Nearly 14,000 patients wait annually for liver transplantation in the U.S. alone. The problem is considerably worse world-wide and represents one of the most challenging hurdles in medicine.
(1) With a universal shortage of organs and limited resources, alternatives to whole organ transplantation are required to address this pandemic. Bioartificial liver devices and repopulation of decellularized liver scaffolds to create bioengineered organs for transplantation have yet to prove effective for the treatment of patients with liver failure. Cell therapy using primary hepatocytes has shown effectiveness in animal models, but the success of this approach has been very limited in the clinical setting. (2) One of the main reasons for this limited success is the inflammation, fibrosis, and scar tissue obstructing blood flow in the failing liver, which constitutes an adverse environment for hepatocyte engraftment and growth. (3) Hereditary tyrosinemia type 1 (HT1) is an ideal disease model to study treatment options for acute and chronic liver failure in the preclinical environment. HT1 is an inborn error of metabolism of the liver caused by a deficiency of the fumarylacetoacetate hydrolase (FAH) enzyme, which is responsible for the last step of tyrosine catabolism and results in the inability to completely metabolize tyrosine.(4) Untreated, HT1 often leads to fulminant liver failure as early as a few months of life. (5) In the chronic form of the disease, FAH deficiency leads to persistent accumulation of toxic metabolites in the liver, causing oxidative damage and subsequent inflammation, fibrosis, cirrhosis along with high rates of hepatocellular carcinoma (HCC). (6, 7) In HT1, inflammatory changes and liver injury are seen within days to weeks without therapy.
Pharmacologic treatment of HT1 exists in the form of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3cyclohexanedione (NTBC), a drug which inhibits tyrosine metabolism upstream of FAH, leading to the build-up of less toxic metabolites. (8) We have previously created and characterized the porcine model of HT1 and showed that this animal is an excellent model of acute and chronic liver failure by reproducing the inflammation, fibrosis and cirrhosis pattern seen in many human liver diseases (9) . We have since demonstrated that ex vivo gene therapy involving lentiviral transfer of a functional Fah cDNA into autologous hepatocytes is curative in both mouse and pig models of HT1. (10) In our previous work, primary hepatocytes were isolated from a partial hepatectomy and transduced ex vivo by a lentiviral vector carrying a functional human Fah gene. Once corrected, cells were transplanted back into the donor animal via portal vein infusion. However, a substantial clinical limitation to our previous approach is that orthotopic hepatocyte transplantation may not be feasible in patients with acute or chronic liver disease, as the diseased liver is often an inadequate and hostile environment for hepatocyte engraftment and expansion. (11) Alternative anatomical sites for transplantation of corrected cells could provide a healthier milieu to enable hepatocyte engraftment and proliferation. Lymph nodes are one such alternative site due to several defining characteristics. For example, they are prepared to harbor rapid expansion of T and B cells to support a swift immune response when needed. (12, 13) Lymph nodes are not only capable of accommodating immune cells, but are also a common metastatic site for many types of cancer. (14) They naturally provide a favorable environment for metastatic cell engraftment and growth. This is due, in part, to their high vascularization potential, which permits neoangiogenesis, (15, 16) as well as to their reticular network of fibroblasts and other stromal cells that provide physical and trophic support. (17, 18) Inherent plasticity, together with the fact that their systemic function is not hampered by the transplanted cells, (19) makes lymph nodes a promising site for ectopic cell delivery. Finally, lymph nodes are also easily accessible for both delivery and monitoring of the transplanted cells.
We reported that, after intraperitoneal transplantation of hepatocytes in mice, these cells colonize the lymph nodes and are able to rescue animals from lethal hepatic failure. (20) We explored the mouse lymph node as an ectopic transplantation site for multiple tissues, including liver, and demonstrated that injection of hepatocytes into a single lymph node generated enough ectopic liver mass to rescue the metabolic disorder in the mouse model of HT1. (21) However, it is unknown whether these promising results are translatable into a larger animal model, where a substantially higher hepatic mass would be required for rescue of liver disease. In this preclinical proof of concept study, we demonstrate the therapeutic potential of ectopic transplantation of ex vivo corrected hepatocytes into lymph nodes in the HT1 pig, a genetic model of liver failure. In tracking the fate of these cells we show the ability of ectopically transplanted hepatocytes to engraft long term in the mesenteric lymph nodes where they recreate important liver architecture that gives rise to multiple cell lineages seen in the native liver, and serve as a reservoir for repopulation of the recovering native liver.
To demonstrate that hepatocytes are able to engraft in lymph nodes in a large animal model, a wildtype pig underwent a partial hepatectomy, and harvested hepatocytes were labeled with 89 Zr (halflife 78.4 h) (22) prior to transplantation into 10-20 mesenteric lymph nodes. Radiolabeling efficiency was ~20% and radioactivity concentration was ~0.1 MBq/10 6 cells. The animal received 6 x 10 8 hepatocytes through direct mesenteric lymph node injection. PET-CT imaging at 6 h posttransplantation demonstrated the presence of radioactivity within mesenteric lymph nodes (261.8 ± 108.7 SUV; Fig 1D, Video S1). Radioactivity remained present within the mesenteric lymph nodes at the 54 and 150 h time points (101.1 ± 34.7 and 70.0 ± 26.4 SUV, respectively). Background activity was measured in the left lumbar paraspinal muscle (0.1 SUV). Interestingly, although no radioactivity was detected within the liver at the 6 h time point, increasing amounts of radiotracer were found in the liver at the 54 and 150 h time points (3.3 ± 0.4 and 5.9 ± 0.8 SUV, respectively), indicating accumulation of 89 Zr-labeled cells or cellular debris. The absence of radioactivity in bone, a known site of uptake for unchelated 89 Zr, suggested that the radiolabel was retained within the dinitrobenzamide DBN chelator construct and not indicative of unincorporated "free" label.
These data at 0.69 and 1.9 half-lives, respectively, suggest possible migration of the transplanted hepatocytes from the lymph nodes to the liver or labeled cellular debris passing through the liver.
No radioactivity above background was found in spleen, lung or other organ systems at either of these time points.
Ex vivo corrected hepatocytes are able to cure a pig model of HT1 after ectopic transplantation into lymph nodes A total of 5 Fah -/animals were maintained on NTBC until the time of ex vivo-corrected autologous hepatocyte transplantation into lymph nodes (Fig 1A-C) , at which point administration of NTBC was discontinued to propagate liver injury and stimulate hepatocyte regeneration. This induced selective expansion of the newly transplanted FAH-positive hepatocytes. Animals were cycled on and off NTBC based on weight parameters until NTBCindependent growth was achieved (Fig 2A, Fig S1) . NTBC-independent growth was attained at a mean of 135 ± 25 days post-transplantation (range: 97 to 161 days), after 3 to 6 cycles of NTBC.
Biochemical cure of the HT1 phenotype was confirmed by normalization of liver specific enzymes known to be elevated in HT1 as well as normalization of tyrosine levels. At the time of euthanasia, mean tyrosine levels (84.2 ± 34.5 µM) in the five treated pigs were within normal limits for wildtype animals, and were significantly lower than untreated Fah -/controls (826.3 ± 277.5 µM) ( Fig   2B) . Similar results were seen in liver function tests in treated animals (AST: 56.8 ± 21.7 U/L; ALP: 186.6 ± 78.8 IU/L; ammonia: 35.8 ± 14.4 µmol/L; albumin: 3.6 ± 0.3g/dL; and total bilirubin: 0.15 ± 0.1 mg/dL) compared to untreated Fah -/controls (AST: 343.7 ±54.5 U/L; ALP: 918.3 ± 282.5 IU/L; ammonia: 553 ± 561.3 µmol/L; albumin: 2.8 ± 0.9 g/dl; total bilirubin: 1 ± 0.7 mg/dl). suggesting that the hepatic insult that occurs during NTBC cycling is reversible with time, as FAHpositive hepatocytes expand to repopulate the liver (Fig 4) .
In order to characterize any differences between the hepatocytes present in the liver and those present in the lymph nodes, we performed next generation sequencing and bioinformatics analysis of both cell populations. Mapping statistics are provided in Table S1 . We found no significant differences in general lentiviral integration profile between these two cell populations. In both cases, integration occurred more often in coding regions than in non-coding regions of the genome, with exons being preferred over introns ( Fig 5A) . Therefore, integration was favored in chromosomes with higher gene densities ( Fig S3B) . In both cell populations, lentiviral integration was rare in CpG-rich islands and was clearly favored downstream of transcription start sites ( Fig   5B and C) , suggesting minimal tumorigenicity potential from oncogene activation. Furthermore, there was no preference for integration in tumor-associated genes in either cell population ( Fig 5D) .
The genes with the highest integration frequency in both groups of cells are presented in Table 2 , and it is here that differences between the two cell populations were found. Fah was the gene with the highest number of distinct integration sites in both groups, although it was only in the top 10 integrated genes in the lymph node population. Interestingly, all but two of the 135 unique integration sites within Fah locus in the liver hepatocyte population were represented in the 206 integration sites present in the lymph node hepatocyte population (Fig 5E) , indicating that the transplanted cells first engrafted in the lymph nodes, divided and then, after clonal expansion, migrated to the liver. The top four genes with the highest integration frequency in the liver population were also within the top 20 genes in the lymph node population, and were all approximately 5-fold enriched in the liver population when evaluated by the total number of reads present for each, suggesting unbiased expansion and enrichment of a subpopulation of lymph node hepatocytes within the liver. These genes were: Mir9799, microRNA; Ndufv2, NADH dehydrogenase ubiquinone flavoprotein 2; Ifrd1, interferon-related developmental regulator 1; and Suclg1, succinyl-CoA ligase (GDP-forming), alpha subunit. In native and ectopic liver samples obtained from our experimental animals after necropsy, expression of Suclg1, Ndufv2, and Ifrd1 was indeed disrupted compared to control liver levels, possibly due to the insertion of the LV construct in at least one allele in some of the corrected hepatocytes ( Fig 5F) . As a control, NIS expression was evaluated and only detectable in 2 of the 3 samples of hepatized lymph nodes analyzed. The fifth most prevalent integration site in the hepatocytes that migrated to the liver was Cenpp, centromere protein P, which was enriched over 78-fold compared to the lymph node population and could be an indication that this disruption caused a favorable migration, engraftment, and/or expansion profile for these clones specifically.
To determine whether hepatocytes transplanted in lymph nodes generated a tissue resembling normal liver, transcriptome profiles of untransplanted and hepatized lymph nodes, as well as native (engrafted) and control (isolated from a healthy, untransplanted pig) livers were compared. Twelve thousand genes were identified as differentially expressed (DE) and contrasting control lymph node to liver tissues including hepatized lymph nodes ( Figure 6A) . T-distributed Stochastic Neighbor Embedding (t-SNE) showed a strong tendency of hepatized lymph node transcripts to cluster with liver tissues samples, whereas control lymph nodes were distinct from the other three groups ( Figure 6B ). These data suggest that a significant proportion of the lymph node transcriptome acquires a liver-like signature after hepatocyte transplantation. To estimate the similarity of hepatized lymph nodes with control liver, we first focused on 24 genes, which were previously described to be liver-specific (LiGEP , Table S2 and Figure S4 ). We then included 20 additional genes in our analysis, which can be classified as cytochrome P450 genes, other enzymes, plasma proteins, transporters, surface molecules and cytokines, and transcription factors (Table S2) .
Cluster heat map showing the RNAseq expression levels of all 44 genes is shown in Figure 6C .
Evaluation of albumin expression demonstrates adoption of a liver phenotype by hepatized lymph nodes relative to control (Fig 6D, left) while analysis of FAH shows transgenic expression in hepatized lymph nodes as well as elevated transgenic expression in repopulated native liver (under TBG promoter) relative to control liver (Fig 6D, right) . Expression levels of all other genes are shown in Fig S4A (LiGEP) and S4B (additional liver-specific genes).
In many human liver diseases, orthotopic hepatocyte transplantation and engraftment is often This timeframe to metabolic correction is comparable to that of our previous study using similar numbers of cells and portal vein delivery into the native FAH-deficient liver.(10) It is worth noting that in our previous study, animals were also maintained on the protective drug NTBC until the time of transplantation, which means that their livers did not have a significant amount of inflammation and fibrosis when they received the corrected hepatocytes. Orthotopic cell transplantation has shown low rates of engraftment, especially when the liver is compromised by inflammation and fibrosis, as is seen in many liver diseases. (29) It has been previously shown that hepatocyte transplantation into lymph nodes is able to generate enough ectopic liver mass to correct HT1 and rescue mice from acute and chronic liver disease. (21) In our study, we show a similar result in the large animal model of HT1, suggesting that hepatocyte transplantation into lymph nodes is scalable and may therefore be a clinically relevant technique that permits the creation of sufficient ectopic liver mass to significantly impact liver function.
There are important differences between these two preclinical studies. Syngeneic and allogeneic hepatocytes were used in the HT1 mouse study by Komori et al, and the HT1 mouse does not exhibit the full extent of liver injury seen in humans, including fibrosis and cirrhosis. (21) The current study was conducted in the HT1 pigs initially supported on NTBC, where liver disease could be delayed until the harvesting of autologous cells was complete. In this study we take advantage of the porcine HT-1 model of liver failure because 1-2 weeks after the withdrawal of NTBC we begin to see rapid and severe liver injury that includes all the same features we see in human liver failure including, inflammation, fibrosis and cirrhosis. This study design allows us to isolate the single variable related to ectopic transplantation of hepatocytes in LNs in the setting of liver failure while avoiding potential confounding variables related to immune suppression or the use of primary hepatocyte alternatives. Now that we have demonstrated the feasibility of targeting LNs for hepatocyte transplantation, subsequent evaluations should be performed in untreated HT1 pigs or other large animal models of liver disease using primary hepatocyte alternatives or allotransplantation and immune suppression.
The fact that our HT1 model, which develops all the acute and chronic liver failure features seen in many other liver diseases, was completely weaned of NTBC requirement and metabolically normalized after the ectopic transplantation procedure indicates the therapeutic potential of this approach. Even though the goal of this study was not to evaluate directly the use of ectopic LN hepatocyte transplantation for the treatment of any specific genetic liver disease, there are some potential applications that should be discussed regarding these diseases. Some optimizations in initial engraftment rates or other modifications (i.e., exogenously applied selective pressure or repeat administrations of corrected cells) would be necessary for diseases that do not inherently propagate healthy cells, such as phenylketonuria or Wilson's disease. However, it is the liver damage itself through the hepatectomy and not something specific to HT1 that provides the stimulus for initial successful engraftment. Additionally, unlike HT1, most liver diseases do not require complete repopulation of the diseased liver, and administration could be catered to effect to see a phenotypic change.
Autologous hepatocyte transplantation could be directly translated into human patients where their condition does not preclude a partial hepatectomy, thereby avoiding immune concerns related to the use of allogeneic cells. However, healthy allogeneic hepatocytes could be transplanted in cases where partial hepatectomy is contraindicated, such as in acute liver failure or severe cirrhosis, which would require additional support via established immune suppression protocols. Possible future sources of hepatocytes include universal donor hepatocytes, IPS-derived hepatocytes, and HLA-matched farmed hepatocytes (30) (31) (32) . In our study hepatocytes were transplanted into pig mesenteric lymph nodes using an open technique due to the paucity of suitable peripheral lymph nodes in the neonatal pig, but the same procedure could foreseeably be performed into central or peripheral lymph nodes in humans via a percutaneous or endoscopic ultrasound-guided technique, as described previously. (28) In our model, we found that although hepatocytes demonstrated long-term survival within lymph nodes, forming appropriate liver architecture including bile ducts, some also subsequently engrafted in the liver after a period of engraftment and expansion in the LNs. This was suggested by bioinformatics data and confirmed by histology. This phenomenon was very limited in previous mouse studies (21) and could be related to the microanatomy and direction of flow in pig lymph nodes, which differ from those of other mammals. Their microarchitecture is inverted, with the germinal centers being located internal to the medulla, which has led authors to suggest that lymphocytes are transported back into a capillary system after passing through a lymph node, as opposed to the lymphatic system as in other animals. (33, 34) Indeed, during the injection procedure it was possible to observe injected fluid communicating from the lymph node into the capillary bed. Based on these results, we also analyzed in detail the overall integration profile of our lentiviral vector in both hepatocytes that remained in the lymph nodes and hepatocytes that migrated to the liver. In both cell populations, the lentiviral vector showed a benign integration profile, with no preference for integration in promoter regions or tumor-associated genes. Analysis of the genes with the highest integration frequency in both populations suggested that hepatocytes that migrated to and expanded in the liver were a subpopulation of hepatocytes that remained in the lymph nodes, although discerning any functional impact of disrupting the integrated genes was beyond the scope of the current study. Interestingly, prevalence of the top four integrated genes in the liver population was approximately five-fold higher than in the lymph node population, a linearity possibly supported by a clonal founder with all four integrations or up to 4 unique clonal founders with identical (unbiased) expansion after migration and liver engraftment. Of note, Fah was within the top twenty genes with the highest integration frequency and was the gene with the highest number of distinct integration sites in both groups. This suggests that homology may guide integration to some extent, a finding that provokes further development to possibly improve safety and predictability of lentiviral genomic disruption.
We also examined relative changes in gene expression, finding no significant differences in general transcriptomes between engrafted and control lymph nodes, as well as engrafted and control liver, In summary, we have shown that ectopic transplantation of corrected, autologous hepatocytes into mesenteric lymph nodes is curative in the HT1 pig model of severe liver failure. Cells transplanted into these ectopic sites engraft and expand creating hepatized LNs, aiding the failing liver.
Hepatocyte transplantation into lymph nodes is a promising approach to the treatment of multiple liver diseases that holds several important advantages over more traditional cell transplantation techniques, especially in patients with pre-existing native liver damage. The NIS reporter should not affect phenotype but is only intended to allow non-invasive tracking of transplanted cells. The finding that ectopic transplantation of corrected hepatocytes seeded subsequent robust orthotopic engraftment was not anticipated, and the additional hypothesis that the hepatocytes that migrated to liver were a subset of those that were initially engrafted in the lymph nodes (as opposed to a unique population that never engrafted ectopically) was further developed and tested using bioinformatics.
These studies in large animals were performed in a limited number of subjects (n=5) due to resource-intensive nature of the involved surgical and diagnostic procedures in large animals, and associated resource requirements for the husbandry, in vivo evaluations, and bioinformatic analyses.
Animals All animal procedures were performed in compliance with Mayo Clinic's Institutional Animal Care and Use Committee regulations and all animals received humane care. For biodistribution experiments, a female wildtype pig was used. For ex vivo gene therapy experiments, male and female Fah -/pigs were used. Fah -/pigs were produced in a 50% Large White and 50% Landrace pig as previously described. (36, 37) NTBC administration NTBC mixed in food was administered at a dose of 1 mg/kg/day with a maximum of 25 mg/day. All animals remained on NTBC until the time of transplantation, after which NTBC administration was discontinued to support expansion of the corrected cells. After hepatocyte transplantation, all animals were monitored daily for loss of appetite or any other clinical signs of morbidity. Animals were weighed daily for the first two weeks post-operatively and weekly thereafter. If loss of appetite, weight loss, or any other signs of morbidity occurred, NTBC treatment was reinitiated for seven days. Animals were cycled on and off NTBC in this fashion to stimulate expansion of corrected FAH-positive cells. Hepatocyte transduction Hepatocytes were co-transduced in suspension at a MOI of 20TUs with two third-generation lentiviral vectors carrying the sodium-iodide symporter (Nis) reporter gene or the pig Fah gene under control of the thyroxine-binding globulin promoter (Fig 1A) . The NIS reporter allows for longitudinal non-invasive monitoring of transplanted cells through single-photon emission computed tomography (SPECT) or positron emission tomography (PET). (42) Transduction occurred over the course of 2 h before transplantation using medium and resuspension techniques previously described.(10) Hepatocytes were resuspended in 0.9% sodium chloride (Baxter Healthcare Corporation, Deerfield, IL).
Hepatocyte transplantation in pigs All pigs received autologous transplantation of hepatocytes through direct mesenteric lymph node injection (Fig 1B) . After partial hepatectomy, animals were kept under general anesthesia until the time of transplantation, approximately 4 h later.. Bowel was exteriorized through the upper midline incision until the root of the mesentery was visible (Fig 1C) . samples; the digested DNA samples were then ligated to linkers and treated with ApoI to limit amplification of the internal vector fragment downstream of the 5′ LTR. Samples were amplified by nested PCR and sequenced using the Illumina HiSeq 2500 Next-Generation Sequencing System (San Diego, CA). After PCR amplification, amplified DNA fragments included a viral, a pig and sometimes a linker segment. The presence of the viral segment was used to identify reads that report a viral mediated integration event in the genome. The reads sequenced from these DNA fragments were processed through quality control, trimming, alignment, integration analysis, and annotation steps.
Quality control of the sequenced read pairs was performed using the FASTQC software. The average base quality of the sequenced reads was >Q30 on the Phred scale. Uniform distribution of A,G,T and C nucleotides was seen across the length of the reads without bias for any specific bases.
The number of unknown "N" bases was less than 1% across the length of the reads. High sequence duplication (> 80%) was observed, however, this was anticipated due to the nature of the experiment and the amplification of specific library fragments.
We used Picard software's (http://broadinstitute.github.io/picard/) insert size metrics function to calculate the average fragment length per sample. This metric averaged across all the samples was 158 base pairs (bps) long with an average standard deviation of 29bps. Since 150bp long reads were sequenced, most of the paired read one (R1) and read two (R2) reads overlapped, providing redundant information. R2 reads were therefore not considered in the analysis.
Reads were trimmed to remove the viral sequence in two separate steps. In step 1, the viral sequence was trimmed from the R1 reads using cutadapt(45) with a mismatch rate (e=0.3) from the 5' end of each read. In step 2, if the linker sequence was present, it was similarly trimmed from the 3'end of the read. Trimmed reads with a length less than 15bps were removed from the rest of the analysis to reduce ambiguous alignments. Untrimmed reads were also removed from the rest of the analysis because they did not contain a viral segment that could be used as evidence of a viral mediated integration.
The remaining R1 reads were aligned to the susScr11 build of the pig reference genome using BWA-MEM in single-end mode. (46) Default BWA-MEM parameters were used. The reads used to identify genomic points of viral integrations had to be uniquely mapped to the genome with a BWA-MEM mapping quality score greater than zero.
An integration point was defined by the position of R1's first aligned base on the susScr1 genome.
Unique integration points were identified across the genome without any constraints on coverage.
However, for downstream annotation and analysis, only those integration points with 5 or more supporting reads were used to minimize calling false positive integration points. RNAs from selected samples were shipped on dry ice to Novogene in Sacramento, CA for library preparation and sequencing. All samples passed Novogene internal quality control with RNA integrity number above eight.
RNAseq data analysis was performed using Partek Genomics Suite software version 7.0. Briefly, quality control was measured considering sequence-read lengths and base-coverage, nucleotide contributions and base ambiguities, quality scores as emitted by the base caller, and overrepresented sequences. All the samples analyzed passed all the QC parameters and were mapped to the annotated pig reference SScrofa11.1_90+nonchromosomal using STAR2.4.1d index standard settings. Estimation of transcript abundance based on the aligned reads was performed by optimization of an expectation-maximization algorithm and expressed as FPKM (Fragments Per Kilobase Million). Differentially expressed (DE) genes were detected using differential gene expression (GSA) algorithm based on p-value of the best model for the given gene, false discovery rate (FDR), ratio of the expression values between the contrasted groups, fold change (FC) between the groups and least-square means (adjusted to statistical model) of normalized gene counts per group. 12000 genes were identified as DE based on log2 |FC| ≥2 and FDR ≥ 0.05 cut-off.
Unsupervised clustering to visualize expression signature was performed using 1-Pearson correlation distance and complete linkage rule and samples were classified using t-SNE (tdistributed stochastic neighbor embedding).
Statistical analysis Numerical data are expressed as mean ± SD (standard deviation) or SEM (standard error of the mean). Statistical significance was determined by Welch's t-test, and established when p ≤ 0.05. Statistical analyses were performed with GraphPad Prism software version 7. 