From pain to gain: Leveraging acetaminophen in hepatocyte transplantation for phenylketonuria
Authors: Ryan J. Schulze, Stephen C. Strom, Scott L. Nyberg
Journal: Hepatology (2023)

OLT has the potential, in theory, to be curative for most, if not all, metabolic liver diseases with a genetic basis. However, the application of OLT as a universal solution will remain a limited clinical option; despite increasingly aggressive acceptance criteria,1 OLT continues to suffer from a shortage of transplantable grafts, underscoring the urgent need for additional therapeutic modalities. Recently, recombinant adeno-associated virus-mediated gene therapies for the liver have offered significant promise but are hampered by immune reactions to the viral capsid, limitations on redosing, and incompatibilities with use in pediatric patients due to episomal transgene dilution.2 Because of these shortcomings, a cell therapy alternative, hepatocyte transplantation (HTx), has gained traction in the cell therapy space over the past 3 decades. Rather than transplanting a whole liver (or even just a single lobe), HTx represents a nuanced approach involving the simple infusion of healthy human hepatocytes into the liver of a recipient to bridge the metabolic gap.3 This strategy could prove curative to thousands of patients either ineligible for or unable to receive OLT or gene therapies and potentially extend the gift of a liver graft from a single recipient to many more. Nonetheless, the successful implementation of allogeneic HTx as a viable clinical therapy has hurdles that must be overcome. These challenges are centered mainly on the need for immunosuppression and the assurance of initial engraftment and subsequent expansion of the transplanted cells. The liver environment, or the "soil" in which the hepatocyte "seeds" are sown, is crucial for the successful integration and long-term potency of the transplanted cells. Several preconditioning regimens have been used clinically (eg, preparative hepatic irradiation) to elicit a proregenerative milieu and render the hepatic parenchyma conducive for cell engraftment.4 Despite best efforts, however, it has been estimated that greater than 70% of the infused cells are cleared within the first 24 hours after HTx by host phagocytes and macrophages.5 Within weeks, it is likely that the initial percentage of transplanted cells taking root in their new home has dwindled substantially, reinforcing a need for strategies to encourage the stable expansion of any newly engrafted cells. One strategy to increase the successful expansion of transplanted hepatocytes is the presence of a selective advantage, either inherent or through genetic manipulation. A classic example of inherent selective advantage is the inborn error of metabolism causing hereditary tyrosinemia type 1. In the hereditary tyrosinemia type 1 example, healthy donor cells, armed with a functional copy of fumarylacetoacetate hydrolase (FAH), a key enzyme in tyrosine (Tyr) metabolism, have the upper hand against the hepatocytes found in an FAH-deficient liver. Without pharmacological intervention, FAH-deficient hepatocytes undergo apoptosis, allowing engrafted cells to take their place. Indeed, this selective advantage has been exploited to encourage the engraftment and expansion of xenotransplanted human hepatocytes into modified severe combined immunodeficient (Fah−/−/Rag2−/−/Il2rγ−/Y) animals.6 This model routinely provides for substantial (>90%) repopulation of the recipient liver by donor hepatocytes. Another inherited metabolic disease of the liver, phenylketonuria (PKU), results from any number of mutations in the gene encoding for an upstream enzyme (phenylalanine hydroxylase, PAH) that catalyzes the hydroxylation of L-phenylalanine (Phe) to L-Tyr. In the absence of a carefully controlled diet, blood Phe levels can quickly become elevated in patients with PKU, resulting in hyperphenylalaninemia. PKU has a largely extrahepatic phenotype, resulting in severe neurological manifestations, intellectual disabilities, and developmental delays.7 Unlike hereditary tyrosinemia type 1, PKU lacks an inherent selective advantage necessary to tip the balance in favor of expansion for donor hepatocytes. How, then, to get donor cells to persist long enough to confer a therapeutic benefit? Can selective advantages be introduced through genetic manipulation or gene editing? Nature has endowed hepatocytes with a robust mechanism to counter xenobiotics. But, in a clever twist of strategy, it may be possible to turn this mechanism against them by genetic manipulation to elicit a survival advantage in donor cells. In 2021, Vonada et al8 demonstrated that transplanted hepatocytes could be rendered resistant to acetaminophen/paracetamol (N-acetyl-p-aminophenol, APAP) toxicity to give them a leg up on the recipient's native cells. With 5 decades of knowledge about its hepatic metabolism, APAP is an analgesic/antipyretic with a lengthy track record as a safe and effective compound for pain control. Moreover, it is inexpensive, readily available, and has documented usage profiles in both pediatric and adult populations. At supratherapeutic concentrations, the metabolism of a small percentage of APAP to N-acetyl-p-benzoquinone imine is hepatotoxic; however, this compound is readily tolerated at lower dosages. In this issue of Hepatology, Vonada et al9 again leverage APAP hepatotoxicity, this time taking advantage of CRISPR gene editing to engineer a selective advantage into transplanted primary hepatocytes and elicit the full phenotypic correction of a mouse model of PKU. The authors isolated fluorescently labeled primary hepatocytes from a reporter (mT/mG) mouse and used CRISPR/Cas9 targeting ex vivo to inactivate Cypor, a cofactor for the P450 machinery with expression primarily restricted to Zone 3 hepatocytes of the hepatic lobule. These pericentral cells are the primary sites of N-acetyl-p-benzoquinone imine production following APAP administration. Cypor-deficient hepatocytes were subsequently transplanted into 2 mouse models of PKU: one that harbors a mutation (Phe263Ser) rendering Pah catalytically inactive (Pahenu2/enu2) and a second containing a homozygous deletion of exon 1 (PahΔexon1/Δexon1). Both models have previously been demonstrated to recapitulate many of the features of human PKU. After less than 11 weeks on an APAP-containing diet, the authors found a therapeutic reduction in circulating Phe levels and reversion of altered coat color compared to untreated control animals. This outward correction was correlated with histological evidence for engraftment and a clear expansion of the transplanted hepatocytes containing the wild-type copy of Pah. Importantly, all animals were ultimately able to be removed from APAP selection, indicating that careful monitoring of Phe levels in tandem with markers of hepatocellular injury allowed for fine-tuning of the APAP dosage to achieve persistent phenotypic correction. Despite a very low initial level of cell engraftment, the APAP selection strategy ultimately allowed for effective therapeutic correction. Nonetheless, the safety of using an APAP-centric approach was understandably a crucial component of the study. At a repopulation level of <20%, phenotypically corrected PKU mice still retained a reservoir of fully functional Zone 3 Cypor-expressing cells and furthermore exhibited no significant elevations in liver enzymes nor evidence for neutral lipid accumulation. In addition, there was no significant impact on the metabolism of Zone 3-metabolized drugs such as caffeine, indicating that this selection strategy does not compromise normal hepatic metabolism. Moving forward, this study represents a significant advance in the versatility of HTx as an alternative to gene therapies for liver disease. For this approach to be viable, however, logistical challenges must be overcome. First is the lack of a high-quality supply of primary human hepatocytes. The use of newly developed animal bioreactors for the in vivo production of large quantities of healthy cells may address this issue. Second is immunosuppression: post-transplantation immune rejection is a hurdle. While the current study does not directly tackle the issue of allogeneic rejection, it references genome engineering approaches that might be adapted to hepatocytes to create "universal donor cells," analogous to strategies in place for allogeneic CAR-T therapies. Integrating such a technique with APAP selection would be a prospective direction, potentially eliminating the rejection challenge altogether. Alternatively, ex vivo gene correction of autologous mutant hepatocytes would serve as another means for avoiding the rejection of transplanted cells.10 In summary, the study by Vonada and colleagues demonstrates that transplantation of APAP-selectable hepatocytes may represent a robust strategy for treating PKU. As HTx is currently limited by the engraftment of only a small percentage of the recipient's liver mass, the absence of expansion would prove insufficient for the full phenotypic correction of many disorders. As demonstrated in the study, the use of APAP as a novel selection agent offers a creative solution to previous issues with cell expansion in HTx. By conferring APAP resistance to wild-type hepatocytes ex vivo through CRISPR gene editing, this approach facilitates the selective expansion of these hepatocytes after transplantation, potentially achieving therapeutic thresholds necessary for disease correction. This innovative application not only surmounts the engraftment challenge but also leverages a well-known, commonly used drug, potentially streamlining clinical translation. One of the most compelling revelations from this study is the mutation-agnostic nature of HTx. The ability of Vonada et al to correct different mouse models of PKU using the same cell therapy underscores the broad-spectrum applicability of this approach, contrasting sharply with mutation-specific gene therapies. As the authors acknowledge, these cells could theoretically be used as a single therapy, making it a viable method for addressing not only PKU but potentially hundreds of other rare and orphan hepatic metabolic disorders as well.