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. 2010 Nov 30;19(5):870–875. doi: 10.1038/mt.2010.270

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

Eduardo Salido 1, Marisol Rodriguez-Pena 2, Alfredo Santana 1, Stuart G Beattie 2, Harald Petry 2, Armando Torres 1
PMCID: PMC3098628  PMID: 21119625

Abstract

Primary hyperoxaluria type I (PH1) is an inborn error of metabolism caused by deficiency of the hepatic enzyme alanine-glyoxylate aminotransferase (AGXT or AGT) which leads to overproduction of oxalate by the liver and subsequent urolithiasis and renal failure. The current therapy largely depends on liver transplantation, which is associated with significant morbidity and mortality. To explore an alternative treatment, we used somatic gene transfer in a mouse genetic model for PH1 (Agxt1KO). Recombinant adeno-associated virus (AAV) vectors containing the human AGXT complementary DNA (cDNA) were pseudotyped with capsids from either serotype 8 or 5, and delivered to the livers of Agxt1KO mice via the tail vein. Both AAV8-AGXT and AAV5-AGXT vectors were able to reduce oxaluria to normal levels. In addition, treated mice showed blunted increase of oxaluria after challenge with ethylene glycol (EG), a glyoxylate precursor. In mice, AGT enzyme activity in whole liver extracts were restored to normal without hepatic toxicity nor immunogenicity for the 50 day follow-up. In summary, this study demonstrates the correction of primary hyperoxaluria in mice treated with either AAV5 or AAV8 vectors.

Introduction

Primary hyperoxaluria type I (PH1) (OMIM #259900) is a rare metabolic disorder, inherited in an autosomal recessive manner. PH1 is characterised by a deficiency of the hepatic enzyme alanine-glyoxylate aminotransferase (AGXT or AGT), which results in the failure to detoxify glyoxylate, with an overproduction of oxalate. AGT converts glyoxylate to glycine, using alanine as the donor of the an amino group, with pyridoxal-phosphate as a cofactor. High levels of oxalate in PH1 patients are excreted by the kidneys, which undergo progressive deterioration as a result of calcium oxalate (CaOx) deposition. After kidney failure, oxalate levels raise to the point of systemic oxalosis, a life-threatening condition. Currently, the most effective treatment for PH1 is pre-emptive liver transplantation, or combined liver and kidney transplantation. However, this treatment has its own limitations including the scarce supply of suitable organs, significant morbidity and mortality, and the life-long requirement for immunosuppressive agents. Thus, new treatments for PH1 are required, and as such, somatic gene therapy is a promising approach, provided that sufficient hepatocytes can be efficiently transduced to limit oxalate production by the liver to amounts that can be excreted into the urine without kidney damage.

In the past decade, recombinant adeno-associated virus (AAV) has emerged as one of the most promising gene transfer vectors for treatment of human diseases based on its ability to transduce both dividing and nondividing cells and to mediate long-term transgene expression without toxicity.1 Preclinical studies using AAV in animal models for different diseases have demonstrated long-term, stable transgene expression in liver, muscle, and central nervous system.2,3,4,5,6,7 In addition, several early phase clinical trials with AAV vectors have shown to be quite safe.8,9,10,11 The host immune response has resulted in limited efficacy in some studies,12 while sustained expression has also been reported even with a T lymphocyte response.13

There are several natural AAV serotypes, with serotype 2 (AAV2) being the most extensively studied in the past two decades. However, the vast majority of the human population have neutralizing antibodies against AAV214 and the relative transduction efficiency of AAV2 in liver is <10% hepatocytes.15 Numerous different AAV serotypes have been demonstrated to mediate diverse tissue tropism16 with the potential to evade anti-AAV2 neutralizing antibodies. In addition, hybrid AAV serotypes have been engineered,17,18 further increasing the AAV vector repertoire for efficient transduction of the liver.

The minimum proportion of hepatocytes that need to be transduced for phenotypic correction varies widely, depending on the mechanisms of disease involved. In PH1, where AGT-deficient hepatocytes would continue to produce oxalate, the therapeutic goal is to reduce oxalate production to levels that can be excreted by the kidney without developing renal failure. In humans, partial liver transplantation, which typically replaces a third of the liver volume, is not sufficient to prevent failure of the simultaneously transplanted kidney.19 Thus, the use of vectors with enhanced hepatocyte tropism is necessary to limit the production of oxalate below levels that can be safely excreted by the kidneys, preventing nephrocalcinosis and, eventually, systemic oxalosis. AAV serotypes 5 and 8 display high tropism to the livers of mice and nonhuman primates after intravenous administration.20,16,17 There is low pre-existing immunity to serotype 5 and 8 AAV in humans,21 making these two vectors sound choices for a gene augmentation strategy in this inborn error of metabolism.

We have developed a mouse model for PH1: Agxt1KO (strain B6;129SvAgxttm1Ull), homozygous for the deletion of exons 4–8 of the Agxt1 gene.22 Agxt1KO mice lack stable Agxt1 mRNA and protein, reproducing key features of the human disease, including severe hyperoxaluria, crystalluria, and CaOx urolithiasis. When challenged with EG, a glyoxylate precursor, Agxt-deficient mice also develop nephrocalcinosis. Here, we show phenotypic correction of Agxt1KO mice by hepatic gene transfer with AAV8 and AAV5 vectors encoding human AGT.

Results

AAV vectors were constructed by inserting the human AGXT cDNA into an pro-AAV2 vector plasmid, under the control of a hybrid EalbAAT liver-specific promoter23 (Figure 1).

Figure 1.

Figure 1

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Adeno-associated virus (AAV) vector structure. The transgene expression cassette is flanked by AAV2 inverted terminal repeats (ITR). All plasmids used contained stuffer and Simian vacuolating virus 40 (SV40)-derived polyA insulator sequences followed by the albumin enhancer-a1-antitrypsin promoter (EalbAATp). Either human alanine-glyoxylate aminotransferase (AGXT), including its 5′ untranslated region, or enhanced green fluorescent protein-complementary DNA (GFP cDNAs) were used, followed by a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) only in those constructs for AAV8 production, and the bovine growth hormone polyadenylation sequence. Thus, the four AAV plasmids used were ssAAV-EalbAAT-AGXT-WPRE-polyA, ssAAV-EalbAAT-AGXT-polyA (expressing the therapeutic gene), ssAAV-EalbAAT-GFP-WPRE-polyA and ssAAV-EalbAAT-GFP-polyA (expressing the reporter gene GFP).

AAV8 vectors were administered at doses of 5×1012 vector genomes per kg body weight (vg/kg), a dose considered sufficient to transduce the vast majority of hepatocytes, in male Agxt1KO mice (n = 10 animals per group), which were followed for 50 days. To compare the effect of gene transfer with AAV5 with respect to AAV8, and also to evaluate possible gender differences in vector-mediated expression we used different doses of AAV5 vectors at either 1.5×1013, 5×1012, or 5×1011 vg/kg body weight, and AAV8 vectors at 5×1012 vg/kg, in both female and male Agxt1KO mice (n = 5 animals per group).

Adult Agxt1KO mice (12–16 weeks old) were placed in metabolic cages, fed an oxalate-free diet and water ad libitum, and allowed to adjust for 3 days. Twenty-four hour urine was collected in acidified tubes and the basal rate of oxalate excretion was determined to be 2.06±0.74 µmol/24 hours. Purified, high 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 (PBS)-sucrose were administered intravenously via the tail vein. An additional group of five Agxt+/+ males, not injected with any vector, were used as wild-type controls.

Urine oxalate excretion was followed for 8 weeks after AAV vector injection. Figure 2a shows the mean oxalate excretion for animals injected with AAV8 vectors encoding either the human AGXT cDNA or green fluorescent protein (GFP). During the first 2 weeks of the study, mice were challenged with 0.25 ml of 0.5 mol/l EG on three occasions, by gavage on days 6, 10, and 13, to follow the response to a discrete overload of the glyoxylate pathway. Both basal oxaluria and the increase in oxalate levels following the administration of the glyoxylate precursor were significantly lower in the treated group, compared with the controls, as evidenced 1 week postinjection. Urine oxalate levels in the treated animals 4 weeks after injection were in the range observed in wild-type mice (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 in mice treated with AAV8-AGXT compared to those receiving AAV8-GFP (increase, Δ = 0.34±0.37 µmol/24 hours versus 1.24±0.57 µmol/24 hours, respectively; P = 0.001; Figure 2b). After 4 weeks, 0.5% EG was supplied continuously into the drinking water, and the increase in oxalate excretion was significantly smaller in AAV8-AGXT treated animals than in controls injected with AAV8-GFP (Δ = 0.18±0.34 versus 2.1,165±1.31 µmol/24 hour, respectively, P < 0.001; Figure 2c), and even smaller than in wild-type mice subjected to the same EG challenge (Δ = 0.50±0.21 µmol/24 hour, P = 0.005; Figure 2c). These data are consistent with the AAV8-AGXT encoded AGT protein accounting for a larger functional reserve in treated mice than wild-type animals. During the last 2 weeks of the experiment, mice were subjected to 0.7% EG in drinking water, which also resulted in significantly blunted increases of oxaluria in AAV8-AGXT treated animals, compared with negative control mice injected with AAV8-GFP (Δ = 0.54±0.56 versus 2.46±0.7 µmol/24 hour, respectively, P < 0.001), a response not significantly different from the one observed in wild-type controls drinking 0.7% EG (Δ = 0.92±0.33, P = 0.06; Figure 2d).

Figure 2.

Figure 2

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Changes in 24-hour oxalate excretion (oxaluria) in a mouse genetic model for primary hyperoxaluria type I (Agxt1KO) after gene therapy. (a) Oxaluria in Agxt1KO mice treated with AAV8. Marked reduction in oxalate excretion is observed in adeno-associated virus 8–alanine-glyoxylate aminotransferase (AAV8-AGXT) treated mice (AGXT cluster of bars) compared with controls [green fluorescent protein (GFP) cluster], already evident at 1 week. Four weeks after injection, AAV8-AGXT treated mice had oxaluria levels not significantly different from the wild-type (Agxt+/+) group (WT). (b) Increase in oxalate excretion after ethylene glycol (EG) gavage. AAV8-AGXT treated mice (AGXT) responded with a mild oxaluria increase (0.34±0.37 µmol/24 hour), while controls (GFP) increased oxalate excretion an average 1.24±0.57 µmol/24 hour (c) Increase in oxalate excretion with 0.5% EG in drinking water. AAV8-AGXT treated mice showed a functional reserve even better than the wild-type mice (WT), resulting in oxaluria elevations of only 0.18±0.34 µmol/24 hour, while the GFP control group increased oxalate excretion by an average 2.65±1.31 µmol/24 hour (d) Increase in oxalate excretion with 0.7% EG in drinking water. AAV8-AGXT treated mice showed an increase in urine oxalate lower than wild-type animals (WT), while the GFP control group responded with a dramatic oxaluria increase. Three animals developed renal failure and died in the GFP control group, while none of the AAV8-AGXT treated mice died. Bars: mean values; Error bars: standard deviation of the means. Nonparametric tests: Mann–Whitney for two unpaired groups, Kruskal–Wallis for three unpaired groups and Friedman test for several repeated measures.

During the last third part of the study period (days 36–50), three animals that received AAV8-GFP died with signs of renal failure, while all the AAV8-AGXT treated mice were healthy during the follow-up. At the end of the study, mice were killed and tissues and blood were collected. All kidneys from the AAV8-GFP injected mice, while none from the mice that received AAV8-AGXT, exhibited some degree of nephrocalcinosis at the end of the study. Four mice showed small CaOx deposits in the medullary region only; 3 mice presented moderate CaOx deposits in the cortex and medulla; and in the three mice that died there was severe and widespread nephrocalcinosis (Figure 5a). Plasma could not be obtained from the mice that died prematurely, but blood urea nitrogen concentrations were significantly elevated among the remaining seven AAV8-GFP treated mice compared to those that received AAV8-AGXT (26.7±9.9 versus 19.1±1.3 mg/dl, respectively, P = 0.04).

Figure 5.

Figure 5

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Kidney histology and liver immunohistochemical detection of alanine-glyoxylate aminotransferase (Agxt). (a) Severe nephrocalcinosis in Agxt−/− mouse treated with control AAV8-green fluorescent protein (GFP), while AAV8-AGXT treated animal shows no calcium oxalate (CaOx) deposits. Bar = 250 µm. (b) Immunohistochemical staining for AGT. Adeno-associated virus 8 (AAV8)-AGXT treated Agxt−/− mice shows 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 intense, homogeneous expression achieved over the entire liver lobule, at the end of the study (8 weeks after injection). (c) Subcellular localization of AGT protein. Agxt−/− mice injected with AAV8-AGXT showed abundant punctated AGT signals, which colocalize with peroxisomal marker PMP70 and not with mitochondria. Bar = 25 µm. (d) AGT immunohistochemistry on male and female livers treated with AAV8-AGXT and AAV5-AGXT. Most of the hepatocytes from male mice injected with 5×1012 vector gemomes (vg)/kg of either AAV8 or AAV5-AGXT are positive. Lower percentages of hepatocytes were detected in female mice treated with the higher dose of AAV5-AGXT. After injection of 5×1011 vg/kg AAV5-AGXT, around 15% hepatocytes showed AGXT immunostaining in males, while the percentage of transduced hepatocytes with this dose was around 6% in females. Bar = 100 µm.

Hepatic AGT enzyme activity was significantly higher in Agxt−/− mice injected with AAV8-AGXT than in the AAV8-GFP controls (24.9±10.6 versus 4.1±1.5 nmol/minute·mg protein, P = 0.002; note that significant residual activity is known to be present in liver extracts of Agxt−/− animals, mainly with alanine as a substrate22).

Figure 3 shows the mean oxalate excretion for animals injected with either the human AGXT expressing vector or the GFP expressing control, at various doses of AAV5, followed over 5 weeks. All AGXT vector dose tested, except for the lower dose used (5×1011 vg/kg) in females, induced a significant reduction of oxalate excretion with respect to basal levels. During the last week of the study (29–35 days after vector administration), mice were challenged with 0.5% EG in drinking water. The increases in oxalate excretion following the administration of the glyoxylate precursor were significantly lower in the groups receiving AAV8-AGXT (0.44±0.49 µmol/24 hours) and the highest dose of AAV5-AGXT (1.41±1.09 µmol/24 hours) than in those injected with AAV5-GFP (4.49±1.41 µmol/24 hours; P = 0.008). Indeed, mice that received AAV8-AGXT and the highest dose of AAV5-AGXT responded to the EG challenge with increases in oxaluria not significantly different to wild-type mice (0.55±0.24 µmol/24 hours; P = 0.15).

Figure 3.

Figure 3

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Changes in 24-hour oxalate excretion (oxaluria) in a mouse genetic model for primary hyperoxaluria type I (Agxt1KO) after gene therapy with adeno-associated virus 5 (AAV5) vectors. (a) Oxaluria in male Agxt1KO mice treated with AAV5–alanine-glyoxylate aminotransferase (AGXT). A marked reduction in oxalate excretion is observed in mice treated with either dose of adeno-associated virus 5–alanine-glyoxylate aminotransferase (AAV5-AGXT) in males, compared with AAV5-green fluorescent protein (GFP) controls. (b) Oxaluria 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 showed a significant decrease in oxalate excretion, compared with AAV5-GFP controls. Bars: mean values; Error bars: standard deviation of the means. Nonparametric tests: Friedman test for repeated measures.

These results are consistent with the analysis of AGT expression in the liver at the end of the study. Western blots of AGT protein in liver (Figure 4) revealed robust AGT expression in AAV8-AGXT-treated mice compared to wild-type, while no protein could be detected in control Agxt1KO mice treated with AAV8-GFP. Higher levels of expression were observed using AAV8 vectors, compared with AAV5 vectors, both in males and females. Nevertheless, the levels of expression achieved with 1.5×1013 vg/kg AAV5-AGXT was consistently higher than those observed in wild-type mice. Lower doses (5×1012 vg/kg, or 5×1011 vg/kg) still resulted in significant AGT expression in male livers, while these doses resulted in relatively low signals in female livers.

Figure 4.

Figure 4

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Alanine-glyoxylate aminotransferase (Agxt) expression (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 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) and female (lower panel) mice injected with 5×1012 vg/kg AAV8-AGXT. The same dose of AAV5-AGXT virus resulted in robust Agxt expression 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.

Transduction and expression of AAV8-AGXT in different tissues of treated animals was also examined by immunoblotting. Brain, lung, spleen, kidney, seminal vesicles, and testicles did not contain detectable AGT protein, but heart samples consistently showed low levels of AGT protein, evident after longer film exposures. The presence of AAV8-AGXT DNA in various tissues was evaluated by real-time PCR, using primers annealing to the 3′ region of the AGXT cDNA and the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) sequence. Standard curves were made with AAV8-AGXT and mouse interleukin 2 plasmids. Variations in the amount of template DNA were corrected by running parallel amplifications of the mouse interleukin 2 gene. In livers of AAV8-AGXT treated mice, we found 899±74 vector copies per diploid genome by real-time PCR analysis. 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 thousand AAV copies per transduced hepatocyte were detected.

Using a rabbit anti-AGT antibody, immunohistochemistry revealed striking differences between livers from Agxt1KO mice treated with either AAV8-AGXT or AAV8-GFP (Figure 5b). Indeed, most of the hepatocytes from mice injected with AAV8-AGXT particles showed some degree of immunostaining, with a characteristic cytoplasmic granular pattern, while no AGT staining was observed in control mice injected with AAV8-GFP. We found 80–90% hepatocytes transduced with AAV8-AGXT [88.6%, 95% confidence interval (CI95): 87.6–89.6 in males and 87.6%, CI95: 86.6–88.6 in females] and the highest dose of AAV5-AGXT (83.7%, CI95: 82.6–84.9 in males and 80.0%, CI95: 78.8–81.2 in females) (Figure 5d). With 5×1012 vg/kg AAV5-AGXT, over half the hepatocytes were positive for AGT immunostaining (55.8%, CI95: 54.3–57.4 in males and 50.3%, CI95: 48.8–51.9 in females), while 5×1011 vg/kg AAV5-AGXT resulted in 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 per group, were used to ascertain the subcellular localization of the expressed AGT protein by confocal microscopy. Intense AGT immunofluorescence was observed in the peroxisomes of AAV8-AGXT transduced hepatocytes, showing colocalization with peroxisomal protein PMP70 (Figure 5c). No mitochondrial mistargeting was observed for the human AGXT (major haplotype) expressed in transduced mouse hepatocytes.

Serum transaminases (alanine aminotransferase and aspartate aminotransferase) were not elevated in AAV8-AGXT treated mice with respect to wild-type mice (58.7±6.2 and 39±6.9 U/L, versus 56.3±10.8 and 37.9±4.1 U/L, respectively, P = 0.46 and 0.64), and no liver lesions or evidence of oncogenesis were detected upon microscopic examination. In addition, we did not detect any significant titer of anti-AGT antibody in serum samples of AAV8-AGXT treated mice, by western blotting.

Discussion

Our experiments provide “proof of principle” that gene transfer with vectors capable of transducing a large proportion of hepatocytes, such as AAV8 and AAV5, is a promising new therapy for PH1. In this type of disease, where nontransduced hepatocytes would continue to produce oxalate, the therapeutic goal can only be achieved if sufficient hepatocytes are transduced so that the overall production of oxalate by the treated liver remains below the capacity of the kidneys to excrete it without suffering irreversible damage. The relatively large glomerular filtration rate of the mouse, might account for the lack of nephrocalcinosis in Agxt1KO mice, unless they are challenged with glyoxylate precursors. The administration of 0.5–0.7% EG increases the oxalate excretion rate to levels resulting in nephrocalcinosis in Agxt1KO mice, but not in wild-type controls.22 Both AAV8 and AAV5 vectors administered at doses above 5×1011 vg/kg are able to blunt the increase of oxalate excretion observed in mice drinking 0.5% EG to levels similar to those observed in wild-type mice.

Differences in biodistribution and vector production technology make AAV5 a good tool for liver transduction in nonhuman primates.24 Although AAV8 and high doses of AAV5 seem to correct the oxalate excretion to similarly levels in males and females, substantially inferior correction was achieved in female mice injected with the lower doses of AAV5 vector. Gender differences in expression of AAV vectors have been reported previously,25,26 pointing to an androgen-dependent pathway involved in AAV transduction.

In summary, we showed that a single tail vein administration of AAV5- or AAV8-AGXT resulted in sustained correction of the PH1 mouse phenotype, without evidence of liver damage or toxicity, using AAV doses similar to the ones previously reported by others.4,27 None of the mice used in our study developed tumors in the liver or other organs, in agreement with a large long-term study that found no evidence of tumorigenesis in AAV-treated mice.28 This AAV-based gene transfer approach resulted in sufficiently robust liver expression of the human AGXT cDNA so as to overcome the potential problem posed by the continued oxalate production of nontransduced hepatocytes. We also demonstrated that the product of the human AGXT gene is correctly targeted to peroxisomes in mouse hepatocytes, and that it complements the deficit in the Agxt1KO model. Although even the lowest dose of AAV5-AGXT tested (5×1011 vg/kg) resulted in some oxaluria reduction in male mice, only 15% hepatocytes stained positive for AGT at this dose. Similar levels of transduction are expected to be insufficient in humans, where partial liver transplantation has been shown to fail to prevent renal failure and oxalosis.19

In general, a good correlation has been found between mice and nonhuman primates as models to test AAV8-mediated gene therapy.29 A recent systematic evaluation of AAV vectors for liver-directed gene transfer30 has shown the potential of novel AAV capsids to deliver high levels and stable transgene expression without toxicity. The feasibility of intravenous injection is encouraging toward the development of a clinical gene therapy procedure for PH1 patients, selecting serotypes with superior efficacy for transduction in humans.

Materials and Methods

Animal experiments, urine and blood analyses. All animal experiments were approved by the animal experimentation ethics committee of the Hospital Universitario de Canarias, and carried out according to the Spanish and European law. B6;129SvAgxttm1Ull mice were genotyped as described,22 bred and maintained in a pathogen-free facility, with free access to standard chow (A04, Safe, France) and water. 12–16 week old mice were restrained and injected into the tail vein using a 1-ml syringe with 27G needle. AAV particles were dissolved in 5% sucrose-PBS solution. Mice were placed in metabolic cages designed for a solitary mouse (model 3600M, Tecniplast, Buguggiate, Italy) and allowed to get acclimatized to a ~4 g/day powdered, oxalate-free diet (TD94045, Harlan, Indianapolis, IN) for 3 days prior to the start of urine collection. Two 24-hour urine collections were performed each week in narrow tubes containing 50 µl 6 N HCl. The oxalate oxidase assay (Greiner, Switzerland, Frickenhausen, Germany) was used to measure urine oxalate, while the Jaffe alkaline picrate test was used to measure urine creatinine. Urine samples below 1 ml/24 hours for 3-month-old mice were typically seen in cages with signs of incomplete urine collection, correlating with low creatinine excretion values, and they were excluded from the study. Similarly, samples with food or fecal contamination were excluded. At the end of the study, the mice were killed, and blood was collected for biochemical analysis. Serum glutamate oxaloacetate transaminase, glutamate pyruvate transaminase and blood urea nitrogen were measured in an autoanalyzer (A25, Biosystems, Barcelona, Spain). Serum samples were also used, diluted 1:100 in PBS, to check for the presence of anti-AGXT mouse antibodies by immunoblotting of recombinant human AGXT protein.

Liver samples were harvested for histology, enzyme AGT activity assay, and western blot analyses. Kidney samples were collected for histological analysis. Five males from the groups receiving higher AAV doses were selected for a more extensive histological study that included brain, lung, heart, stomach, liver, spleen, pancreas, kidney, testis, and seminal vesicles. DNA and protein were also extracted from these tissues.

Histological analysis. Tissues were fixed in 4% buffered paraformaldehyde and either embedded in paraffin or cryoprotected in 20% sucrose and snap frozen in liquid nitrogen. Hematoxylin and eosin staining was performed for in all tissues collected, and CaOx staining31 was performed on kidney sections. Kidney sections from a PH1 patient with nephrocalcinosis were used as positive control for the CaOx staining.

AGT enzyme assay, western blot and immunohistochemistry. AGT activity and western blot analyses were carried out as described.22 Immunohistochemistry was performed on frozen sections, incubating with 1:5,000 rabbit antihuman AGXT antibody (a gift from Dr C. Danpure, UCL, UK) for 2 hours, followed by 3, 5 minutes each, PBS washes and a 30-minute incubation in horseradish peroxidase–conjugated anti-rabbit serum (Dako, Carpinteria, CA). After another 3 PBS washes, a 3,3′-diaminobenzidine-H2O2 solution was used as chromogen, and some sections were counterstained with hematoxylin. Serial sections were also stained with 1:5,000 anti-glutamine synthetase antibody (Santa Cruz Biotechnologies, Santa Cruz, CA) to label perivenular zones. To ascertain the percentages of hepatocytes expressing AGT, three areas of one slide per animal were digitized, at ×200 magnification, in a microscope fitted with digital camera. ImagePro plus v.6 software was used to count the number of cells where immunostaining was above background levels. Background levels were established with sections from AAV8-GFP injected mice processed in parallel. Since the mouse liver has a significant number of binucleated hepatocytes, a single cell was counted when two nuclei were in close proximity.

Recombinant AAV construction, production, and DNA analysis. The AAV plasmids used in this study contain the expression cassette flanked by two ITRs from the AAV2 and an appropriate stuffer sequence to adjust genome size to the optimal packaging capacity (4.1–4.9 kb) described for AAV. The expression cassette has the following elements: the 5′ inverted terminal repeats from AAV2, a liver-specific EalbAAT promoter with regulatory sequences from the albumin enhancer,23 either human AGXT cDNA or enhanced GFP cDNA, the bovine growth hormone polyadenylation sequence, WPRE, and the 3′ inverted terminal repeats from AAV2. Similar expression cassettes were also made without the WPRE sequence. The four AAV plasmids used (Figure 1) were ssAAV-EalbAAT-AGXT-WPRE-polyA, ssAAV-EalbAAT-AGXT-polyA (expressing the therapeutic AGXT gene), ssAAV-EalbAAT-GFP-WPRE-polyA and ssAAV-EalbAAT-GFP-polyA (expressing the reporter gene GFP).

Recombinant AAV8 vectors were produced by calcium phosphate-mediated cotransfection in 293 cells of three different plasmids: pAdDeltaF6, p5E18-VD2/8 and the therapeutic (ssAAV-EalbAAT-AGXT- WPRE-polyA) or reporter gene (ssAAV -EalbAAT-GFP-WPRE-polyA) plasmid.16,32 Similarly, AAV5 vectors were produced using p5E18-VD2/5 instead. AAV was harvested from transfected 293 cells by three cycles of freeze-thaw 48 hours later. The virus was purified by ion exchange column chromatography and iodixanol gradient centrifugation followed by filtration and further concentration against PBS–5% sucrose. Virus titers (vg/ml) were determined by quantitative-PCR performed in triplicate, using TaqMan (Applied Biosystems) protocols, and primers pr300fw (5′-CCCTGTTTGCTCCTCCGATAA-3′) and pr301rv (5′-GTCCGTATTTAAGCAGTGGATCCA-3′), which amplify a 95 bp fragment from the hAAT promoter region. AAV capsid composition and purity was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis.

The number of AAV vector genomes per cell from different tissues was determined by real-time PCR analysis with primers corresponding to the AGXT and WPRE sequences. Genomic DNA from different mouse tissues (liver, heart, brain, kidney, lung, spleen, testis, and seminal vesicle) was isolated by proteinase K digestion followed by phenol-chloroform extraction. Fifty nanograms of DNA were used as template for PCR. Dilutions of the rAAV vector plasmid were used to generate a standard curve for determination of vector genome copies.

Statistical analysis. SPSS v.16 software was used, with no assumption of normal distribution. Mann–Whitney and Kruskal–Wallis tests were used to compare two or more unpaired groups, respectively, while Friedman test was used to compare several repeated measures. Statistical significance was assumed when P < 0.05.

Acknowledgments

The authors acknowledge the excellent work of Eric J. Timmermans with vector production, Cristina Paz with animal care. We are also thankful to Drs Gloria González-Aseguinolaza, Antonio Fontanellas, Larry Shapiro and Sander van Deventer for helpful discussions. This work was supported by grant SAF2007-62343 from the Spanish Ministry of Science. S.G.B. and H.P. work for Amsterdam Molecular Therapeutics (AMT), a private company dedicated to gene therapy, while M.R.P. is a former employee of AMT.

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