of animals that had been selected as best candidates for transplantation experiments. To this aim, 16 sera were analyzed in vitro by a neutralizing antibody assay based on the infectivity inhibition of the AAV luciferase vector by a test serum in HELA cells. AAV2-Luciferase (5 × 103 MOI) or AAV9-Luciferase (3 × 105 MOI) was preincubated with serial dilutions (from 1/2 to 1/64) of sera or with culture medium as control. The percentage of inhibition was calculated by quantifying the luciferase activity in cellular extracts 24h postinfection in relation to the control without serum. The output of the measure has been considered the dilution able to inhibit the cellular transduction of 50%.

In vivo studies in non-human primates

Cynomolgus monkeys (M. fascicularis, supplied by Sicombrec, Makati, Philippines), 3–4-year-old purpose-bred males, 3–6kg and of known ABO blood type, were used as donor and recipient in autotransplantation experiments (n=4). All experiments and procedures were conducted in accordance with the Italian Animals Act (D.L.n.26, 4 March 2014) and were authorized by a special Decree of the Italian Ministry of Health. After adequate sedation with ketamine (5–10mgkg−1) and metedomidine (10μgkg−1), the non-human primate was anesthetized with propofol (2.5mgkg−1) and midazolam (0.2mgkg−1) and maintained under general anesthesia with isofluorane (0.5–2% end tidal), O2 (0.8–2.5lmin−1) and N2O (0–2.5lmin−1). Analgesia was provided with buprenorphine (0.01mgkg−1). The abdominal aorta and lower vena cava were isolated and clamped. The left kidney was removed and injected with 1 × 1013 vg per kidney of AAV9-LEA29Y and maintained at 4°C for 1h. Following the cold ischemia time, the kidney was transplanted in the same animal. Longitudinal arteriotomy and venotomy were performed, followed by anastomosis end to end of the renal artery and of the renal vein. The surgery time was approximately 3h. All animals were housed under standard environmental conditions (12h light–dark cycle, temperature: 22±1°C and humidity: 50%) with free access to food and water. All efforts were put in place to minimize discomfort and animal care was provided by veterinarians and animal-care technicians with extensive expertise in the health care and housing of non-human primate. Animals were followed for 2 months and then killed for the evaluation of LEA29Y mRNA and protein expression.

Statistical analysis

Results of mixed lymphocyte reaction experiments are given as mean±s.d. The significance of differences between groups was analyzed using non-parametric Kruskal–Wallis test. All data were analyzed using the MedCalc 10.0.1 statistical software (Ostend, Belgium). Statistical significance was defined as P<0.05.

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Conflict of interest

The authors declare no conflict of interest.

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Acknowledgements

We thank Kerstin Mierke for editing the manuscript. We are also grateful to the staff of CORIT and of the University of Padua for the assistance provided during the in vivo studies. This work was supported by Fondazione CARIPLO and Fondazione Cassa di Risparmio di Padova e Rovigo (CARIPARO).