original article © The American Society of Gene & Cell Therapy

Phenotypic Correction of a Mouse Model for Primary With Adeno-associated Virus Gene Transfer

Eduardo Salido1, Marisol Rodriguez-Pena2, Alfredo Santana1, Stuart G. Beattie2, Harald Petry2 and Armando Torres1

1Centre for Biomedical Research on Rare Diseases (CIBERER), Hospital Universitario Canarias, Instituto Tecnologías Biomédicas, University of La Laguna, Tenerife, Spain; 2Department of Research and Development, Amsterdam Molecular Therapeutics, Amsterdam, North Holland, The Netherlands

Primary hyperoxaluria type I (PH1) is an inborn error of , or combined liver and kidney transplanta- metabolism caused by deficiency of the hepatic enzyme tion. However, this treatment has its own limitations including the alanine-glyoxylate aminotransferase (AGXT or AGT) scarce supply of suitable organs, significant morbidity and mortal- which leads to overproduction of oxalate by the liver ity, and the life-long requirement for immunosuppressive agents. and subsequent urolithiasis and renal failure. The current Thus, new treatments for PH1 are required, and as such, somatic therapy largely depends on liver transplantation, which gene therapy is a promising approach, provided that sufficient is associated with significant morbidity and mortality. To hepatocytes can be efficiently transduced to limit oxalate produc- explore an alternative treatment, we used somatic gene tion by the liver to amounts that can be excreted into the urine transfer in a mouse genetic model for PH1 (Agxt1KO). without kidney damage. Recombinant adeno-associated virus (AAV) vectors con- In the past decade, recombinant adeno-associated virus (AAV) taining the human AGXT complementary DNA (cDNA) has emerged as one of the most promising gene transfer vectors for were pseudotyped with capsids from either serotype 8 treatment of human diseases based on its ability to transduce both or 5, and delivered to the livers of Agxt1KO mice via dividing and nondividing cells and to mediate long-term transgene the tail vein. Both AAV8-AGXT and AAV5-AGXT vectors expression without toxicity.1 Preclinical studies using AAV in animal were able to reduce oxaluria to normal levels. In addi- models for different diseases have demonstrated long-term, stable tion, treated mice showed blunted increase of oxaluria transgene expression in liver, muscle, and central nervous system.2–7 after challenge with ethylene glycol (EG), a glyoxylate In addition, several early phase clinical trials with AAV vectors have 8–11 precursor. In mice, AGT enzyme activity in whole liver shown to be quite safe. The host immune response has resulted 12 extracts were restored to normal without hepatic tox- in limited efficacy in some studies, while sustained expression has 13 icity nor immunogenicity for the 50 day follow-up. In also been reported even with a T lymphocyte response. summary, this study demonstrates the correction of pri- There are several natural AAV serotypes, with serotype 2 mary hyperoxaluria in mice treated with either AAV5 or (AAV2) being the most extensively studied in the past two decades. AAV8 vectors. However, the vast majority of the human population have neu- tralizing antibodies against AAV214 and the relative transduction Received 16 September 2010; accepted 6 November 2010; efficiency of AAV2 in liver is <10% hepatocytes.15 Numerous dif- published online 30 November 2010. doi:10.1038/mt.2010.270 ferent AAV serotypes have been demonstrated to mediate diverse tissue tropism16 with the potential to evade anti-AAV2 neutral- Introduction izing antibodies. In addition, hybrid AAV serotypes have been Primary hyperoxaluria type I (PH1) (OMIM #259900) is a rare engineered,17,18 further increasing the AAV vector repertoire for metabolic disorder, inherited in an autosomal recessive manner. efficient transduction of the liver. PH1 is characterised by a deficiency of the hepatic enzyme alanine- The minimum proportion of hepatocytes that need to be glyoxylate aminotransferase (AGXT or AGT), which results in the transduced for phenotypic correction varies widely, depending on failure to detoxify glyoxylate, with an overproduction of oxalate. the mechanisms of disease involved. In PH1, where AGT-deficient AGT converts glyoxylate to glycine, using alanine as the donor hepatocytes would continue to produce oxalate, the therapeutic of the an amino group, with pyridoxal-phosphate as a cofactor. goal is to reduce oxalate production to levels that can be excreted High levels of oxalate in PH1 patients are excreted by the kidneys, by the kidney without developing renal failure. In humans, partial which undergo progressive deterioration as a result of calcium liver transplantation, which typically replaces a third of the liver oxalate (CaOx) deposition. After , oxalate levels volume, is not sufficient to prevent failure of the simultaneously raise to the point of systemic oxalosis, a life-threatening condition. transplanted kidney.19 Thus, the use of vectors with enhanced Currently, the most effective treatment for PH1 is pre-emptive hepatocyte tropism is necessary to limit the production of oxalate

Correspondence: Eduardo Salido, Hospital Universitario de Canarias, Ofra s/n, La Laguna, Tenerife 38320, Spain. E-mail: [email protected]

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below levels that can be safely excreted by the kidneys, preventing (PBS)- were administered intravenously via the tail vein. nephrocalcinosis and, eventually, systemic oxalosis. AAV sero- An additional group of five Agxt+/+ males, not injected with any types 5 and 8 display high tropism to the livers of mice and non- vector, were used as wild-type controls. human primates after intravenous administration.20,16,17 There is Urine oxalate excretion was followed for 8 weeks after AAV low pre-existing immunity to serotype 5 and 8 AAV in humans,21 vector injection. Figure 2a shows the mean oxalate excretion for making these two vectors sound choices for a gene augmentation animals injected with AAV8 vectors encoding either the human strategy in this inborn error of metabolism. AGXT cDNA or green fluorescent protein (GFP). During the first We have developed a mouse model for PH1: Agxt1KO (strain 2 weeks of the study, mice were challenged with 0.25 ml of 0.5 mol/l B6;129SvAgxttm1Ull), homozygous for the deletion of exons 4–8 of EG on three occasions, by gavage on days 6, 10, and 13, to fol- the Agxt1 gene.22 Agxt1KO mice lack stable Agxt1 mRNA and low the response to a discrete overload of the glyoxylate pathway. protein, reproducing key features of the human disease, including Both basal oxaluria and the increase in oxalate levels following the severe hyperoxaluria, crystalluria, and CaOx urolithiasis. When administration of the glyoxylate precursor were significantly lower challenged with EG, a glyoxylate precursor, Agxt-deficient mice in the treated group, compared with the controls, as evidenced 1 also develop nephrocalcinosis. Here, we show phenotypic correc- week postinjection. Urine oxalate levels in the treated animals 4 tion of Agxt1KO mice by hepatic gene transfer with AAV8 and weeks after injection were in the range observed in wild-type mice AAV5 vectors encoding human AGT. (0.37 ± 0.11 versus 0.31 ± 0.13 μmol/24 hour, respectively, P = 0.28). The response to the administration of EG was significantly blunted Results

AAV vectors were constructed by inserting the human AGXT Increase (∆) after EG gavage a 4 Baseline b cDNA into an pro-AAV2 vector plasmid, under the control of a 1 week 1.5 hybrid EalbAAT liver-specific promoter23 (Figure 1). 3 4 weeks AAV8 vectors were administered at doses of 5 × 1012 vector 1.0 2 P = 0.001 genomes per kg body weight (vg/kg), a dose considered sufficient P < 0.001 to transduce the vast majority of hepatocytes, in male Agxt1KO 1 0.5 µ mol/24 hour oxalate mice (n = 10 animals per group), which were followed for 50 days. 0 µ mol/24 hour oxalate 0.0 To compare the effect of gene transfer with AAV5 with respect to T W GFP GFP AAV8, and also to evaluate possible gender differences in vector- AGXT AGXT mediated expression we used different doses of AAV5 vectors at c Increase (∆) after continuous 0.5% EG d Increase (∆) after continuous 0.7% EG 13 12 11 either 1.5 × 10 , 5 × 10 , or 5 × 10 vg/kg body weight, and AAV8 4.0 P = 0.005 3 vectors at 5 × 1012 vg/kg, in both female and male Agxt1KO mice 3.5 3.0 (n = 5 animals per group). 2.5 2 Adult Agxt1KO mice (12–16 weeks old) were placed in met- 2.0 P = 0.003 P < 0.001 1.5 P = 0.002 P < 0.001 1 abolic cages, fed an oxalate-free diet and water ad libitum, and 1.0 0.5 µ mol/24 hour oxalate allowed to adjust for 3 days. Twenty-four hour urine was col- µ mol/24 hour oxalate lected in acidified tubes and the basal rate of oxalate excretion 0.0 0 T T W W GFP GFP was determined to be 2.06 ± 0.74 μmol/24 hours. Purified, high AGXT AGXT titer (1.7–6.3 × 1011 vg/ml for AAV8 and 4.5–10 × 1011 vg/ml for AAV5) preparations of vectors in 0.2 ml phosphate buffered-saline Figure 2 changes in 24-hour oxalate excretion (oxaluria) in a mouse genetic model for primary hyperoxaluria type I (Agxt1KO) after gene Stuffer therapy. (a) Oxaluria in Agxt1KO mice treated with AAV8. Marked reduc- tion in oxalate excretion is observed in adeno-associated virus 8–alanine- EAIbAATp WPRE glyoxylate aminotransferase (AAV8-AGXT) treated mice (AGXT cluster of 5′ ITR 3′ ITR bars) compared with controls [green fluorescent protein (GFP) cluster], already evident at 1 week. Four weeks after injection, AAV8-AGXT treated cDNA mice had oxaluria levels not significantly different from the wild-type (Agxt+/+) group (WT). (b) Increase in oxalate excretion after ethylene SV40 polyA insulator bGH-polyA glycol (EG) gavage. AAV8-AGXT treated mice (AGXT) responded with a mild oxaluria increase (0.34 ± 0.37 μmol/24 hour), while controls (GFP) Figure 1 Adeno-associated virus (AAV) vector structure. The trans- increased oxalate excretion an average 1.24 ± 0.57 μmol/24 hour (c) gene expression cassette is flanked by AAV2 inverted terminal repeats Increase in oxalate excretion with 0.5% EG in drinking water. AAV8-AGXT (ITR). All plasmids used contained stuffer and Simian vacuolating virus treated mice showed a functional reserve even better than the wild-type 40 (SV40)-derived polyA insulator sequences followed by the albumin mice (WT), resulting in oxaluria elevations of only 0.18 ± 0.34 μmol/24 enhancer-a1-antitrypsin promoter (EalbAATp). Either human alanine- hour, while the GFP control group increased oxalate excretion by an ­glyoxylate aminotransferase (AGXT), including its 5′ untranslated region, average 2.65 ± 1.31 μmol/24 hour (d) Increase in oxalate excretion with or enhanced green fluorescent protein-complementary DNA (GFP cDNAs) 0.7% EG in drinking water. AAV8-AGXT treated mice showed an increase were used, followed by a woodchuck hepatitis virus post-transcriptional in urine oxalate lower than wild-type animals (WT), while the GFP control regulatory element (WPRE) only in those constructs for AAV8 production, group responded with a dramatic oxaluria increase. Three animals devel- and the bovine growth hormone polyadenylation sequence. Thus, the oped renal failure and died in the GFP control group, while none of the four AAV plasmids used were ssAAV-EalbAAT-AGXT-WPRE-polyA, ssAAV- AAV8-AGXT treated mice died. Bars: mean values; Error bars: standard EalbAAT-AGXT-polyA (expressing the therapeutic gene), ssAAV-EalbAAT- deviation of the means. Nonparametric tests: Mann–Whitney for two GFP-WPRE-polyA and ssAAV-EalbAAT-GFP-polyA (expressing the reporter unpaired groups, Kruskal–Wallis for three unpaired groups and Friedman gene GFP). test for several repeated measures.

Molecular Therapy vol. 19 no. 5 may 2011 871 © The American Society of Gene & Cell Therapy Primary Hyperoxaluria Gene Therapy

in mice treated with AAV8-AGXT compared to those receiv- These results are consistent with the analysis of AGT expres- ing AAV8-GFP (increase, ∆ = 0.34 ± 0.37 μmol/24 hours versus sion in the liver at the end of the study. Western blots of AGT 1.24 ± 0.57 μmol/24 hours, respectively; P = 0.001; Figure 2b). After protein in liver (Figure 4) revealed robust AGT expression in 4 weeks, 0.5% EG was supplied continuously into the drinking water, AAV8-AGXT-treated mice compared to wild-type, while no and the increase in oxalate excretion was significantly smaller in protein could be detected in control Agxt1KO mice treated with AAV8-AGXT treated animals than in controls injected with AAV8- AAV8-GFP. Higher levels of expression were observed using AAV8 GFP (∆ = 0.18 ± 0.34 versus 2.1,165 ± 1.31 μmol/24 hour, respec- vectors, compared with AAV5 vectors, both in males and females. tively, P < 0.001; Figure 2c), and even smaller than in wild-type mice Nevertheless, the levels of expression achieved with 1.5 × 1013 vg/ subjected to the same EG challenge (∆ = 0.50 ± 0.21 μmol/24 hour, kg AAV5-AGXT was consistently higher than those observed in P = 0.005; Figure 2c). These data are consistent with the AAV8- wild-type mice. Lower doses (5 × 1012 vg/kg, or 5 × 1011 vg/kg) still AGXT encoded AGT protein accounting for a larger functional resulted in significant AGT expression in male livers, while these reserve in treated mice than wild-type animals. During the last 2 doses resulted in relatively low signals in female livers. weeks of the experiment, mice were subjected to 0.7% EG in drink- Transduction and expression of AAV8-AGXT in different ing water, which also resulted in significantly blunted increases of tissues of treated animals was also examined by immunoblot- oxaluria in AAV8-AGXT treated animals, compared with nega- ting. Brain, lung, spleen, kidney, seminal vesicles, and testicles tive control mice injected with AAV8-GFP (∆ = 0.54 ± 0.56 versus did not contain detectable AGT protein, but heart samples con- 2.46 ± 0.7 μmol/24 hour, respectively, P < 0.001), a response not sistently showed low levels of AGT protein, evident after longer significantly different from the one observed in wild-type controls film exposures. The presence of AAV8-AGXT DNA in various drinking 0.7% EG (∆ = 0.92 ± 0.33, P = 0.06; Figure 2d). tissues was evaluated by real-time PCR, using primers annealing During the last third part of the study period (days 36–50), to the 3′ region of the AGXT cDNA and the woodchuck hepatitis three animals that received AAV8-GFP died with signs of renal failure, while all the AAV8-AGXT treated mice were healthy dur- a Males AAV5 ing the follow-up. At the end of the study, mice were killed and 5 Baseline tissues and blood were collected. All kidneys from the AAV8-GFP P = 0.004 P = 0.023 4 Day 27 injected mice, while none from the mice that received AAV8- P = 0.006 AGXT, exhibited some degree of nephrocalcinosis at the end of the 3 study. Four mice showed small CaOx deposits in the medullary region only; 3 mice presented moderate CaOx deposits in the cor- 2

tex and medulla; and in the three mice that died there was severe µ mol/24 hour oxalate 1 and widespread nephrocalcinosis (Figure 5a). Plasma could not be obtained from the mice that died prematurely, but blood urea 0 nitrogen concentrations were significantly elevated among the GFP remaining seven AAV8-GFP treated mice compared to those that 5e12AGXT 5e11AGXT 1.5e13AGXT received AAV8-AGXT (26.7 ± 9.9 versus 19.1 ± 1.3 mg/dl, respec- b Females AAV5 tively, P = 0.04). 2.5 P = 0.002 Hepatic AGT enzyme activity was significantly higher in Baseline Agxt−/− mice injected with AAV8-AGXT than in the AAV8-GFP 2.0 Day 27 controls (24.9 ± 10.6 versus 4.1 ± 1.5 nmol/minute·mg protein, P = 0.002 1.5 P = 0.002; note that significant residual activity is known to be present in liver extracts of Agxt−/− animals, mainly with alanine as 1.0 a substrate22). µ mol/24 hour oxalate Figure 3 shows the mean oxalate excretion for animals 0.5 injected with either the human AGXT expressing vector or the 0.0 GFP expressing control, at various doses of AAV5, followed over GFP 5 weeks. All AGXT vector dose tested, except for the lower dose 5e12AGXT 5e11AGXT used (5 × 1011 vg/kg) in females, induced a significant reduction of 1.5e13AGXT oxalate excretion with respect to basal levels. During the last week Figure 3 changes in 24-hour oxalate excretion (oxaluria) in a mouse of the study (29–35 days after vector administration), mice were genetic model for primary hyperoxaluria type I (Agxt1KO) after challenged with 0.5% EG in drinking water. The increases in oxalate gene therapy with adeno-associated virus 5 (AAV5) vectors. (a) excretion following the administration of the glyoxylate precur- Oxaluria in male Agxt1KO mice treated with AAV5–alanine-glyoxylate aminotransferase (AGXT). A marked reduction in oxalate excretion is sor were significantly lower in the groups receiving AAV8-AGXT observed in mice treated with either dose of adeno-associated virus (0.44 ± 0.49 μmol/24 hours) and the highest dose of AAV5-AGXT 5–alanine-glyoxylate aminotransferase (AAV5-AGXT) in males, com- (1.41 ± 1.09 μmol/24 hours) than in those injected with AAV5-GFP pared with AAV5-green fluorescent protein (GFP) controls. (b) Oxaluria (4.49 ± 1.41 μmol/24 hours; P = 0.008). Indeed, mice that received in femaleAgxt1KO mice treated with AAV5-AGXT. Only females injected with either 1.5 × 1013 vg/kg or 5 × 1012 vg/kg but not 5 × 1011 vg/kg AAV8-AGXT and the highest dose of AAV5-AGXT responded to showed a significant decrease in oxalate excretion, compared with the EG challenge with increases in oxaluria not significantly differ- AAV5-GFP controls. Bars: mean values; Error bars: standard deviation of ent to wild-type mice (0.55 ± 0.24 μmol/24 hours; P = 0.15). the means. Nonparametric tests: Friedman test for repeated measures.

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AgxtKO injected with AGXT-AAV particles

12 12 11 WT AAV8 - 5 × 10 vg/kg AAV5 - 5 × 10 vg/kg AAV5 - 5 × 10 vg/kg Males

Agt

Gapdh

Females

Agt

Gapdh

Figure 4 Alanine-glyoxylate aminotransferase (Agxt) expres- sion (western blot) in liver of adeno-associated virus (AAV)-AGXT treated mice. Fifty μg liver protein from Agxt−/− mice treated with either 5 × 1012 vg/kg AAV8-AGXT, 5 × 1012 vg/kg AAV5-AGXT or 5 × 1011 vg/ kg AAV5-AGXT were probed with affinity-purified rabbit antibody raised d Males Females against recombinant mouse AGT and reprobed with rabbit anti-Gapdh serum as a loading control. High levels of AGT protein, higher than those observed in wild-type mice (wt), are seen in both male (upper panel) AAV8 and female (lower panel) mice injected with 5 × 1012 vg/kg AAV8-AGXT. 5 × 1012 The same dose of AAV5-AGXT virus resulted in robust Agxt expression vg/kg in males, also higher than in wt mice, but significantly lower levels of expression were seen in females. Injection of 5 × 1011 vg/kg AAV5-AGXT resulted in lower Agxt expression in males, while it was detectable only after longer exposures in females. Three representative samples from each group are presented. Five minutes exposure. AAV5 5 × 1012 virus post-transcriptional regulatory element (WPRE) sequence. vg/kg Standard curves were made with AAV8-AGXT and mouse inter- leukin 2 plasmids. Variations in the amount of template DNA were corrected by running parallel amplifications of the mouse inter- leukin 2 gene. In livers of AAV8-AGXT treated mice, we found AAV5 11 899 ± 74 vector copies per diploid genome by real-time PCR analy- 5 × 10 vg/kg sis. The estimation of vector copy number per mouse genome in other tissues were: 4.4 ± 0.8 (kidney), 1.6 ± 0.6 (lung), 0.7 ± 0.1 (spleen), 1.3 ± 0.1 (testis), and 3.3 ± 0.6 (heart). Thus, 50 days after injection of 1011 viral particles per mouse (5 × 1012 vg/kg), close to a Figure 5 Kidney histology and liver immunohistochemical detection thousand AAV copies per transduced hepatocyte were detected. of alanine-glyoxylate aminotransferase (Agxt). (a) Severe nephrocal- cinosis in Agxt−/− mouse treated with control AAV8-green fluorescent pro- Using a rabbit anti-AGT antibody, immunohistochemistry tein (GFP), while AAV8-AGXT treated animal shows no calcium oxalate revealed striking differences between livers from Agxt1KO mice (CaOx) deposits. Bar = 250 μm. (b) Immunohistochemical staining for treated with either AAV8-AGXT or AAV8-GFP (Figure 5b). Indeed, AGT. Adeno-associated virus 8 (AAV8)-AGXT treated Agxt−/− mice shows most of the hepatocytes from mice injected with AAV8-AGXT par- high levels of expression while no immunostaining is seen in the liver of mice injected with AAV8-GFP. Slides were scanned together to show the ticles showed some degree of immunostaining, with a characteristic intense, homogeneous expression achieved over the entire liver lobule, cytoplasmic granular pattern, while no AGT staining was observed at the end of the study (8 weeks after injection). (c) Subcellular local- in control mice injected with AAV8-GFP. We found 80–90% hepato- ization of AGT protein. Agxt−/− mice injected with AAV8-AGXT showed cytes transduced with AAV8-AGXT [88.6%, 95% confidence interval abundant punctated AGT signals, which colocalize with peroxisomal marker PMP70 and not with mitochondria. Bar = 25 μm. (d) AGT immu- (CI95): 87.6–89.6 in males and 87.6%, CI95: 86.6–88.6 in females] and nohistochemistry on male and female livers treated with AAV8-AGXT the highest dose of AAV5-AGXT (83.7%, CI95: 82.6–84.9 in males and AAV5-AGXT. Most of the hepatocytes from male mice injected with 12 5 × 1012 vector gemomes (vg)/kg of either AAV8 or AAV5-AGXT are posi- and 80.0%, CI95: 78.8–81.2 in females) (Figure 5d). With 5 × 10 vg/ kg AAV5-AGXT, over half the hepatocytes were positive for AGT tive. Lower percentages of hepatocytes were detected in female mice treated with the higher dose of AAV5-AGXT. After injection of 5 × 1011 vg/ immunostaining (55.8%, CI95: 54.3–57.4 in males and 50.3%, CI95: kg AAV5-AGXT, around 15% hepatocytes showed AGXT immunostain- 48.8–51.9 in females), while 5 × 1011 vg/kg AAV5-AGXT resulted in ing in males, while the percentage of transduced hepatocytes with this dose was around 6% in females. Bar = 100 μm. only 15.1% (CI95: 14.0–16.2) male hepatocytes and 6.5% (CI95: 5.8– 7.3) female hepatocytes positive for AGT immunostaining. Primary cultures of hepatocytes, isolated from one mouse peroxisomal protein PMP70 (Figure 5c). No mitochondrial mis- per group, were used to ascertain the subcellular localization of targeting was observed for the human AGXT (major haplotype) the expressed AGT protein by confocal microscopy. Intense AGT expressed in transduced mouse hepatocytes. immunofluorescence was observed in the peroxisomes of AAV8- Serum transaminases (alanine aminotransferase and aspartate AGXT transduced hepatocytes, showing colocalization with aminotransferase) were not elevated in AAV8-AGXT treated mice

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with respect to wild-type mice (58.7 ± 6.2 and 39 ± 6.9 U/L, ver- without toxicity. The feasibility of intravenous injection is encour- sus 56.3 ± 10.8 and 37.9 ± 4.1 U/L, respectively, P = 0.46 and 0.64), aging toward the development of a clinical gene therapy proce- and no liver lesions or evidence of oncogenesis were detected dure for PH1 patients, selecting serotypes with superior efficacy upon microscopic examination. In addition, we did not detect any for transduction in humans. significant titer of anti-AGT antibody in serum samples of AAV8- AGXT treated mice, by western blotting. Materials and Methods Animal experiments, urine and blood analyses. All animal experi- Discussion ments were approved by the animal experimentation ethics committee Our experiments provide “proof of principle” that gene transfer of the Hospital Universitario de Canarias, and carried out according to the Spanish and European law. B6;129SvAgxttm1Ull mice were genotyped with vectors capable of transducing a large proportion of hepa- as described,22 bred and maintained in a pathogen-free facility, with free tocytes, such as AAV8 and AAV5, is a promising new therapy access to standard chow (A04, Safe, France) and water. 12–16 week old for PH1. In this type of disease, where nontransduced hepato- mice were restrained and injected into the tail vein using a 1-ml syringe cytes would continue to produce oxalate, the therapeutic goal with 27G needle. AAV particles were dissolved in 5% sucrose-PBS solution. can only be achieved if sufficient hepatocytes are transduced so Mice were placed in metabolic cages designed for a solitary mouse (model that the overall production of oxalate by the treated liver remains 3600M, Tecniplast, Buguggiate, Italy) and allowed to get acclimatized to a below the capacity of the kidneys to excrete it without suffering ~4 g/day powdered, oxalate-free diet (TD94045, Harlan, Indianapolis, IN) irreversible damage. The relatively large glomerular filtration rate for 3 days prior to the start of urine collection. Two 24-hour urine collec- of the mouse, might account for the lack of nephrocalcinosis in tions were performed each week in narrow tubes containing 50 µl 6 N HCl. The oxalate oxidase assay (Greiner, Switzerland, Frickenhausen, Germany) Agxt1KO mice, unless they are challenged with glyoxylate pre- was used to measure urine oxalate, while the Jaffe alkaline picrate test was cursors. The administration of 0.5–0.7% EG increases the oxalate used to measure urine creatinine. Urine samples below 1 ml/24 hours for excretion rate to levels resulting in nephrocalcinosis in Agxt1KO 3-month-old mice were typically seen in cages with signs of incomplete mice, but not in wild-type controls.22 Both AAV8 and AAV5 vec- urine collection, correlating with low creatinine excretion values, and they tors administered at doses above 5 × 1011 vg/kg are able to blunt were excluded from the study. Similarly, samples with food or fecal con- the increase of oxalate excretion observed in mice drinking 0.5% tamination were excluded. At the end of the study, the mice were killed, EG to levels similar to those observed in wild-type mice. and blood was collected for biochemical analysis. Serum glutamate oxalo- Differences in biodistribution and vector production technol- acetate transaminase, glutamate pyruvate transaminase and blood urea nitrogen were measured in an autoanalyzer (A25, Biosystems, Barcelona, ogy make AAV5 a good tool for liver transduction in nonhuman 24 Spain). Serum samples were also used, diluted 1:100 in PBS, to check primates. Although AAV8 and high doses of AAV5 seem to correct for the presence of anti-AGXT mouse antibodies by immunoblotting of the oxalate excretion to similarly levels in males and females, sub- recombinant human AGXT protein. stantially inferior correction was achieved in female mice injected Liver samples were harvested for histology, enzyme AGT activity assay, with the lower doses of AAV5 vector. Gender differences in expres- and western blot analyses. Kidney samples were collected for histological sion of AAV vectors have been reported previously,25,26 pointing to analysis. Five males from the groups receiving higher AAV doses were an androgen-dependent pathway involved in AAV transduction. selected for a more extensive histological study that included brain, lung, In summary, we showed that a single tail vein administration heart, stomach, liver, spleen, pancreas, kidney, testis, and seminal vesicles. DNA and protein were also extracted from these tissues. of AAV5- or AAV8-AGXT resulted in sustained correction of the PH1 mouse phenotype, without evidence of liver damage or tox- Histological analysis. Tissues were fixed in 4% buffered paraformaldehyde icity, using AAV doses similar to the ones previously reported by and either embedded in paraffin or cryoprotected in 20% sucrose and snap 4,27 others. None of the mice used in our study developed tumors frozen in liquid nitrogen. Hematoxylin and eosin staining was performed in the liver or other organs, in agreement with a large long-term for in all tissues collected, and CaOx staining31 was performed on kidney study that found no evidence of tumorigenesis in AAV-treated sections. Kidney sections from a PH1 patient with nephrocalcinosis were mice.28 This AAV-based gene transfer approach resulted in suf- used as positive control for the CaOx staining. ficiently robust liver expression of the human AGXT cDNA so as to overcome the potential problem posed by the continued oxalate AGT enzyme assay, western blot and immunohistochemistry. AGT 22 production of nontransduced hepatocytes. We also demonstrated activity and western blot analyses were carried out as described. Immunohistochemistry was performed on frozen sections, incubat- that the product of the human AGXT gene is correctly targeted ing with 1:5,000 rabbit antihuman AGXT antibody (a gift from Dr C. to peroxisomes in mouse hepatocytes, and that it complements Danpure, UCL, UK) for 2 hours, followed by 3, 5 minutes each, PBS the deficit in the Agxt1KO model. Although even the lowest dose washes and a 30-minute incubation in horseradish peroxidase–conjugated of AAV5-AGXT tested (5 × 1011 vg/kg) resulted in some oxaluria anti-rabbit serum (Dako, Carpinteria, CA). After another 3 PBS washes, reduction in male mice, only 15% hepatocytes stained positive for a 3,3′-diaminobenzidine-H2O2 solution was used as chromogen, and AGT at this dose. Similar levels of transduction are expected to some sections were counterstained with hematoxylin. Serial sections were be insufficient in humans, where partial liver transplantation has also stained with 1:5,000 anti-glutamine synthetase antibody (Santa Cruz been shown to fail to prevent renal failure and oxalosis.19 Biotechnologies, Santa Cruz, CA) to label perivenular zones. To ascertain the percentages of hepatocytes expressing AGT, three areas of one slide per In general, a good correlation has been found between mice animal were digitized, at ×200 magnification, in a microscope fitted with and nonhuman primates as models to test AAV8-mediated gene digital camera. ImagePro plus v.6 software was used to count the number 29 therapy. A recent systematic evaluation of AAV vectors for of cells where immunostaining was above background levels. Background ­liver-directed gene transfer30 has shown the potential of novel levels were established with sections from AAV8-GFP injected mice AAV capsids to deliver high levels and stable transgene expression processed in parallel. Since the mouse liver has a significant number of

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binucleated hepatocytes, a single cell was counted when two nuclei were 4. Gregorevic, P, Blankinship, MJ, Allen, JM, Crawford, RW, Meuse, L, Miller, DG et al. (2004). Systemic delivery of genes to striated muscles using adeno-associated viral in close proximity. vectors. Nat Med 10: 828–834. 5. Grimm, D, Zhou, S, Nakai, H, Thomas, CE, Storm, TA, Fuess, S et al. (2003). Preclinical Recombinant AAV construction, production, and DNA analysis. The in vivo evaluation of pseudotyped adeno-associated virus vectors for liver gene therapy. Blood 102: 2412–2419. AAV plasmids used in this study contain the expression cassette flanked 6. Paterna, JC, Feldon, J and Büeler, H (2004). Transduction profiles of recombinant by two ITRs from the AAV2 and an appropriate stuffer sequence to adjust adeno-associated virus vectors derived from serotypes 2 and 5 in the nigrostriatal genome size to the optimal packaging capacity (4.1–4.9 kb) described for system of rats. J Virol 78: 6808–6817. 7. Sarkar, R, Tetreault, R, Gao, G, Wang, L, Bell, P, Chandler, R et al. (2004). Total AAV. The expression cassette has the following elements: the 5′ inverted correction of hemophilia A mice with canine FVIII using an AAV 8 serotype. Blood 103: terminal repeats from AAV2, a liver-specific EalbAAT promoter with 1253–1260. 23 8. Flotte, TR, Zeitlin, PL, Reynolds, TC, Heald, AE, Pedersen, P, Beck, S et al. (2003). regulatory sequences from the albumin enhancer, either human AGXT Phase I trial of intranasal and endobronchial administration of a recombinant adeno- cDNA or enhanced GFP cDNA, the bovine growth hormone polyadenyla- associated virus serotype 2 (rAAV2)-CFTR vector in adult cystic fibrosis patients: a tion sequence, WPRE, and the 3′ inverted terminal repeats from AAV2. two-part clinical study. Hum Gene Ther 14: 1079–1088. 9. Kay, MA, Manno, CS, Ragni, MV, Larson, PJ, Couto, LB, McClelland, A et al. (2000). Similar expression cassettes were also made without the WPRE sequence. Evidence for gene transfer and expression of factor IX in haemophilia B patients The four AAV plasmids usedFigure ( 1) were ssAAV-EalbAAT-AGXT- treated with an AAV vector. Nat Genet 24: 257–261. 10. Manno, CS, Chew, AJ, Hutchison, S, Larson, PJ, Herzog, RW, Arruda, VR et al. (2003). WPRE-polyA, ssAAV-EalbAAT-AGXT-polyA (expressing the therapeutic AAV-mediated factor IX gene transfer to skeletal muscle in patients with severe AGXT gene), ssAAV-EalbAAT-GFP-WPRE-polyA and ssAAV-EalbAAT- hemophilia B. Blood 101: 2963–2972. 11. Wagner, JA, Nepomuceno, IB, Messner, AH, Moran, ML, Batson, EP, Dimiceli, S GFP-polyA (expressing the reporter gene GFP). et al. (2002). A phase II, double-blind, randomized, placebo-controlled clinical Recombinant AAV8 vectors were produced by calcium phosphate- trial of tgAAVCF using maxillary sinus delivery in patients with cystic fibrosis with mediated cotransfection in 293 cells of three different plasmids: antrostomies. Hum Gene Ther 13: 1349–1359. 12. Manno, CS, Pierce, GF, Arruda, VR, Glader, B, Ragni, M, Rasko, JJ et al. (2006). pAdDeltaF6, p5E18-VD2/8 and the therapeutic (ssAAV-EalbAAT-AGXT- Successful transduction of liver in hemophilia by AAV-Factor IX and limitations WPRE-polyA) or reporter gene (ssAAV -EalbAAT-GFP-WPRE-polyA) imposed by the host immune response. Nat Med 12: 342–347. 16,32 13. Brantly, ML, Chulay, JD, Wang, L, Mueller, C, Humphries, M, Spencer, LT et al. (2009). plasmid. Similarly, AAV5 vectors were produced using p5E18-VD2/5 Sustained transgene expression despite T lymphocyte responses in a clinical trial of instead. AAV was harvested from transfected 293 cells by three cycles of rAAV1-AAT gene therapy. Proc Natl Acad Sci USA 106: 16363–16368. freeze-thaw 48 hours later. The virus was purified by ion exchange column 14. McKeon, C and Samulski, RJ (1996). NIDDK Workshop on AAV Vectors: Gene Transfer into Quiescent Cells. Hum Gene Ther 7: 1615–1619. chromatography and iodixanol gradient centrifugation followed by filtration 15. Nakai, H, Thomas, CE, Storm, TA, Fuess, S, Powell, S, Wright, JF et al. (2002). A limited and further concentration against PBS–5% sucrose. Virus titers (vg/ml) were number of transducible hepatocytes restricts a wide-range linear vector dose–response in recombinant adeno-associated virus-mediated liver transduction. J Virol 76: determined by quantitative-PCR performed in triplicate, using TaqMan 11343–11349. (Applied Biosystems) protocols, and primers pr300fw (5′-CCCTGTTTGCT 16. Gao, GP, Alvira, MR, Wang, L, Calcedo, R, Johnston, J and Wilson, JM (2002). Novel CCTCCGATAA-3′) and pr301rv (5′-GTCCGTATTTAAGCAGTGGATC adeno-associated viruses from rhesus monkeys as vectors for human gene therapy. Proc Natl Acad Sci USA 99: 11854–11859. CA-3′), which amplify a 95 bp fragment from the hAAT promoter region. 17. Wu, Z, Asokan, A and Samulski, RJ (2006). Adeno-associated virus serotypes: vector AAV capsid composition and purity was determined by sodium dodecyl toolkit for human gene therapy. Mol Ther 14: 316–327. 18. Büning, H, Perabo, L, Coutelle, O, Quadt-Humme, S and Hallek, M (2008). Recent sulfate polyacrylamide gel electrophoresis. developments in adeno-associated virus vector technology. J Gene Med 10: 717–733. The number of AAV vector genomes per cell from different tissues 19. Danpure, CJ (1995). Advances in the enzymology and molecular genetics of primary hyperoxaluria type 1. Prospects for gene therapy. Nephrol Dial Transplant 10 Suppl 8: was determined by real-time PCR analysis with primers corresponding 24–29. to the AGXT and WPRE sequences. Genomic DNA from different mouse 20. Gao, G, Vandenberghe, LH and Wilson, JM (2005). New recombinant serotypes of tissues (liver, heart, brain, kidney, lung, spleen, testis, and seminal vesicle) AAV vectors. Curr Gene Ther 5: 285–297. 21. Boutin, S, Monteilhet, V, Veron, P, Leborgne, C, Benveniste, O, Montus, MF et al. was isolated by proteinase K digestion followed by phenol-chloroform (2010). Prevalence of serum IgG and neutralizing factors against adeno-associated extraction. Fifty nanograms of DNA were used as template for PCR. virus (AAV) types 1, 2, 5, 6, 8, and 9 in the healthy population: implications for gene therapy using AAV vectors. Hum Gene Ther 21: 704–712. Dilutions of the rAAV vector plasmid were used to generate a standard 22. Salido, EC, Li, XM, Lu, Y, Wang, X, Santana, A, Roy-Chowdhury, N et al. (2006). curve for determination of vector genome copies. Alanine-glyoxylate aminotransferase-deficient mice, a model for primary hyperoxaluria that responds to adenoviral gene transfer. Proc Natl Acad Sci USA 103: 18249–18254. Statistical analysis. SPSS v.16 software was used, with no assumption of 23. Kramer, MG, Barajas, M, Razquin, N, Berraondo, P, Rodrigo, M, Wu, C et al. (2003). In vitro and in vivo comparative study of chimeric liver-specific promoters. Mol Ther 7: normal distribution. Mann–Whitney and Kruskal–Wallis tests were used 375–385. to compare two or more unpaired groups, respectively, while Friedman test 24. Nakai, H, Fuess, S, Storm, TA, Muramatsu, S, Nara, Y and Kay, MA (2005). Unrestricted hepatocyte transduction with adeno-associated virus serotype 8 vectors was used to compare several repeated measures. Statistical significance was in mice. J Virol 79: 214–224. assumed when P < 0.05. 25. Bell, P, Wang, L, Lebherz, C, Flieder, DB, Bove, MS, Wu, D et al. (2005). No evidence for tumorigenesis of AAV vectors in a large-scale study in mice. Mol Ther 12: 299–306. 26. Nathwani, AC, Gray, JT, McIntosh, J, Ng, CY, Zhou, J, Spence, Y et al. (2007). Safe and ACKNOWLEDGMENTS efficient transduction of the liver after peripheral vein infusion of self-complementary The authors acknowledge the excellent work of Eric J. Timmermans AAV vector results in stable therapeutic expression of human FIX in nonhuman with vector production, Cristina Paz with animal care. We are also primates. Blood 109: 1414–1421. 27. Davidoff, AM, Ng, CY, Zhou, J, Spence, Y and Nathwani, AC (2003). Sex significantly thankful to Drs Gloria González-Aseguinolaza, Antonio Fontanellas, influences transduction of murine liver by recombinant adeno-associated viral vectors Larry Shapiro and Sander van Deventer for helpful discussions. This through an androgen-dependent pathway. Blood 102: 480–488. work was supported by grant SAF2007-62343 from the Spanish 28. Pañeda, A, Vanrell, L, Mauleon, I, Crettaz, JS, Berraondo, P, Timmermans, EJ et al. (2009). Effect of adeno-associated virus serotype and genomic structure on liver Ministry of Science. S.G.B. and H.P. work for Amsterdam Molecular transduction and biodistribution in mice of both genders. 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