Hereditary tyrosinemia type 1 (HT1) is a recessive genetic disease of amino acid metabolism. It is caused by the loss of functional fumarylacetoacetate hydrolase (FAH), an enzyme critical for phenylalanine and tyrosine metabolism (Thompson et al., (2020)). FAH deficiency results in toxic and carcinogenic metabolites that accumulate in liver hepatocytes and kidney proximal tubules, leading to morbidity and mortality.
The prevalence of HT1 varies across regions worldwide but is estimated at 1 in 100,000 (NIH Medline Plus). Regional differences include Norway, where the prevalence is 1 in 60,000, and Quebec, Canada, at 1 in 16,000. HT1 can lead to liver and kidney failure, liver cancer, and neurological crises. Affected infants fail to gain weight and grow slowly due to poor tolerance to high-protein foods causing diarrhea and vomiting. Untreated children with HT1 usually do not survive past the age of 10 years.
The current standard of care for HT1 involves taking an oral drug (NTBC, 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione) twice daily and maintaining a strict life-long diet low in phenylalanine and tyrosine. NTBC blocks the accumulation of the metabolites leading to HT1 symptoms by a mechanism of action serendipitously discovered during studies on small-molecule herbicides (Chinsky et al., 2017).
According to Cacicedo et al. (2022), some adult patients fail to respond to NTBC treatment, and some NTBC-treated children do not develop normally and can be afflicted with neurocognitive problems. Cacicedo et al. investigated an alternative treatment option that replaces NTBC treatment with the administration of mRNA encoding the wild-type mRNA for FAH. As discussed in this blog, there are three key features of their novel approach to protein replacement therapy for HT1 disease:
- Use of TriLink’s CleanCap® reagent to prepare highly pure FAH mRNA efficiently
- Target hepatocytes with FAH mRNA in a lipid nanoparticle (LNP)
- Use of a well-established FAH-deficient mouse model of human HT1 disease
FAH mRNA Sequence Design, Preparation, and LNP Delivery
Advances in mRNA technology have established that translational efficiency and half-life of an in vitro translated (IVT) mRNA are significantly influenced by codon selection, nucleoside composition, and both the 5’- and 3’-untranslated regions (UTRs) flanking the mRNA of interest (Leppek et al.). Although particulars such as high GC content/U-depletion and modified versus unmodified nucleoside composition are relatively straightforward to optimize empirically, selecting 5’- and 3’-UTRs relies more on adopting experimental findings reported in the scientific literature or patents (e.g., Baumhof, 2019).
With these factors in mind, and as depicted here, Cacicedo et al. employed an optimized DNA template to synthesize IVT FAH mRNA. They maximized GC content and utilized previously successful 5’ and 3’ UTR sequences (Gebre et al., 2021). They employed TriLink’s CleanCap® reagent and unmodified NTPs for co-transcriptional IVT synthesis (Henderson et al.) of FAH mRNA. 
Formulation of mRNAs in LNPs was performed at Acuitas Therapeutics (Thess et al. and Pardi et al.) using a self-assembly process: a solution of mRNA is rapidly mixed with a mixture of lipids comprised of an ionizable cationic lipid, phosphatidylcholine, cholesterol, and PEG-lipid. These LNPs are known to undergo ApoE-mediated endocytosis by liver hepatocytes in vivo and release mRNA cargo into the cytoplasm (Thess et al.).
To determine liver hepatocyte uptake in FAH-deficient mice, which have compromised liver function (see below), Cacicedo et al. first used luciferase (Luc) mRNA formulated in LNPs. Luc LNP signals after 6 hours were observed predominantly in the liver following tail vein injection but were also seen after intramuscular injection, albeit to a lesser extent. Importantly, this demonstrated transport of Luc mRNA-LNPs to FAH-deficient compromised liver hepatocytes via the bloodstream and protein expression at a distant site, consistent with delivery to hepatocytes in healthy mice (Pardi et al.).
HT1 Disease Model in FAH-Deficient Mice
Development of a FAH-deficient mouse model that closely mimics the metabolic disorder and lethality of HT1 in humans was reported in 2001 by Aponte et al.
The FAH-deficient mouse harbor a single N-ethyl-N-nitrosourea–induced point mutation in the FAH gene that alters FAH mRNA splicing, ultimately leading to the formation of a truncated, unstable FAH protein that is degraded. Heterozygous breeding is used to expand the mouse colony, and then homozygote males and females are bred for experiments. To avoid early postnatal lethality of FAH-deficient offspring, pregnant and nursing females drink water supplemented with NTBC. FAH-deficient neonates die within 24 hours of birth if NTBC is not continually administered in the mothers’ drinking water when pregnant due to acute liver failure.
This mouse model is now well-established for studies of human HT1 disease. Cacicedo et al. used it to investigate the feasibility of FAH protein replacement therapy by administration of LNP-encapsulated FAH mRNA.
Stable FAH Protein Synthesis After FAH mRNA-LNP Administration
First, the livers of FAH-deficient and WT mice were dissected 24 hours after single tail vein injections to demonstrate FAH protein production in the target organ. FAH was quantified by Western blot analysis. Approximately 50% of WT FAH protein levels were observed in livers of FAH-deficient mice injected with FAH mRNA-LNP. Notably, a substantial quantity of FAH protein (~30% of WT levels) was still detected in livers 4 days post-injection. Control livers (PBS-injected, FAH-deficient mice ± NTBC) lacked detectable FAH protein.
After establishing successful FAH liver expression, FAH-deficient mice were subjected to one of the following three regimens:
- FAH-deficient mice received NTBC supplementation without interruption and received a sham injection of PBS (NTBC+PBS+);
- NTBC supplementation was withdrawn on day 5 prior to treatment, and PBS was injected on day 0 (NTBC-PBS+);
- NTBC supplementation was stopped on day 5 prior to treatment, and a single dose of LNP-formulated therapeutic FAH mRNA (NTBC-mRNA+) was injected intravenously on day 0.
After injections, blood and liver samples were collected on days 1, 2, and 4. The therapeutic effect of FAH mRNA administration was shown by downstream metabolite differences. Two metabolites typically elevated in HT1 disease (SA and TYR) were reduced in the experimental group. Importantly, levels of these metabolites were also equivalent to physiological levels found in WT mice. These findings highlight the efficacy of FAH mRNA-LNP in promoting physiologic metabolism.
Additional experiments further evaluated the mRNA-LNP treatment. Over a 21-day period, repeated mRNA-LNP injections 5 days apart successfully maintained body weight and significantly improved levels of multiple metabolites: serum SA levels were comparable with NTBC-treated mice; TYR reached physiologic levels (which, interestingly, is not seen with just NTBC treatment). Comparisons of intramuscular and intravenous injections showed that as little as ≤2.5 % of WT FAH protein levels was achieved after repeated intramuscular administration, yet this amount was sufficient to rescue FAH-deficient mice from premature death.
Taken together, these findings demonstrate FAH protein expression in livers of FAH-deficient mice following a single FAH mRNA-LNP injection and a concomitant reduction in multiple toxic serum metabolites. Importantly, the mRNA-LNP treatment affected TYR levels, which was not observed in mice on an NTBC diet. Further, the findings demonstrate that repeated injections were safe, tolerated, and effective at normalizing metabolism and that intramuscular mRNA-LNP injections may also be amenable as a treatment for HT1.
Concluding Comments
Cacicedo et al. concluded that the above findings support a clinical trial of periodic low-dose intramuscular administration of FAH mRNA-LNP versus standard of care NTBC treatment of HT1 patients.
Others have used gene therapy for HT1 to achieve WT FAH-gene insertion into genomic DNA of FAH-deficient mice using an adeno-associated virus (AAV) vector (Paulk et al.).
The use of IVT mRNA obviates possible insertional mutagenesis by AAV, which is of continuing concern to the gene therapy community (Mullard 2021).
The practicality of life-long HT1 therapy with synthetic FAH mRNA could benefit from advances in strategies for lower doses (e.g., self-amplifying RNA), longer duration of protein expression (e.g., modified nucleosides or circular RNA), and oral administration (e.g., synthetic nanoparticles). Such advancements would improve the clinical utility of mRNA-based protein replacement therapies for other inherited metabolic diseases (Prieve et al., 2018).
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