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Curative ex vivo liver-directed gene therapy in a pigmodel of hereditary tyrosinemia type 1Raymond D. Hickey,1,2* Shennen A. Mao,1 Jaime Glorioso,1 Faysal Elgilani,1 Bruce Amiot,3

Harvey Chen,1 Piero Rinaldo,4 Ronald Marler,5 Huailei Jiang,6 Timothy R. DeGrado,6

Lukkana Suksanpaisan,6,7 Michael K. O’Connor,6 Brittany L. Freeman,8 Samar H. Ibrahim,8

Kah Whye Peng,2 Cary O. Harding,9 Chak-Sum Ho,10 Markus Grompe,11 Yasuhiro Ikeda,2

Joseph B. Lillegard,1,12 Stephen J. Russell,2 Scott L. Nyberg1

We tested the hypothesis that ex vivo hepatocyte gene therapy can correct the metabolic disorder in fumarylacetoa-cetate hydrolase–deficient (Fah−/−) pigs, a large animalmodel of hereditary tyrosinemia type 1 (HT1). Recipient Fah−/−

pigs underwent partial liver resection and hepatocyte isolation by collagenase digestion. Hepatocytes were trans-duced with one or both of the lentiviral vectors expressing the therapeutic Fah and the reporter sodium-iodide sym-porter (Nis) genes under control of the thyroxine-binding globulin promoter. Pigs received autologous transplants ofhepatocytes by portal vein infusion. After transplantation, the protective drug 2-(2-nitro-4-trifluoromethylbenzyol)-1,3 cyclohexanedione (NTBC) was withheld from recipient pigs to provide a selective advantage for expansion ofcorrected FAH+ cells. Proliferation of transplanted cells, assessed by both immunohistochemistry and noninvasivepositron emission tomography imaging of NIS-labeled cells, demonstrated near-complete liver repopulation bygene-corrected cells. Tyrosine and succinylacetone levels improved to within normal range, demonstrating completecorrection of tyrosine metabolism. In addition, repopulation of the Fah−/− liver with transplanted cells inhibited theonset of severe fibrosis, a characteristic of nontransplanted Fah−/− pigs. This study demonstrates correction of diseasein a pig model of metabolic liver disease by ex vivo gene therapy. To date, ex vivo gene therapy has only been suc-cessful in small animal models. We conclude that further exploration of ex vivo hepatocyte genetic correction is war-ranted for clinical use.

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INTRODUCTION

Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive inbornerror ofmetabolism, caused bydeficiency in fumarylacetoacetate hydro-lase (FAH), an enzyme that catalyzes the last step of tyrosinemetabolism(1, 2). Absence of FAH causes accumulation of fumarylacetoacetate, re-sulting in mutagenic, cytostatic, and acutely apoptotic events within thecell (3–5). Acute onset of HT1 is characterized by severe liver dys-function (6), most frequently leading to death, if untreated. The chronicform of HT1 is characterized by progressive liver disease, typically se-vere fibrosis and hepatocellular carcinoma. The most common treat-ment for HT1 is a low-tyrosine diet combined with administration of2-(2-nitro-4-trifluoromethylbenzyol)-1,3 cyclohexanedione (NTBC)(7), a potent inhibitor of 4-hydroxyphenylpyruvate dioxygenase, an en-zyme in the tyrosine metabolic pathway. However, some patients arerefractory to NTBC therapy, and those on NTBC remain at increasedrisk of several conditions, including cirrhosis, hepatocellular carcinoma,

1Department of Surgery, Mayo Clinic, Rochester, MN 55905, USA. 2Department ofMolecularMedicine, Mayo Clinic, Rochester, MN 55905, USA. 3Brami Biomedical Inc., Coon Rapids, MN55433, USA. 4Division of Laboratory Genetics, Department of Laboratory Medicine and Pa-thology, Mayo Clinic, Rochester, MN 55905, USA. 5Department of Comparative Medicine,Mayo Clinic, Scottsdale, AZ 85259, USA. 6Department of Radiology, Mayo Clinic, Rochester,MN 55905, USA. 7Imanis Life Sciences, Rochester, MN 55902, USA. 8Division of PediatricGastroenterology, Mayo Clinic, Rochester, MN 55905, USA. 9Department of MolecularandMedical Genetics and Department of Pediatrics, Oregon Health and ScienceUniversity,Portland, OR 97239, USA. 10Histocompatibility Laboratory, Gift of Life Michigan, Ann Arbor,MI 48108, USA. 11Papé Family Pediatric Research Institute, Oregon Health and Science Uni-versity, Portland, OR 97239, USA. 12Midwest Fetal Care Center, Children’s Hospitals andClinics of Minnesota, Minneapolis, MN 55404, USA.*Corresponding author. Email: [email protected]

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and impaired intellectual development (8). Therefore, liver transplanta-tion remains the only curative therapy for this disease.

We have recently demonstrated that transplantation of wild-typesyngeneic hepatocytes can correct metabolic disease in FAH-deficient(Fah−/−) mice, a model of HT1 (9). We were able to show that regen-erating hepatocytes in this mouse model can be noninvasively imagedusing the sodium-iodide symporter (Nis) gene to label the cells. NIS-positive cells can take up radiolabeled iodine, or its analogs, and there-fore be imaged using single-photon emission computed tomography(SPECT) or positron emission tomography (PET). This study in ro-dents therefore corroborated previous data demonstrating the potentialefficacy of hepatocyte transplantation in treating metabolic liver dis-eases (10, 11). To further test the efficacy of cell transplantation in aclinically relevant setting, we looked toward a large animal model ofHT1: the previously generated and characterized Fah−/− pig (12, 13).In the absence of NTBC, Fah−/− pigs undergo acute liver failure thatis characterized by diffuse and severe hepatocellular damage (12).WhenFah−/−pigs are cycled on and off low-doseNTBC, animals undergo pro-gressive clinical decline that results in chronic liver failure and severefibrosis (14). Fah−/− pigs therefore provide a unique genetically engi-neered large animal model of a metabolic disease to test regenerativetherapies, such as ex vivo gene therapy.

After some successful ex vivo gene therapy interventions in smallanimal models of liver disease, including a rabbit model of familial hy-percholesterolemia (15), a single clinical trial of ex vivo gene therapywasundertaken nearly two decades ago using a gammaretroviral vector totreat five patients with familial hypercholesterolemia by modifying au-tologous primary hepatocytes (16). Although amodest therapeutic ben-efit was observed in some of the patients, this approach was limited by

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poor transduction efficiencies using gammaretroviruses and limited en-graftment of corrected cells. As a result, no further clinical trials using exvivo gene therapy for liver disease have occurred. More recently, how-ever, the success of ex vivo gene therapy in hematopoietic stem cells fortreating hematological disorders using lentiviral vectors (LV) (17, 18)raises the possibility that similar success can be achieved in treating

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metabolic liver diseases. LVs have a number of key advantages for he-patocyte gene therapy, including the ability to infect nondividing cells,sustained gene delivery through integration, and safer integration siteprofiles than those of gammaretroviruses (19). The efficacy of suchan approach has been demonstrated in a small animal model of HT1(20). Here, we demonstrate that ex vivo gene therapy in autologous he-

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patocytes can prevent liver failure andcorrect metabolic function in Fah−/− pigs,setting the stage for translation of this ap-proach to humanmetabolic liver diseases.

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RESULTS

Allogeneic hepatocytetransplantation cannot correctmetabolic disease in Fah−/− pigsTo date, the vast majority of human hepa-tocyte transplantations for metabolic liverdiseases have been of allogeneic source(21, 22). To simulate clinical human hepa-tocyte transplantation, we tested the effi-cacy of allogeneic hepatocyte transplantin Fah−/− pigs. We first characterized thegenetic loci governing the pig swine leuko-cyte antigen (Sla) and Fah genes (23, 24).The Sla gene complex is located on Susscrofa chromosome 7 (SSC7), with classI on the short (p) arm and class II on thelong (q) arm, spanning the centromere.Our data predicted that Fah is located onthe long arm of SSC7, telomeric to the Slaclass II region, about 27 million base pairsfrom the major histocompatibility complexclass II antigen Sla-Drb1 gene (fig. S1A).

On the basis of SLA typing results (fig.S1B), we performed a transplant of Sla-matched allogeneic FAH+ hepatocytesfrom a heterozygote Fah+/− pig (Y501)into a single Fah−/− pig (Y502) (table S1).Isolated hepatocytes were injected throughthe portal vein, and NTBC was periodicallyremoved from the diet to stimulate expan-sion of transplanted FAH+ cells. The animaldid not show clinical improvement nor didit become NTBC-independent (fig. S1C).Histological analysis demonstrated the ab-sence of any transplanted FAH+ cells(fig. S1D). On the basis of these results, wefocused the remainingexperimentsonusinggene therapy in autologous hepatocytes.

Ex vivo hepatocyte gene therapycorrects HT1 in miceA vesicular stomatitis virus glycoproteinG–pseudotyped LV was produced thatexpressed the porcine Fah cDNA underthe control of a liver-specific promoter

Fig. 1. Ex vivogene therapy in Fah−/−miceusing LV-Fah is curative. (A) Schematic representation of theLV used for mouse and pig studies in which the porcine Fah cDNA is under control of the human TBG
promoter and two copies of a human a1-microglobulin/bikunin enhancer. LTR, long terminal repeat; Y,psi packaging sequence; RRE, Rev-responsive element; cPPT, central polypurine tract; WPRE, Woodchuckhepatitis virus posttranscriptional regulatory element; LTR/DU3, 3′ long terminal repeat with deletion inU3 region. (B) Mouse experiment timeline, where ex vivo transduced hepatocytes are transplanted intosyngeneic recipient mice. Livers from recipients were collected at three time points: day 14 (n = 2), day40 (n = 2), and day 148 (n = 2). d, day. (C) FAH immunohistochemistry (IHC) at days 14, 40, and 148 aftertransplantation. A nontransplanted mouse (−LV) is included as a negative control. Scale bars, 300 mm.(D) FAH IHC at lower magnification than (C) at day 148 after transplantation. A nontransplanted mouse(−LV) is included as a negative control. Scale bars, 4 mm. (E) Biochemical analysis of serum on day 148comparing alkaline phosphatase (ALP), alanine aminotransferase (ALT), total bilirubin (TBIL), and albumin(ALB) in Fah−/− mice that underwent transplantation with hepatocytes from Fah−/− mice that received novector (−LV; n = 4) or were transduced ex vivo with LV-Fah (+LV; n = 2). Data are means ± SD. P values weredetermined using a two-tailed t test.
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containing the human thyroxine-binding globulin (TBG) promoter andtwo copies of the a1-microglobulin/bikunin enhancer (referred to asTBG throughout) (Fig. 1A) (25). To test the efficacy of this vectorin vivo, we first transplanted donor Fah−/−mouse hepatocytes that weretransduced with LV-Fah into syngeneic Fah−/− mice by intrasplenicinjection (Fig. 1B). NTBC was withheld periodically (mice were onNTBC from days 15 to 20 and 43 to 48 after transplant) to allow expan-sion of transplanted cells (26). Twomice were euthanized, and the liverswere harvested at three time points: days 14, 40, and 148 after trans-plantation (Fig. 1B). All recipient Fah−/−mice showed engraftment ofLV-transduced hepatocytes, and there was substantial, albeit non-homogeneous, expansion of the cells at the two later time points, in-dicating differences in initial engraftment of cells between lobes (Fig. 1,C and D). In mouse livers harvested at day 148, there was almost

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complete repopulation of the host tissue by LV-transduced hepato-cytes. Liver cell repopulation correspondedwith normalization of bio-chemical indicators of liver function in Fah−/− mice, whereas micereceiving no cell transplants had severely impaired liver functionand disease (Fig. 1E).

Ex vivo hepatocyte gene therapy was tested in four Fah−/− pigsOn the basis of promising results in mouse models of HT1, we proceededto testing ex vivo gene therapy in the pig model of HT1 (Fig. 2A). Ourgroup had previously demonstrated the benefit of dexamethasone inincreasing LV transduction in mouse hepatocytes ex vivo (9). Using thisprotocol, ~80% of primary pig hepatocytes were transduced with an LVat a multiplicity of infection (MOI) of 2000 physical lentiviral particles(LPs), and ~60% of cells were transduced at an MOI of 500 LPs (Fig. 2B).



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A total of four Fah−/− pigs underwentex vivo gene therapy for this study (table S1and Fig. 2A). Animals underwent a partialliver resection by midline laparotomy.About10%of the liverwas removedandhe-patocytes were isolated, with cell viability af-ter purification ranging from 88 to 92%.Hepatocytes were transduced with one orboth of LV-Fah and LV-Nis (to use to trackthe cells noninvasively) in suspension. FAHexpression was confirmed in transducedhepatocytes from all four donors (Fig. 2C).Viability and plateability of hepatocyteswere confirmed after LV transduction, andno differences were observed betweencontrol and transduced cells (Fig. 2D). Af-ter LV transduction, between 350 and 864million live cells were injected through theportal vein, either by direct catheterizationof the portal vein or using ultrasound guid-ance to inject hepatocytes percutane-ously (fig. S2 and movie S1). No adverseevents were encountered with the sur-gery, and all animals were ambulatoryand feeding within 24 hours.

LV-transduced hepatocytes engraftand proliferate in vivo in pigsAll animals remained on NTBC for48hoursafter surgery (Fig. 2A).At thispoint,NTBCwas removed from the diet to stimu-late expansion of transplanted cells. Animalswere cycled on and off NTBC until weightstabilization occurred. From this pointon, animals did not receive NTBC. Twoanimals (Y707 and Y846) were euthanizedearly in the study to test the efficacy ofthe ex vivo gene therapy approach in pigsand to determine whether a selective ad-vantage for FAH+ cells exists in this model.Both animals appeared to achieve weightstability off NTBC (Fig. 3A), indicating thatexpansion of transplanted cells had oc-curred. In contrast to a nontransplanted

Fig. 2. Ex vivo gene therapy approach in Fah−/− pigs. (A) Schematic of the six steps involved in ex vivogene therapy and liver repopulation in Fah−/− pigs: (1) partial hepatectomy was performed, and (2) hepa-
tocyteswere isolatedby collagenase digestion. (3) Ex vivo gene therapywith an LVwas performedbefore (4)retransplantation of the autologous cells. (5) NTBC was periodically withdrawn from the diet to stimulateexpansion of the FAH+ cells. (6) Clinical evaluation, serum biochemistry analysis, and liver histopath-ological analysis were performed 2 (n = 1 pig), 4 (n = 1 pig), and 12 (n = 2 pigs) months after transplanta-tion. (B) Percentage of green fluorescent protein (GFP)–positive hepatocytes detected by flow cytometryafter transduction of cells with an LV expressing GFP at MOIs of 0, 500, and 2000 LPs. Flow cytometry wasperformed 72 hours after transduction. Data are means ± SD (n = 3 replicates). P values versus 0MOI weredetermined using a two-tailed t test. Representative fluorescent images are also shown. Scale bars, 100 mm.(C) In vitroWestern blot data confirming expression of FAH protein in pig hepatocytes after transductionex vivo with LV-Fah (+) or untransduced cells (−) from the same animal. b-Actin served as loading con-trol. (D) Representative images of plated hepatocytes from pig Y842 that were untransduced with LV(−LV) or were transduced with LV (+LV-Fah + LV-Nis). Scale bars, 100 mm.
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Fah−/− pig (L768), robust FAH expressionwas detected in Y707 and Y846 at time ofeuthanasia (Fig. 3B). Individual clusters ofFAH+ cellswere evidentwithin the lobulesof the pig parenchyma, consistent withexpansion of single engrafted cells (Fig.3C). Masson’s trichrome staining revealedno evidence of severe fibrosis in either an-imal, and nomajor pathology was observedby hematoxylin and eosin (H&E) staining(Fig. 3B). On the basis of these results, weelected to determine the long-term effectof ex vivo gene therapy in the pig modelof HT1.

Ex vivo hepatocyte gene therapycorrects metabolic deficiency inFah−/− pigsPigs Y842 and Y845 were studied for12 months, with one animal euthanized atthis time point. The pigs exhibited NTBC-relatedweight fluctuations previously notedin Fah−/− mice (27). Both animals becamecompletely independent of NTBC 95 days(Y842) and 140 days (Y845) after ex vivogene therapy and hepatocyte transplan-tation (Fig. 4A). Consistent with NTBC-independence, these two pigs gainedweight and did not manifest any failure-to-thrive phenotype that was previouslynoted in control Fah−/− pigs off NTBC (12).

Biochemical analysis was performed at12months. Reference values for wild-typeand Fah−/− pigs off NTBC (Fah−/−) werecompiled from previous studies (12).Fah−/− pigs receiving LV-Fah had normalliver function that was characterized bymarkedly reduced plasma levels of totalbilirubin and ammonia, compared toFah−/−pigs offNTBC (12) (Fig. 4B). Com-parable to wild-type animals (12), Fah−/−

pigs that underwent ex vivo gene therapyhad considerably reduced levels of bothtyrosine and succinylacetone, which arediagnostic for HT1 in humans and pigs(Fig. 4B). Additionally, plasma concentra-tions of methionine were reduced inanimals Y842 and Y845.

LV-Fah–transduced hepatocytescompletely repopulateFah−/− pig liversTo determine the degree of repopulationof LV-Fah–transduced hepatocytes, pigY845 was euthanized at 12 months aftertransplantation, and FAH IHC was per-formed. FAH staining revealed near-complete repopulation of the recipient

Fig. 3. LV-Fah–transduced hepatocytes proliferate extensively in vivo in pigs.Animals Y707 and Y846were euthanized at 2 and 4 months after transplantation, respectively. Control Fah−/− pig L768 did not re-
ceive any gene-corrected hepatocytes but was cycled on and off NTBC. (A) Weight stabilization of pigs Y707and Y846. Gray areas are times on NTBC (for Y707, from day 24 to day 32; for Y846, from days 14 to 21, 35 to42, 57 to 64, and 86 to 93). (B) Representative liver tissues stained for FAH are shown at low and high mag-nification for each animal; the dotted rectangle indicates the area of enlargement. Serial sections of H&E–and Masson’s trichrome (MT)–stained liver are also provided. Scale bars, 4 mm (low-magnification FAH);800 mm (all other images). (C) FAH staining of liver tissue from pigs Y707 and Y846, identifying individualexpanding hepatocyte nodules (black arrows).
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liver by FAH+ cells (Fig. 5A). Individual lobules appeared to be en-grafted by one to two FAH+ hepatocytes that subsequently proliferatedextensively to repopulate the entire lobule. Individual hepatocyte cloneswere uniquely identified by variations in FAH-staining intensity be-tween and within lobules (Fig. 5A).

We next determined the average number of LV integrations in therepopulated liver tissue from Y842 and Y845 using quantitative poly-merase chain reaction (qPCR). On the basis of this analysis, wecalculated an average of 0.13 copies of the LV vector per haploid ge-nome (Fig. 5B). Given the ploidy content of hepatocytes and the pro-portion of hepatocytes in the liver, this integration frequency indicatesan average of one LV integration per hepatocyte, consistent with the invitro transduction frequencies (Fig. 2B). Last, the proportion of cells un-dergoing proliferation and apoptosis was determined in livers from allpigs using Ki67 and terminal deoxynucleotidyl transferase–mediateddeoxyuridine triphosphate nick end labeling (TUNEL) staining, respec-

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tively. All transplanted livers had increased numbers of proliferatingcells compared to control pigs, consistent with FAH+ cells repopulatingthe Fah−/− liver (Fig. 5C and fig. S3). No significant increase in apoptosiswas detected in transplanted livers compared to controls (Fig. 5D andfig. S3).

NIS-labeled hepatocytes can be imagednoninvasively in vivoEight months after hepatocyte transplantation in pig Y842, we per-formed noninvasive imaging of repopulating cells using PET/CT imag-ing with the iodide analog [18F]tetrafluoroborate ([18F]TFB). In thisanimal, autologous hepatocytes were transduced with both LV-Fahand LV-Nis (table S1). On the basis of studies with GFP and red fluo-rescent protein (RFP), we estimate that 50 to 60% of cells were doublyFAH+NIS+ (Fig. 6A). Because cells in the liver do not express Nis (9),only LV-Nis–transduced hepatocytes should take up iodine and be de-



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tected by PET imaging with [18F]TFB.The ability of LV-Nis–transduced hepa-tocytes to take up radiolabeled iodide(125I) intracellularly was confirmed ini-tially in vitro (Fig. 6B).

In vivo, in a control wild-type pig, up-take of [18F]TFBwas detected only in thestomach and not in the liver (Fig. 6C),consistent with data from mouse studies(28). In contrast, pig Y842 demonstratedsignificant uptake of [18F]TFB in the liver,thus allowing noninvasive evaluation of re-populating clusters of FAH+ cells (Fig. 6C).These PET/CT data were validated by lap-aroscopic biopsies demonstrating robustexpansion of the LV-Fah–transduced he-patocytes in the liver of Y842 (Fig. 5A).

Repopulation by FAH+ cellsprevents onset of severe fibrosisAlthough livers from pigs Y707 (analyzed2 months after transplant) and Y846 (an-alyzed 4 months after transplant) dem-onstrated periportal or lobular fibrousconnective tissue within normal limits(Fig. 3B), the long-term effect of ex vivogene therapy on the onset of fibrosis wasunknown. Fah−/− pigsmaintained on verylow doses of NTBC exhibit significant in-creases in fibrosis and portal hypertension(14). In contrast, in both pigs that receivedFAH-corrected hepatocyte transplants,repopulation of the Fah−/− liver preventedthe progression of severe fibrosis (Fig. 7).In these two pigs, there was a significantdecrease in fibrosis, asquantifiedbyMasson’strichrome staining, compared to Fah−/− pigscycled on and off NTBC (Fig. 7).

Variation in staining using Masson’strichrome did exist between differentlobes in Y845 (fig. S4), in which the sever-ity grade for fibrosis was scored by the

Fig. 4. Ex vivo hepatocyte gene therapy and transplantation in Fah−/− pigs corrects metabolic dis-ease. (A) Weight charts of Y842 and Y845 in the proceeding days after transplantation. Time on NTBC is
depicted in the gray shading (for Y842, from days 14 to 21, 34 to 41, 55 to 62, and 85 to 92; for Y845, fromdays 14 to 21, 37 to 44, 62 to 69, 94 to 101, and 131 to 138). (B) Plasma and serumbiochemical analyseswereperformed at 12 months on pigs Y842 and Y845. Reference values for wild-type (WT) and Fah−/− pigs offNTBC (Fah−/−) are compiled from previous studies (12). Individual data points for each animal are plottedwith themeanof each group depictedwith a line. P valueswere determined using a two-sided independentt test.
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pathologist from minimal (majority of the liver) to moderate (mi-nority of the liver) fibrosis. Variations in the degree of fibrosisappeared to be associated with differences in cell engraftment in dif-ferent lobes, a characteristic that was also noted between lobes in themouse experiments (Fig. 1D). In addition, histopathological analysisof liver tissue revealed no evidence of hepatocellular carcinoma (Figs.3 and 7)—an important consideration given the potential risk of in-sertional mutagenesis that has been reported by ex vivo gene therapyapproaches with gammaretroviral vectors for treating some primaryimmunodeficiency diseases (29).


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DISCUSSION

Although the individual incidence of each inborn error of metabolismdisorder is rare, it is estimated that 22% of all pediatric liver transplantsresulted from metabolic disease (30). As the shortage of donor organscontinues to limit organ transplantation, other therapeutic strategies areurgently required to treat this patient population with debilitating dis-eases. One potential alternative is hepatocyte transplantation. Theresults presented here from a large animal model of liver failure dem-onstrate that ex vivo gene therapy is indeed a potential alternative ther-apeutic strategy to whole-organ transplantation. Similar to the mousemodel (26) and as has been reported in humanHT1 patients (31), FAH+

cells had the proliferative advantage to expand and repopulate theFah−/− pig liver under selective NTBC withdrawal. Repopulation ofthe liver with FAH+ cells produced a marked clinical outcome in whichamelioration of the HT1 phenotype and prevention of fibrosis wereachieved—a compelling finding forHT1patientswho experience severefibrosis.

Several small-animal models of metabolic liver diseases havebeen treated successfully by hepatocyte transplantation (10, 11),paving the way for human hepatocyte transplantations to occur(22). However, nearly all human hepatocyte transplantations todate have involved allogeneic cells. Therefore, in addition to theneed for long-term immunosuppression in the patient, results todate suggest that an immune response is still mounted against thetransplanted allogeneic cells, causing loss of engrafted cells (32, 33).Successful application of ex vivo gene therapy in small animal mod-els paved the way for an autologous ex vivo cell therapy approachto treat five patients with familial hypercholesteremia (16). In re-cent years, the advent of self-inactivating LV has resulted in the safeand efficacious treatment of several primary immunodeficienciesusing ex vivo gene therapy (17, 18). Here, we confirmed that suchan approach could be used to treat metabolic liver disorders. A ma-jor restraint of current transplantation techniques is the inability tomonitor cells noninvasively and longitudinally after injection, partic-ularly given the ability of hepatocytes to engraft and expand outsideof the liver (34). We previously demonstrated noninvasive three-dimensional imaging of regenerating tissue in individual animalsover time using NIS as a reporter gene in mouse hepatocytes (9). Here,we used PET/CT imaging combined with the iodide analog [18F]TFBto detect FAH+ hepatocytes noninvasively in one pig. Our imaginganalysis, in combination with liver staining, showed expansion ofindividual FAH+ hepatocytes throughout the parenchyma. Althoughimaging was only performed in one animal, the result validates NISas a suitable noninvasive reporter for future cell transplantationstudies.

Fig. 5. FAH+ hepatocytes completely repopulate the Fah−/− liver.(A) Representative liver tissues from pigs Y842 and Y845 stained for FAH
at the time of hepatectomy (left panels) and 12 months after transplan-tation with LV-Fah–transduced hepatocytes (middle and right panels). Anage-matched, nontreated control WT pig is shown for comparison. High-magnification images of the regions outlined by dotted squares are pro-vided. (B) DNA from eight different lobes from pig Y845 (1 to 8) and twobiopsies from pig Y842 (1 and 2) were analyzed for integration of LV byqPCR. Total copies of integrated LV per 150 ng of genomic DNA were cal-culated based on a standard curve. The “integration events per haploid ge-nome” were calculated based on the pig haploid genome size of 2800 Mb.(C) Ki67-positive nuclei were quantified by counting nuclei in 10 randomsections per slide in paraffin-embedded tissue sections of pigs Y707, Y846,Y842, and Y845 and a controlWT pig. Data aremeans ± SEM. P values versuscontrol were determined using a two-tailed t test. (D) Hepatocyte apoptosiswas quantified in three random paraffin-embedded tissue sections of pigsY707, Y846, Y842, and Y845 and a control WT pig. Apoptotic nuclei werequantified by counting nuclei in 10 random20×microscopic slides per tissuesection. Data are means ± SEM, with statistical significance versus controldetermined using one-way analysis of variance (ANOVA), followed byBonferroni’s multiple comparisons test.



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Fig. 6. NIS labeling of cells permits noninvasive detec-tion of repopulating hepatocytes. (A) The percentage of
GFP+ and/or RFP+ hepatocytes after transduction with orwithout LV-GFP and LV-RFP at anMOI of 500 LPs. Flow cytom-etry was performed 72 hours after transduction. Data aremeans ± SD (n = 3 replicates). (B) In vitro uptake of 125I,measured in counts per minute (cpm), in LV-Nis–transducedhepatocytes and control hepatocytes frompig Y842. Data aremeans ± SD (n= 3 replicates). P valuewas determined using atwo-tailed t test. (C) Representative transverse PET/CT imagesof a control WT pig and pig Y842 after injection of [18F]TFB.Different positions of the liver are shown from head (left side)to feet (right side) of the animals. The relative intensity of [18F]TFB uptake is representedby low (blue) to high (red). The liveris outlined in purple, and the spleen is outlined in white.
Fig. 7. Ex vivo gene therapy prevents onset of severe fibrosis in Fahpigs. Representative Masson’s trichrome–stained liver sections from pigs

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Y842 and Y845 are shown at the time of hepatectomy (before transplant)and 12 months after transplantation. A control fibrotic liver from a Fah−/−

pig (14) and WT control are included for comparison. Scale bars, 700 mm.Fibrosis (collagen) in pigs Y842 and Y845 before transplantation (pre) and12 months after transplantation (post) was quantified and compared to un-treated Fah−/− animals with fibrosis (n = 3) and normal WT (n = 3) controls[data for controls are from Hickey et al. (12) and Mao et al. (14)]. Data aremeans ± SD. P values were determined using a two-tailed t test.





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There are some limitations to this study. First, although the results ofthese studies demonstrate the efficacy of an ex vivo gene therapy ap-proach, we used only four experimental animals; only two were forthe same amount of time. These data need to be reproduced in a largercohort of animals because pigs, similar to humans—and unlike rodents—are outbred, and phenotypic differences do exist between differentanimals. Second, in future studies, there is a need to perform more rig-orous testing of the insertion site profile of the LV (20). Although thiswas not yet possible given the current incomplete annotation status ofthe pig genome, it would be interesting to compare the insertion profilein pig cells to those of mouse and human hepatocytes, to determinewhether any evidence of clonal selection occurs in this model. Thereare several challenges that will need to be overcome before achievingsuccessful clinical translation formetabolic diseases, in which a selectiveadvantage does not exist for the corrected cell to proliferate. The twoprincipal limitations are cell engraftment and proliferation. Althoughmany protocols have been developed to attempt to overcome thesebarriers in small and large animals, including preparative irradiationand portal embolization (35–37), these protocols have yet to be appliedto a suitable preclinicalmodel ofmetabolic disease. It is our opinion thatlarge animal models, such as the Fah−/− pigs used in this study, the dogmodel of hemophilia B, and the pigmodel of familial hypercholesterolemia(38, 39), should be used to a greater degree in preclinical translational workto accelerate clinical translation of novel regenerative therapies, includ-ing cell transplantation.

For clinical translation, we hypothesize that pediatric patients withinborn errors of metabolism of the liver are most likely to benefit fromthe significant advantages of ex vivo LV gene therapy when comparedto other approaches being examined today. In contrast toAAV-mediatedgene therapy, LV integration provides stable gene expression that isoptimal for the treatment of pediatric patients in whom hepatocytes areactively dividing and growing. Furthermore, the recent clinical successin treating several primary immunodeficiency disorders using ex vivogene therapy in hematopoietic cells sets a precedent for the use of LV-mediated gene therapy in children (17, 18). However, despite the cleardemonstration of safety to date of LV in the clinic for primary immuno-deficiencies, further work to test genotoxicity of LV in human hepato-cytes is required before initiation of any clinical trials, particularly giventhe recent data on genotoxicity of other viral vectors, including AAV, ininducing hepatocellular carcinoma in rodentmodels (40). Once this hasbeen demonstrated, we believe that ex vivo hepatocyte gene therapywith LV will provide a lasting impact for the treatment of inborn errorsofmetabolismof the liver in children before their disease has a chance toreach an advanced state.

MATERIALS AND METHODS

Study designOur study was designed to test the hypothesis that ex vivo gene therapyin primary hepatocytes could correct and ameliorate disease in a largeanimal model of metabolic liver disease.We chose to use a pig model ofHT1 for this purpose for clinical relevance. As a preliminary proof ofconcept, we used the mouse model of HT1 to test the efficacy of the LVexpressing Fah to correct disease. Mice were randomly assigned to eachexperimental group. For the primary study, we used four pigs. Bio-chemical and histopathology data for wild-type and Fah−/− pigs offNTBC were compiled from previous studies (12). The use of historical

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controls and the low number of animals used were determined by theavailability of thismodel and the need to comply with the regulations ofthe Institutional Animal Care and Use Committee at Mayo Clinic tolimit the number of experimental animals. Investigators involved in his-topathological and biochemical analyses were blinded. Investigatorsperforming animal handling, sampling, euthanasia, and rawdata collec-tion were not blinded.

Animals and animal careAll animals received humane care in compliance with the regulations ofthe Institutional Animal Care and Use Committee at Mayo Clinic.Fah−/− mice (41) on the C57Bl/6J background were administeredNTBC (Yecuris) in the drinkingwater at 8mg/liter to prevent acute liverinjury before transplantation. Fah−/− pigs were produced in a 50%LargeWhite and 50% Landrace pig (12, 13). NTBC was administered to theanimals orally, mixed within a portion of daily chow rations. Pregnantsows were given 25 to 50 mg of NTBC per day for the duration of ges-tation. Initial testing of dose and efficacy determined that there was nodifference between 25 and 50 mg per day; as a result, two pregnantanimals received 50 mg of NTBC per day, whereas the later pigs re-ceived only 25 mg per day. Weaned piglets were administered NTBC(1 mg/kg) per day until hepatocyte transplantation. All animals wereobserved at least daily for clinical signs and symptoms consistent withHT1 and acute liver failure. All animals were weighed daily for the firstmonth, twice weekly up to 3 months, and then weekly until the end ofthe study. Control animals, including wild-type and Fah−/− pigs, havebeen described previously (12, 14).

Hepatocyte isolation, transduction, and transplantationAll animals undergoing cell transplantation received a femoral arterialline placement to monitor blood pressure. In addition, body tempera-ture, respiration rate, and heart rate were continually monitored andrecorded. For ex vivo gene therapy, after a midline laparotomy, pigsunderwent a segmental liver resection using aCavitronUltrasonic Sur-gical Aspirator to complete the parenchymal transection. About 10%of the liver was removed. The liver specimen underwent an ex vivotwo-step perfusion, and hepatocytes were isolated as described pre-viously (42). Hepatocytes were transduced in suspension for 2 to16 hours at the MOI indicated in table S1. Hepatocyte medium wasidentical to that described previously (9), which included 10 mM dexa-methasone (Sigma-Aldrich). After two centrifugation washes, hepato-cytes were resuspended in saline at a concentration of 10 × 106 to20 × 106 cells/ml and transplanted through a portal vein infusion.For percutaneous injection, the portal vein was identified by surfaceultrasound using a 2- to 5-MHz transducer (SonoSite). Under ultra-sound guidance, an 18-gauge needle was directed percutaneouslyinto the portal vein for injection of hepatocytes (movie S1). For trans-plantation of allogeneic cells in pig Y502, hepatocytes were isolatedand transplanted as above, except that no transduction with LVoccurred. After transplantation, all animals received continuous mo-nitoring of vital signs until they recovered from anesthesia andbecame fully ambulatory. For the sham control pig L768 who re-ceived no cells, no surgery was performed, and the animal was cycledon and off NTBC.

Mouse experiments were performed as described previously (9).Briefly, hepatocytes were harvested from Fah−/− donor mice using astandard two-step collagenase digestion and centrifugal purification.Hepatocytes were cultured for 24 hours in six-well Primaria culture





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plates (BDBiosciences) during which LV-Fahwas added at anMOI of2000 LPs. Hepatocytes were trypsinized, and 0.5 × 106 cells wereinjected through the spleen into syngeneic Fah−/− mice.

PET imaging and analysisImaging was performed on the high-resolution GEDiscovery 690 ADCPET/CT system (GE Healthcare). [18F]TFB was synthesized as de-scribed previously (43). A single injection of [18F]TFB was infused in-travenously 75 min before scanning (dose of ~11 mCi at time ofinjection). CTwas performed at 120 kV and 150mA,with tube rotationof 0.5 s and pitch of 0.516. PET was performed as a two-bed acquisitionwith 10min per bed and 17 slice overlap, resulting in a 27-cm axial fieldof view. Coregistered images were rendered and visualized using thePMOD software (PMOD Technologies). Location of liver and spleenwas determined from the CT image.

Histopathological analysisFor tissue analysis, samples were fixed in 10% neutral buffered formalin(PROTOCOL, Fisher Scientific) and processed for paraffin embeddingand sectioning. For H&E staining andMasson’s trichrome staining, slideswere prepared using standard protocols. Quantification of fibrosis wasperformed on Masson’s trichrome–stained slides by analysis of digitalimages using Aperio ImageScope software (version 12.1). Quantificationof collagen positivity in 10 random areasmeasuring 2mm3 each was per-formed in each slide using the Positive Pixel Count algorithm that isaltered with a hue value of 0.62, hue width of 0.40, and color saturationthreshold of 0.005. IHC for FAH (44) and Ki67 (MIB-1; Dako) was per-formed with a BOND-III automatic stainer (Leica), with a 20-min an-tigen retrieval step using BOND Epitope Retrieval Solution 2 (Leica),and stained with diaminobenzidine (Leica). Quantification of Ki67+ cellswas performedusingAperio ImageScope softwareNuclear algorithm (ver-sion 9) in10 random areas measuring 0.25 mm3 each. Terminal TUNELstaining was performed as described previously (45) using the ApopTagPeroxidase In Situ Apoptosis Detection Kit (Millipore).

Quantitative analysis of vector integrationDNA was isolated from total liver using a DNeasy Blood and TissueKit (Qiagen). Primers (5′-CGAGATCTGAGTCCGGTAGC-3′ and 5′-CCGACCTCCCTAACCCTATG-3′) were designed to amplify integratedvectors in the pig genome. qPCR was performed with Bullseye EvaGreenqPCRMasterMix (MidSci) using an initial denaturation step at 95°C for10 min, followed by 40 cycles at 95°C for 15 s and at 60°C for 60 s. Ab-solute copy number was determined using a standard curve rangingfrom 3.21 × 105 to 2.05 × 101 copies of the vector plasmid. All sampleswere run in triplicate using 150 ng of DNA.

Statistical analysisResults are expressed as means ± SD or SEM, as stated in figure captions.Statistical analyses were conducted with GraphPad Prism software version6. Experimental differences between two groups were evaluated by Stu-dent’s two-tailed t test, assuming equal variance. For comparison of bio-chemical parameters between pigs, because of the lack of availableanimals required to perform a randomized study, differences betweenexperimental animals were compared to historical controls using a two-sided, independent t test with 5% type 1 error rate. Differences betweenmultiple groups were compared using one-way ANOVA followed byBonferroni’s multiple comparisons test. P < 0.05 was considered statis-tically significant.

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SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/8/349/349ra99/DC1MethodsFig. S1. Allogeneic transplanted hepatocytes were not detected in recipient pig Y502.Fig. S2. Ultrasound guidance allows a less invasive method to inject cells in the portal vein.Fig. S3. Assessment of cell proliferation and apoptosis in transplanted Fah−/− pigs.Fig. S4. Variation in fibrosis is associated with differences in cell repopulation in pig Y845.Table S1. Summary of cell transplantations.Movie S1. Ultrasound-guided percutaneous portal vein injection.

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Acknowledgments: We thank Exemplar Genetics for management of the Fah−/− herd; I. Ivanov(Mayo Clinic, Rochester) for biochemical analyses; L. Gross and T. Blahnik (Mayo Clinic, Rochester)and J. Pattengill (MayoClinic, Arizona) for histology support; L. Linscheid, D. McConnell, and B. Kemp(Mayo Clinic, Rochester) for PET imaging support; P. Novotny (Mayo Clinic, Rochester) for statisticalanalyses; D. Meixner for assistance with portal vein injections; M. Curry for figure preparation; andG. Gores (Mayo Clinic, Rochester) for thoughtful discussion. Funding: R.D.H. was funded throughanNIH K01 DK106056 award, an American Liver Foundation Postdoctoral Fellowship Award, and aMayo Clinic Center for Regenerative Medicine Career Development Award. S.L.N. was fundedthrough an NIH R41 DK092105 award, the Wallace H. Coulter Foundation, Marriot Foundation,Darwin Deason Family Foundation, and Mayo Foundation. S.H.I. was funded through the Centerfor Clinical and Translational Science KL2 programKL2TR000136-09 and theMayo Clinic Center forCell Signaling in Gastroenterology, Pilot and Feasibility Award, program P30DK084567. Authorcontributions: R.D.H., S.A.M., J.G., F.E., and S.L.N. designed experiments, performed experiments,interpreted results, and wrote the paper. B.A. performed hepatocyte isolations. H.C. providedsurgical expertise. P.R. and C.O.H. facilitated biochemical data collection and analysis. R.M.provided pathology reports. H.J. and T.R.D. provided radioisotope for PET/CT imaging. L.S. andM.K.O. analyzed PET/CT data. B.L.F. and S.H.I. performed TUNEL assay and analysis. C.-S.H. conductedthe SLA typing and interpretation. K.W.P., M.G., Y.I., J.B.L., and S.J.R. provided intellectual input,designed experiments, and interpreted data.Competing interests: The authors declare that theyhave no competing interests. Data and materials availability: All data are shown, and all mate-rials are commercially available or are available from the corresponding author upon request.

Submitted 4 February 2016Accepted 5 July 2016Published 27 July 201610.1126/scitranslmed.aaf3838

Citation: R. D. Hickey, S. A. Mao, J. Glorioso, F. Elgilani, B. Amiot, H. Chen, P. Rinaldo, R. Marler,H. Jiang, T. R. DeGrado, L. Suksanpaisan, M. K. O’Connor, B. L. Freeman, S. H. Ibrahim,K. W. Peng, C. O. Harding, C.-S. Ho, M. Grompe, Y. Ikeda, J. B. Lillegard, S. J. Russell,S. L. Nyberg, Curative ex vivo liver-directed gene therapy in a pig model of hereditarytyrosinemia type 1. Sci. Transl. Med. 8, 349ra99 (2016).

nceTranslationalMedicine.org 27 July 2016 Vol 8 Issue 349 349ra99 10


1Curative ex vivo liver-directed gene therapy in a pig model of hereditary tyrosinemia type

Lillegard, Stephen J. Russell and Scott L. NybergSamar H. Ibrahim, Kah Whye Peng, Cary O. Harding, Chak-Sum Ho, Markus Grompe, Yasuhiro Ikeda, Joseph B.Ronald Marler, Huailei Jiang, Timothy R. DeGrado, Lukkana Suksanpaisan, Michael K. O'Connor, Brittany L. Freeman, Raymond D. Hickey, Shennen A. Mao, Jaime Glorioso, Faysal Elgilani, Bruce Amiot, Harvey Chen, Piero Rinaldo,

DOI: 10.1126/scitranslmed.aaf3838, 349ra99349ra99.8Sci Transl Med

move into patients, to regenerate their own livers and spare them the long wait times on the liver transplant list.demonstrated in large animals the use of materials that are safe for use in people, the technology is now poised toliver failure and fibrosis and also restored metabolic function, which is deteriorated in HT1 disease. Having

, and then put the cells back into the same animals. The ex vivo gene therapy approach preventedFahthe correct ), transduced them withFahHT1. The authors took liver cells from pigs that have HT (through a defect in the gene

. turned to gene therapy as a way to cureet altransplant. However, owing to the shortage of liver donors, Hickey is a liver−−an inherited metabolic disease−−The only cure for hereditary tyrosinemia type 1 (HT1)

Skipping the waiting list

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http://stm.sciencemag.org/content/scitransmed/9/418/eaam6375.full

http://stm.sciencemag.org/content/8/349/349ra99#BIBL

http://www.sciencemag.org/help/reprints-and-permissions

http://www.sciencemag.org/about/terms-service


curative ex vivo liver-directed gene therapy in a pig ... · gene therapy curative ex vivo...

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