Gene Therapy in a Murine Model of Methylmalonic Acidemia Using Raav9-Mediated Gene Delivery

ORIGINAL ARTICLE Gene therapy in a murine model of methylmalonic acidemia using rAAV9-mediated gene delivery

JS Se´nac1,4, RJ Chandler1,2,4, JR Sysol1,LLi3 and CP Venditti1

Methylmalonic acidemia (MMA), an inherited metabolic disorder caused by deﬁcient activity of methylmalonyl-CoA mutase, carries a poor prognosis for long-term survival. While administration of a recombinant adeno-associated virus serotype 8 vector (rAAV8) can rescue MutÀ/À mice from neonatal lethality and provide sustained phenotypic correction, translation of gene therapy to human subjects will likely require multiple rounds of systemic administration and, ideally, the use of a vector that transduces the kidney. To examine the effectiveness of alternative rAAVs in the treatment of MMA, a serotype 9 rAAV expressing the Mut cDNA was constructed and delivered to newborn MutÀ/À mice (n¼11). rAAV9 gene therapy directed hepatic transgene expression within 24 h and effectively rescued the MutÀ/À mice from lethality, conferred long-term survival, markedly improved metabolism and resulted in striking preservation of renal function and histology. Systemic readministration of the vector at a dose similar to that used in human clinical trials (2.5Â109 GC of rAAV9 per gram) to older, treated MutÀ/À mice (n¼5) lowered circulating metabolites, increased in vivo propionate oxidative capacity and produced transgene expression in the kidney and liver. Our data support the use of an rAAV9 vector in the acute and chronic treatment of MMA, and highlight the renal tropism afforded by this novel serotype. Gene Therapy (2012) 19, 385–391; doi:10.1038/gt.2011.108; published online 21 July 2011

Keywords: AAV9; MMA; methylmalonic acidemia; stable isotope; renal disease

INTRODUCTION lethality and provide long-term phenotypic correction.22 Although Methylmalonic acidemia (MMA) is an autosomal recessive inborn neonatal rAAV8 gene therapy effectively targeted numerous tissues in error of organic acid metabolism characterized by severe metabolic MutÀ/À mice, particularly the liver and skeletal muscle, transgene instability, multiorgan pathology and a poor prognosis for long-term expression was not detectable in the kidney and decreased in other survival.1–7 This disorder is most commonly caused by deficient tissues over time. This suggests that translation to human subjects activity of methylmalonyl-CoA mutase,1 the 5¢-deoxyadenosylcobala- would likely require multiple rounds of gene delivery throughout life. min-dependent enzyme that isomerizes L-methylmalonyl-CoA into Alternative rAAV vectors that could provide enhanced renal tropism succinyl-CoA in the mitochondrial inner space. with the potential to be administered in the setting of an immune Current management approaches for vitamin B12 (hydroxoco- response to rAAV8 may be required for effective gene therapy balamin) non-responsive MMA patients include dietary restriction of application in humans. propiogenic amino acids, nutritional supplement administration and AAV serotypes are numerous, exhibit distinct tissue tropism and vigilant monitoring. Liver or combined liver/kidney transplantation offer the potential for optimization of gene delivery to specific target have been used to treat those with the most severe clinical manifesta- tissues and/or cell types.23,24 Among the naturally occurring adeno- tions.8–17 Patients with MMA, even those who have received liver associated viruses isolated from simian and human tissues, serotype 9 transplants, can develop progressive renal dysfunction,11 pathologically has recently been recognized as superior in the ability to direct hepatic characterized by tubulointerstitial nephritis,18,19 and eventually require and renal gene transfer in a mouse model of renal tubulointerstitial kidney transplantation.20 Whether circulating metabolite(s) and/or a fibrosis.25 These properties, coupled with previous studies showing cell intrinsic defect underlies the renal pathology of MMA remains that rAAV9 vectors exhibit rapid and widespread transgene expression unknown.21 The disease manifestations seen in the patient population, in mice and larger animals,26–28 suggest that a serotype 9 rAAV vector even those who have been intensively treated, demonstrate the need for would be promising in gene therapy applications for MMA.25–31 To new therapies, ideally ones that could target both the liver and kidney, explore the use of this novel serotype in the treatment of mice with to increase stability and protect from renal insufficiency. MMA, we developed and characterized an AAV serotype 9 vector, We have previously demonstrated that a recombinant adeno- rAAV9-CBA-mMut, to express Mut from the enhanced, chicken associated virus serotype 8 (rAAV8) vector expressing the murine b-actin promoter.32 Intrahepatic delivery of rAAV9-CBA-mMut Mut gene under the control of the cytomegalovirus enhancer/chicken effectively rescued MutÀ/À mice from neonatal lethality, provided b-actin promoter could rescue newborn MutÀ/À mice from neonatal immediate and persistent expression of the transgene, and after

1Organic Acid Research Section, Genetics and Molecular Biology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA; 2Institute for Biomedical Sciences, George Washington University, Washington, DC, USA and 3Kidney Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA Correspondence: Dr CP Venditti, Organic Acid Research Section, Genetics and Molecular Biology Branch, National Human Genome Research Institute, National Institutes of Health, Building 49 Room 4A18, Bethesda, MD 20892, USA. E-mail: [email protected] 4These authors contributed equally to this study. Received 14 February 2011; revised 13 June 2011; accepted 15 June 2011; published online 21 July 2011 Gene therapy for MMA with AAV9 JS Se´nac et al 386

100 Mut -/- Mut -/-

+/- -/- rAAV9-CBA-mMut rAAV2-CBA-mMut

80 *** Mut Mut 24 hrs 72 hrs 24 hrs 72 hrs

Mut alive

-/- 60 Mut C III 40

Percent Percent Mut -/- rAAV9-CBA-mMut 1x1010 GC (n =11) 20 Figure 2 Mut expression in liver tissues 1 and 3 days after neonatal Untreated Mut -/- (n =58) intrahepatic injection of 1Â1010 GC of rAAV9-CBA-mMut or rAAV2-CBA- 0 mMut. Immunoreactive Mut enzyme (labeled Mut) is present in the untreated Mut+/À extract and in MutÀ/À mice that were treated with rAAV9- 0 100 200 300 400 CBA-mMut, but not in the untreated MutÀ/À mouse or MutÀ/À mice that Days received rAAV2-CBA-mMut. The mitochondrial loading control (labeled Figure 1 Survival of MutÀ/À mice after rAAV9-CBA-mMut gene therapy complex III) shows approximately the same intensity in each sample. treatment. Survival is shown in days between untreated MutÀ/À mice (n¼58) and MutÀ/À mice treated with a single intrahepatic dose of 1Â1010 GC of rAAV9-CBA-mMut (n¼11), 82% (n¼9) of treated MutÀ/À mice were alive at Growth improvement after gene therapy day of life (DOL) 365. All untreated MutÀ/À mice perished by DOL 72, with Although untreated MutÀ/À and Mut+/À mice are indistinguishable at the majority of deaths occurring before DOL 24. ***rAAV9-CBA-mMut- birth, the rare MutÀ/À animals that survive neonatal lethality present treated MutÀ/À mice survive significantly longer than untreated MutÀ/À mice signs of severe growth retardation.34 By 2 months of age, untreated (Po0.001). MutÀ/À mice weigh less than one third as much as sex-matched Mut+/À littermates, appear grossly abnormal and uniformly perish by low-dose systemic reinjection at 1 year, directed expression of the 72 days.22 In contrast, MutÀ/À mice treated with rAAV9-CBA-mMut transgene in the liver and kidney. These studies highlight the novel were significantly larger than untreated MutÀ/À mice (Figure 3a; characteristics of rAAV9-mediated gene delivery as a treatment for ***Po0.001). They appeared and behaved in an identical manner as MMA, particularly rapid transgene expression in vivo and tropism for treated control littermates, but were slightly smaller than heterozygous the kidney, an organ involved in the pathophysiology of many littermates, even after vector readministration (Supplementary hereditary and acquired disorders. Figure 1). Furthermore, treated MutÀ/À mice thrive on a regular diet, unlike untreated MutÀ/À mice that have to be maintained on a RESULTS high-fat and carbohydrate diet. Gene therapy with low-dose AAV9 rescues the lethal MutÀ/À phenotype Restoration of Mut function and activity MutÀ/À mice received a single dose of 1Â1010 vector genome copies of In order to study the efficacy of gene therapy, we analyzed various rAAV9-CBA-mMut by intrahepatic injection at birth (n¼11). While parameters that reflect the Mut enzymatic activity and function, two treated MutÀ/À mice died within the first 48 h, 82% of the mice including metabolite concentrations and ability of the treated À/À 13 13 that received rAAV9-CBA-mMut gene therapy survived 1 year post Mut mice to oxidize 1- C-sodium propionate into CO2. treatment (Figure 1). All of the untreated MutÀ/À mice died by day Compared with controls, plasma MMA levels remained significantly of life 72, with more than 90% mortality occurring by day of life elevated at all times in the treated MutÀ/À mice, and stabilized at 24 (Figure 1). In contrast, at 1 year, 89% of the injected hetero- 323 mmol lÀ1 (±30 mmol lÀ1) by 1 year of life (Figure 3b). zygous control littermates were alive. Deceased mice were autolysed, Mice with functional hepatic Mut activity can oxidize 1-13C-sodium 13 22 cannibalized or entirely missing from their cages. propionate into CO2. This activity is markedly reduced in untreated MutÀ/À mice (Figure 3c). At 9 months, rAAV9-CBA- rAAV9 gene therapy vector leads to rapid and high-level expression mMut-treated MutÀ/À mice and heterozygous littermates were of the mutase transgene injected with 1-13C-sodium propionate and breath samples were +/À To examine the expression kinetics of the viral transgene in the collected while the CO2 production rate was measured. Mut À/À 13 13 immediate pre-injection period, livers from treated newborn Mut mice convert B50% of 1- C-sodium propionate into CO2 in mice were collected at 24 and 72 h after treatment with either 25 min (n¼3) whereas untreated MutÀ/À mice (n¼6) oxidize only 1Â1010 GC of rAAV9-CBA-mMut or rAAV2-CBA-mMut, a serotype B10% of the injected dose (Figure 3c). At 9 months post treatment, that has well-studied uncoating properties in the murine liver.33 1-13C-propionate oxidation was significantly increased above the Untreated Mut+/À and MutÀ/À littermate livers were harvested in parallel untreated state in the rAAV9-CBA-mMut-treated MutÀ/À mice and served as controls. There was no detectable level of immunoreactive (n¼5; Figure 3c), demonstrating that the Mut transgene was mutase enzyme in untreated MutÀ/À mice (Figure 2). However, as early persistently expressed and functional in vivo. as 24 h after injection, rAAV9-CBA-mMut-treated MutÀ/À mice demonstrated hepatic Mut protein at levels similar to those seen in untreated rAAV9 gene therapy preserves the glomerular filtration rate and Mut+/À animals (Figure 2). By 72 h, Mut expression continued to kidney cytoarchitecture increase in the rAAV9-CBA-mMut-treated MutÀ/À mice (Figure 2). Because human MMA patients suffer from kidney failure and previous In contrast, Mut was absent in MutÀ/À mice similarly treated with rAAV2- studies have established that older, untreated MutÀ/À mice exhibit CBA-mMut (Figure 2) at both times, demonstrating the influence of severe and widespread tubulointerstitial changes,34 we examined the serotype and tropism on early transgene expression in the liver. the effect of gene therapy on kidney function and histology in the

Gene Therapy Gene therapy for MMA with AAV9 JS Se´nac et al 387

a Mut +/- (n =8) b Mut -/- rAAV9-CBA-mMut 1x1010 GC (n =9) Mut -/- rAAV9-CBA-mMut 120 -/- Mut (n =3) 1x1010 GC (n =9)

100 1500 ***

*** M)

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40 *** 500

20 Percent weight of

0 Plasma methylmalonic acid ( 0 60 270 365 60 270 365 Days Days

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60 L/min) C propionate

µ 400 13 40 dose oxidized GFR ( 200 20 Percent 1-

0 0 Mut +/- (n =3) Mut -/- rAAV9-CBA-mMut 1x1010 GC (n =3) Figure 3 Growth and metabolic effects after rAAV9 gene therapy. (a) Percent weight at day of life (DOL) 60, 270 and 365 between Mut+/À diet and gender- matched littermates (n¼8,9) compared with untreated MutÀ/À mice on DOL 60 (n¼3) or MutÀ/À mice treated via an intrahepatic injection of 1Â1010 GC of rAAV9-CBA-mMut at birth (n¼9) is depicted. The rAAV9-CBA-mMut-treated MutÀ/À mice showed significant growth improvement compared with the untreated MutÀ/À mice (***Po0.001) but were smaller than Mut+/À diet and gender-matched littermates. Error bars represent ±1s.d.(b) Plasma / methylmalonic acid levels (mM) were measured at DOL of 60, 270 and 365 in the rAAV9-CBA-mMut-treated MutÀ À mice (n¼9) as a reflection of Mut activity. Error bars represent ±1 s.d. The rAAV9-CBA-mMut-treated mutant mice have increased plasma methylmalonic acid concentrations that are reduced +/ on DOL 270 (399 mM (±40 mM)) and 365 (323 mM (±30 mM)) compared with the values at DOL 60 (1196 mM (±78 mM)). Untreated and treated Mut À mice have plasma methylmalonic acid levels between 5–10 mM and are not depicted in this graph. (***Po0.001; P¼0.2, NS (not significant)) Error bars represent ±1 s.d. All untreated MutÀ/À mice perished by DOL 72. (c)1-13C-propionate oxidation 1 year after rAAV9-CBA-mMut treatment. In all, 200 mgof1-13C- sodium propionate was injected i.p. into Mut+/À (n¼3), 1Â1010 GC rAAV9-CBA-mMut-treated MutÀ/À (n¼5) or untreated MutÀ/À (n¼6) mice. 13C 13 enrichment in expired CO2 was measured and used to determine the percent of the administered 1- C-propionate dose that was oxidized. Error bars surround the 95% confidence intervals. The rAAV9-CBA-mMut-treated MutÀ/À mice show a significant increase in the ability to oxidize 1-13C-propionate compared with the untreated MutÀ/À mice at 25 min post injection (***Po0.001; **Po0.01). (d) Glomerular filtration rate measured by FITC-inulin clearance at 1 year of life between rAAV9-CBA-mMut-treated Mut+/À (n¼3) and treated MutÀ/À mice (n¼3). There is no significant difference between treated MutÀ/À mice and heterozygous littermates. Error bars represent ±1s.d. rAAV9-CBA-mMut-treated MutÀ/À mice. The measured glomerular Measurement of the concentration of methylmalonic acid in the filtration rate (GFR) did not significantly differ between the rAAV9-CBA- plasma, and whole-body 1-13C-sodium propionate oxidative capa- mMut-treated MutÀ/À (n¼3) and Mut+/À mice (n¼3; Figure 3d). As the city monitored the metabolic effects of reinjection. Within 3 days untreated MutÀ/À littermates perish, GFR measurements values for after readministration of rAAV9-CBA-mMut, the plasma methyl- untreated, matched mutant controls are not available for comparison. malonic acid concentrations significantly decreased to 126 mM Kidney histology was next examined in rAAV9-CBA-mMut-treated (±11 mM; Po0.001) compared with pretreatment levels of 323 mM À/À +/À 13 Mut mice (n¼6) and compared with Mut littermates (n¼3). (±30 mM;Figure4a),and1-C-sodium propionate oxidative Consistent with the GFR measurements, renal histology in the rAAV9- capacity increased (Figure 4b). The extent of metabolic improve- CBA-mMut-treated MutÀ/À mice was unremarkable, without ment remained stable 1 month after reinjection, the plasma characteristic tubulointerstitial changes, and appeared identical to methylmalonic acid was unchanged (Figure 4a) and 1-13C-sodium the rAAV9-CBA-mMut-treated Mut+/À control mice (Supplementary propionate oxidation was equivalent between the reinjected rAAV9- Figure 2). CBA-mMut MutÀ/À mice (n¼5) and heterozygote controls (n¼3; Figure 4b). Low-dose systemic reinjection of rAAV9-CBA-mMut at 1 year results in metabolic improvements and transgene expression in the kidney Expression of Mut after reinjection with rAAV9-CBA-mMut One year after neonatal injection, a set of treated rAAV9-CBA-mMut A set of rAAV9-CBA-mMut MutÀ/À mice (n¼2) were killed 1 month MutÀ/À mice (n¼5) were reinjected with a low dose of rAAV9-CBA- after reinjection to examine transgene expression compared with a mMut (2.5Â109 GC gramÀ1) delivered by the retro-orbital route. rAAV9-CBA-mMut MutÀ/À mouse injected at birth and killed at

Gene Therapy Gene therapy for MMA with AAV9 JS Se´nac et al 388

Mut -/- rAAV9-CBA-mMut Mut +/- (n =3) 10 1x10 GC (n =5) -/- 10 *** Mut rAAV9-CBA-mMut 1x10 GC (n =5) 400 80 M)

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200 C propionate 40 13 dose oxidized 100 20 Percent 1- Percent Plasma methylmalonic acid ( Plasma methylmalonic 0 0 365 3 30 01330 Days pre post

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Mut -/- rAAV9-CBA-mMut re-administered Mut +/- Mut -/- expression at 2 months injected at birth expression at 1 month

Mut Mut +/- Mut -/- expression at 2 months injected at birth expression at 1 month Mut C III C III

Figure 4 Metabolic improvement after systemic rAAV9-CBA-mMut readministration. In all, 2.5Â109 GC rAAV9-CBA-mMut per gram body weight was delivered by retro-orbital injection to Mut+/À (n¼3) and MutÀ/À mice (n¼5) that had previously received rAAV9-CBA-mMut by intrahepatic delivery on day of / life (DOL) 1. (a) Methylmalonic acid concentrations in the rAAV9-CBA-mMut-treated MutÀ Àmice on DOL 365 were 323 mM (±30 mM; n¼5) and dropped to 126 mM (±11 mM; n¼5) 3 days after readministration of rAAV9-CBA-mMut, and remained at approximately the same level 30 days later (136 mM (±16 mM), n¼5). Error bars represent ±1 s.d. (***Po0.001; P¼0.6, NS (not significant)); (b)1-13C-propionate oxidation of treated rAAV9-CBA-mMut MutÀ/À mice +/À 13 (n¼5) and Mut littermates (n¼3) before and at 1, 3 and 30 days after systemic readministration of rAAV9-CBA-mMut. C enrichment in expired CO2 was measured and used to determine the percent of the administered 1-13C-propionate dose that was oxidized. Error bars surround the 95% confidence intervals. The rAAV9-CBA-mMut re-treated MutÀ/À mice had diminished oxidative capacity at 25 min on day 1 after reinjection compared with treated heterozygous controls (**Po0.01; *Po0.05) that increased to heterozygote levels by day 30 (P¼0.3, NS (not significant)); (c) Mut expression in liver extracts prepared from Mut+/À and MutÀ/À mice after systemic readministration of rAAV9-CBA-mMut. In all, 30 mg of clarified whole-liver extracts was analyzed for transgene expression by western blotting. The same membranes were probed with either anti-methylmalonyl-CoA mutase antibody (labeled Mut) or an anti- ubiquinol-cytochrome c oxidoreductase antibody (labeled complex III) to control for loading and mitochondrial content. Immunoreactive Mut enzyme is present in the untreated Mut+/À extract, and in MutÀ/À mouse that was treated with rAAV9-CBA-mMut 1 year after neonatal injection (lane 4), 1 month after reinjection (lane 5) and 2 months after reinjection (lanes 6 through 9). The mitochondrial loading control shows approximately the same intensity in each sample. (d) Mut expression in kidney extracts prepared from Mut+/À and MutÀ/À mice after systemic readministration of rAAV9-CBA-mMut at 1 year of life. The animals are the same group studied in (c). In all, 30 mg of clarified whole-kidney extracts was analyzed for transgene expression by western blotting in exactly the same manner as for the liver extracts. Immunoreactive Mut enzyme is present in the untreated Mut+/À extract, but not in an untreated MutÀ/À animal (lane 3) or MutÀ/À mouse that was studied 1 year after receiving a single neonatal injection of rAAV9-CBA-mMut (lane 4). However, in the MutÀ/À mice that received a second injection of rAAV9-CBA-mMut 1 year after birth, immunoreactive enzyme was detected 1 month (lane 5) and 2 months (lanes 6, 7 and 9) after re-treatment. The mitochondrial loading control shows approximately the same intensity in each sample.

1 year of age. After 1 year, the level of immunoreactive Mut in the liver DISCUSSION of a rAAV9-CBA-mMut-treated MutÀ/À mouse was similar to that In this study, we demonstrate the efficacy of an rAAV9 vector to treat seen in an untreated Mut+/À control (Figure 4c), and showed MMA in a mouse model that replicates the features of severe human persistent expression at 1 and 2 months after reinjection with disease. Consistent with reports showing that serotype 9 AAV vectors rAAV9-CBA-mMut (Figure 4c). Mut was not present in a kidney are highly efficacious gene delivery agents in mice,26 we have achieved extract prepared from a MutÀ/À mouse that was treated only with a long-term correction of the neonatal lethal phenotype seen in the single neonatal injection of rAAV9-CBA-mMut, but was readily untreated MutÀ/À mice using a 10-fold lower dose than that pre- detected 1 month after systemic reinjection (Figure 4d). Further viously used with rAAV8 vectors.22,35 Early and long-term transgene analysis using quantitative PCR confirmed that Mut mRNA was expression, proven by expression of Mut gene in the liver of MutÀ/À present in the kidney of the re-treated rAAV9-CBA-mMut MutÀ/À mice as early as 24 h post treatment with persistence to 1 year and mice, but at levels well below those measured in heterozygous beyond, likely contributes to the biological efficacy of this gene therapy littermates (data not shown). Western analysis using liver and kidney approach. The rescued MutÀ/À mice were vigorous and appeared extracts from reinjected rAAV9-CBA-mMut MutÀ/À mice were also well, tolerated a non-restricted diet and had markedly improved examined 2 months after reinjection and yielded similar results to metabolic parameters compared with the untreated state. Like those seen at the earlier 1 month time point. This supports the patients who receive liver and combined liver/kidney transplants,8 observation that there is Mut expression in the kidney following the circulating metabolites were not normalized in the treated mice. systemic gene therapy with rAAV9-CBA-mMut (Figure 4d). However, the clinical effects of rAAV9-CBA-mMut gene therapy

Gene Therapy Gene therapy for MMA with AAV9 JS Seńac et al 389 endured throughout life, as the animals appeared and acted in a The studies presented here clearly demonstrate the therapeutic manner identical to control littermates. potential of a serotype 9 rAAV vector to effectively deliver the Mut Recently, the potential of rAAV9 to transduce the kidney and gene in a mouse model of MMA that faithfully replicates the severe provide biological efficacy in a genetic mouse model of tubulointer- human phenotype. The rapid hepatic transgene expression we have stitial renal disease has been established.25 As kidney failure is a well- documented in rAAV9-CBA-mMut-treated neonatal MutÀ/À mice recognized and devastating complication of MMA, we also examined suggests that a rapidly uncoating rAAV vector33 might be used to fluorescein isothiocyanate (FITC)-inulin clearance in the treated treat MMA patients acutely in the setting of metabolic decompensa- Mut/À mice to measure the GFR, a sensitive measure of renal function. tion, perhaps as early as the newborn period. Transduction of the liver, No significant differences were observed between Mut+/À and rAAV9- and especially the kidney, further supports the consideration of AAV CBA-mMut-treated MutÀ/À mice in our GFR study. Although the serotype 9 gene therapy vectors for future use in the treatment of manifestations of renal disease in mouse models of MMA are not patients with MMA. fully explored, previous studies have established that tubulointerstitial nephritis, similar to what has been documented in MMA patients, MATERIALS AND METHODS À/À 34 does occur in older, untreated Mut mice that escape lethality. Murine model of MMA À/À In the small number of rAAV9-treated Mut mice that were killed MutÀ/À mice contain a deletion of exon 3 in the methylmalonyl-CoA mutase after GFR studies were performed at 1 year of age, renal histology gene, display neonatal lethality and exhibit massive elevations of methylmalonic was similar to that seen in treated controls and showed no tubuloin- acid in the blood, urine and tissues.38 Mut RNA, protein and enzymatic activity terstitial changes (Figure 3d and Supplementary Figure 2). That are undetectable in MutÀ/À mice. Mut+/À mice have biochemical parameters +/+ 22,38 the treated MutÀ/À mice studied here tolerated a non-restricted identical to Mut mice and were used as controls throughout the study. diet, have normal glomerular kidney function and have no evidence of tubulointerstitial changes suggest that rAAV9 gene therapy has rAAV9 construction, production and delivery ameliorated the progression of MMA-associated kidney damage, The murine Mut cDNA was cloned into the pAAV2-CI-CB7-RBG expression either by transduction of a critical renal cellular element, such plasmid to generate rAAV9-CBA-mMut. pAAV2-CI-CB7-RBG contains the as the proximal tubule, or by reducing the ‘nephrotoxic’ metabolite transcriptional control elements from the cytomegalovirus enhancer/chicken b load. -actin promoter, cloning sites for the insertion of a complementary DNA and the rabbit b-globin polyA signal flanked by AAV2 terminal repeats.32 rAAVs In light of the published data showing that rAAV9 can be read- were subsequently packaged with either AAV9 or AAV2 capsid, purified by ministered and still mediate high levels of transgene expression, cesium chloride centrifugation or column chromatography, and titered by 36 even in the presence of neutralizing antibodies, asubsetoftreated quantitative PCR as previously described.39 rAAV9-CBA-mMut had a titer À/À Mut mice were reinjected at 1 year of age. A dose of rAAV9-CBA- of 1.38Â1012 GC mlÀ1; rAAV2-CBA-mMut had a titer of 5.52Â1012 GC mlÀ1. mMut similar to that used in human clinical trials with other rAAVs In all, 1Â1010 GC of the viral vector was delivered to neonatal mice via (2.5Â109 GC gramÀ1)37 was systemically delivered. As early as 3 days intrahepatic injection immediately after birth.22 At 1 year of age, a subset of post injection, the reinjected MutÀ/À mice exhibited improved mice that had been injected in the neonatal period were re-treated with 9 metabolism and the response persisted for at least 1 month post 2.5Â10 GC per gram body weight rAAV9-CBA-mMut delivered via treatment, despite the fact that the circulating metabolites remained retro-orbital injection. The National Human Genome Research Institute substantially increased in re-treated MutÀ/À mice. These findings Animal User Committee approved all animal studies. demonstrate that a second round of gene delivery can substantially Western blotting replenish Mut enzymatic activity in vivo and can be readily measured Tissue samples were homogenized in T-PER tissue protein extraction buffer using a combination of circulating and whole-body metabolic (Pierce Biotechnology, Rockford, IL, USA) in the presence of Halt protease parameters. inhibitor cocktail (Pierce Biotechnology) using a 2-ml Tenbroeck tissue grinder In order to determine whether a systemic rAAV9-CBA-mMut (Wheaton, Millville, NJ, USA). Protein extracts were analyzed by western blot readministration resulted in a greater transgene expression in the and probed with rabbit polyclonal antibodies against the Mut enzyme.22 liver and kidney, tissues were harvested 1 or 2 months after reinjection, Mitochondrial loading control was detected with mouse monoclonal antibo- analyzed by western blotting and quantitative PCR, and the results dies against complex III core II (SKU# A-11143, Invitrogen, Carlsbad, CA, were compared with that of mice that had received only a single USA). The anti-Mut antibodies were used at a dilution of 1:1000 and the anti- rAAV9-CBA-mMut treatment at birth. MutÀ/À mice that received only Complex III Core II antibody was used at a dilution of 1:3000. Horseradish a single injection at birth had hepatic Mut protein levels comparable peroxidase–conjugated anti-rabbit IgG (NA934; GE Healthcare Life Sciences, À/À Piscataway, NJ, USA) or anti-mouse IgG (NA931; GE Healthcare Life Sciences) to treated heterozygous littermates. In Mut mice that received a were used as secondary antibodies and visualized after chemiluminescence second injection of rAAV9-CBA-mMut at 1 year, transgene expression detection (Pierce Biotechnology). further increased in the liver (Figure 4c). Although one otherwise À/À asymptomatic treated Mut mouse had elevated LFTs before vector Metabolic studies readministration (Supplementary Figure 3) for an unknown reason, Blood samples were collected from treated mice by orbital bleeding. Plasma was liver function tests in the multiply treated animals did not reveal isolated by centrifugation, diluted in water and stored at À801C in a screw-top AAV9-associated transaminitis. As rAAV9 has been reported to trans- tube. Methylmalonic acid concentrations in plasma were measured by gas duce the kidney in mice,25,26 we next examined whole-kidney extracts chromatography–mass spectrometry.40,41 To determine 1-13C-propionate oxi- prepared from mice at 1 month and 2 months after systemic dation in vivo, mice were placed into a respiratory chamber after intraperitoneal 13 22,42 reinjection. Immunoreactive enzyme was apparent in the MutÀ/À injection of 200 mgof1- C-sodium propionate. Breath samples from mutant, control and treated mice were collected at 5, 15 and 25 min after kidneys studied after re-treatment, compared with undetectable levels injection and the isotope ratio (13C/12C) of the expired gas was measured with a present before reinjection. These data establish that a second injection gas isotope ratio mass spectrometer (Metabolic Solutions, Nashua, NH, USA). of rAAV9-CBA-mMut, systemically delivered at a dose equal to that The percent dose oxidized at each time point was calculated as: used in human trials for other AAV serotypes, resulted in significantly increased Mut expression in both the liver and the kidney. % dose oxidized ¼ total 13Cexcretedðmmol=doseðmmolÞÂ100%Þ

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Glomerular filtration rate 11 Nyhan WL, Gargus JJ, Boyle K, Selby R, Koch R. Progressive neurologic disability in The GFR was measured using the FITC-labeled inulin clearance method.43 methylmalonic acidemia despite transplantation of the liver. Eur J Pediatr 2002; 161: Experiments were performed on age- and sex-matched Mut+/À (n¼3) and 377–379. 12 Hsui JY, Chien YH, Chu SY, Lu FL, Chen HL, Ho MJ et al. Living-related liver À/À Mut mice (n¼3) at 1 year of age. All of the mice had received a single transplantation for methylmalonic acidemia: report of one case. Acta Paediatr Taiwan neonatal intrahepatic injection of rAAV9-CBA-mMut. Under isoflurane 2003; 44: 171–173. anesthesia, mice were given a single retro-orbital bolus injection of 5% 13 Nagarajan S, Enns GM, Millan MT, Winter S, Sarwal MM. Management of methylma- FITC-labeled inulin (3.74 ml per gram body weight). Heparinized serial blood lonic acidaemia by combined liver-kidney transplantation. J Inherit Metab Dis 2005; 28: 517–524. collections were performed from tail cuts at 3, 7, 10, 15, 35, 55 and 75 min after 14 Kaplan P, Ficicioglu C, Mazur AT, Palmieri MJ, Berry GT. Liver transplantation is not inulin administration and the plasma was subsequently removed. As pH affects curative for methylmalonic acidopathy caused by methylmalonyl-CoA mutase defi- FITC fluorescence values, each plasma sample was buffered by mixing 1 ml ciency. Mol Genet Metab 2006; 88: 322–326. 15 Kasahara M, Horikawa R, Tagawa M, Uemoto S, Yokoyama S, Shibata Y et al. Current plasma with 9 mlof500mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid role of liver transplantation for methylmalonic acidemia: a review of the literature. solution (pH 7.4). The amount of FITC label present in the samples was then Pediatr Transplant 2006; 10: 943–947. measured using a fluorospectrometer at 538 nm emission (NanoDrop 3300, 16 Morioka D, Kasahara M, Horikawa R, Yokoyama S, Fukuda A, Nakagawa A. Efficacy of Thermo Scientific, Wilmington, DE, USA). A two-compartment clearance model living donor liver transplantation for patients with methylmalonic acidemia. Am J was used to calculate the GFR. Plasma fluorescence data were fit to a two-phase Transplant 2007; 7: 2782–2787. 17 Mc Guire PJ, Lim-Melia E, Diaz GA, Raymond K, Larkin A, Wasserstein MP et al. exponential decay curve using nonlinear regression (GraphPad Prism, GraphPad Combined liver-kidney transplant for the management of methylmalonic aciduria: a À1 Software, San Diego, CA, USA). GFR (mlmin ) was calculated using the case report and review of the literature. Mol Genet Metab 2008; 93:22–29. equation: GFR¼I/(A/a+B/b). I is the amount of FITC-inulin delivered by 18 Oberholzer VG, Levin B, Burgess EA, Young WF. Methylmalonic aciduria. An inborn injection, A and B are the y-intercept values of the two decay rates, and a and b error of metabolism leading to chronic metabolic acidosis. Arch Dis Child 1967; 42: 492–504. are the decay constants for the distribution and elimination phases, respectively. 19 Walter JH, Michalski A, Wilson WM, Leonard JV, Barratt TM, Dillon MJ. Chronic renal failure in methylmalonic acidaemia. Eur J Pediatr 1989; 148:344–348. 20 Van Calcar SC, Harding CO, Lyne P, Hogan K, Banerjee R, Sollinger H et al. Renal Statistical analyses transplantation in a patient with methylmalonic acidaemia. JInheritMetabDis1998; Differences in the survival between treated and untreated groups were analyzed 21: 729–737. using a w2-test. Differences in weight, metabolite levels and GFRs were assessed 21 Kolker S, Okun JG. Methylmalonic acid—an endogenous toxin? Cell Mol Life Sci 2005; 62: 621–624. using a Student’s t-test. The Kruskal–Wallis test was used to determine the 22 Chandler RJ, Venditti CP. Long-term rescue of a lethal murine model of methylmalonic statistical significance in measured propionate oxidation rates between groups acidemia using adeno-associated viral gene therapy. Mol Ther 2010; 18:11–16. at 25 min after injection. P-values o0.05 were considered significant. 23 Wu Z, Asokan A, Samulski RJ. Adeno-associated virus serotypes: vector toolkit for human gene therapy. Mol Ther 2006; 14:316–327. 24 Gao G, Vandenberghe LH, Wilson JM. New recombinant serotypes of AAV vectors. Curr Gene Ther 2005; 5: 285–297. CONFLICT OF INTEREST 25 Schievenbusch S, Strack I, Scheffler M, Nischt R, Coutelle O, Ho¨sel M et al. Combined The authors declare no conflict of interest. paracrine and endocrine AAV9 mediated expression of hepatocyte growth factor for the treatment of renal fibrosis. Mol Ther 2010; 18: 1302–1309. 26 Zincarelli C, Soltys S, Rengo G, Rabinowitz JE. Analysis of AAV serotypes 1–9 mediated ACKNOWLEDGEMENTS gene expression and tropism in mice after systemic injection. Mol Ther 2008; 16: JSS, RJC, JRS and CPV were supported, in part, by the Intramural Research 1073–1080. 27 Yue Y, Ghosh A, Long C, Bostick B, Smith BF, Kornegay JN et al. A single intravenous Program of the National Human Genome Research Institute, National injection of adeno-associated virus serotype-9 leads to whole body skeletal muscle Institutes of Health. LL was supported, in part, by the Intramural Research transduction in dogs. Mol Ther 2008; 16: 1944–1952. Program of the National Institute of Diabetes and Digestive and Kidney 28 Yue Y, Shin JH, Duan D. Whole body skeletal muscle transduction in neonatal dogs with Diseases, National Institutes of Health. JRS also received support from the AAV-9. Methods Mol Biol 2011; 709:313–329. 29 Inagaki K, Fuess S, Storm TA, Gibson GA, Mctiernan CF, Kay MA et al. Robust systemic Angels for Alyssa MMA Research Foundation. transduction with AAV9 vectors in mice: efficient global cardiac gene transfer superior to that of AAV8. 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