1. Introduction
Fabry disease is the second-most prevalent lysosomal storage disorder (LSD) after Gaucher disease [
1]. The disease is caused by mutations in the alpha-galactosidase (
GLA) gene, which result in a deficiency of the encoded lysosomal enzyme α-galactosidase A (α-Gal A) [
2]. Abnormal enzymatic activity leads to the progressive accumulation of globotriaosylsphingosine (Lyso-Gb3) and other glycosphingolipids in the plasma and various tissues [
3]. This ultimately triggers a series of cellular and tissue responses that cause damage to multiple organs, such as the heart, liver, spleen, and kidneys [
4].
Enzyme replacement therapy (ERT) and pharmacologic chaperones have been developed for Fabry disease [
5,
6]. ERT with either agalsidase beta (1 mg/kg every other week) or agalsidase alfa (0.2 mg/kg every other week) is available for the treatment of Fabry patients [
7]. However, ERT is also subject to significant drawbacks. For example, considerable clinical variability is observed among treated patients, and efficacy is largely dependent on the age at which the therapy is initiated and the development of antibodies against infused enzymes [
8]. Pharmacologic chaperone, on the other hand, is only effective for certain patients with specific mutant isoforms of α-Gal A [
6]. As Fabry disease is a genetic disease, recent advances in gene therapy provide more options for treating the disease [
9]. In murine and non-human primate models of Fabry disease, the delivery of an mRNA encoding α-Gal A has been shown to reduce Lyso-Gb3 and globotriaosylceramide (Gb3) levels in the heart and kidney [
10], suggesting the potential of using the mRNA technique for Fabry disease treatment. Metabolic disorders were observed in Fabry patients, accompanied by some complications such as malignant ventricular arrhythmias, sudden cardiac death, end-stage renal failure, and stroke [
11]. Fabry’s disease patients also experience oxidative stress, impaired energy metabolism, changes in membrane lipids, disrupted cell transport, and impaired autophagy [
2]. Previous studies with patients injected with mRNA vaccines such as those for COVID-19 have shown metabolome alterations [
12]. On the other hand, whether the metabolic profile is changed with the treatment of mRNA injection-based protein supplementation is largely unclear.
In the present study, the LNP-mRNA strategy was adapted by preparing an mRNA encoding α-Gal A for the treatment of Fabry disease in mice. To clarify the mechanistic basis for the effects of such LNP-mRNA treatment, the plasma metabolome was analyzed. A total of 20 differential metabolites, which are mainly enriched in the arachidonic acid metabolism, alanine, aspartate and glutamate metabolism, and tricarboxylic acid cycle pathway, were identified. The results revealed that mRNA therapy can sufficiently reduce inflammation, alter amino acid metabolism and energy metabolic pathways, and alleviate oxidative stress in these Fabry model mice.
2. Materials and Methods
2.1. Chemicals
Dulbecco’s Modified Eagle Medium (DMEM), glutamine, glucose, fetal bovine serum, and antibiotics were purchased from Gibco (Gibco, Grand Island, NY, USA). Anti-α-Gal A (ab168341, 1:5000), anti-β-actin (ab8226, 1:1000), and secondary antibodies (ab6785, 1:10,000) were purchased from Abcam (Abcam, Cambridge, UK). α-Gal A activity assay kit was purchased from Biovision (Milpitas, CA, USA). Globotriaosylsphingosine (Lyso-Gb3) was purchased from Matreya (Pleasant Gap, PA, USA). Lipofectamine 2000 was purchased from Invitrogen (Carlsbad, CA, USA). Liquid chromatography–mass spectrometry (LC–MS)-grade methanol and MS-grade acetonitrile were purchased from Thermo Fisher Scientific (Waltham, MA, USA). OASIS MCX cartridges (30 mg, 60 mm) were purchased from Waters Corp. (Milford, MA, USA). Formic Acid (98.0% for LC–MS) was purchased from Tokyo Chemical Industry (Shanghai, China). The HEK293 cell line was obtained from the American Type Culture Collection (ATCC). ALT, AST, NF-κB, IL-6, and TNF-α ELISA kits were obtained from Enzyme Link Biotechnology Co., Ltd. (Shanghai, China).
2.2. Synthesis and Characterization of mRNA and Lipid Nanoparticles
The mouse version of the
GLA gene was from the mouse database in the NCBI (accession number NM_013463.3). The
GLA gene was used in Fabry mouse experiments. The mRNA and LNPs used for this study were produced based on the Liverna Therapeutics platform [
13]. Briefly, the mRNA molecules were synthesized using an optimized T7 RNA polymerase-mediated transcription reaction with complete replacement of uridine by N1-methyl-pseudouridine in vitro. The reaction included a DNA template containing the open reading frame of the α-Gal A gene flanked by 5′/3′ untranslated region (UTR) sequences and a poly-A tail. In vitro transcribed (IVT) mRNAs were encapsulated in LNPs according to a modified procedure wherein an ethanolic lipid mixture of ionizable cationic lipids, phosphatidylcholine, cholesterol, and polyethylene glycol-lipid was rapidly mixed with an aqueous solution containing the mRNA products. Analytical characterization of the resultant product was conducted, including the determination of particle size, polydispersity, encapsulation, endotoxin levels, and bioburden.
2.3. Cell Culture and Transfection
HEK293 cells were cultured in DMEM supplemented with 2 mM glutamine, 4.5 g/L glucose, 10% fetal bovine serum, and antibiotics. The transfection of these cells with cDNA or mRNA (4 μg) (human version GLA gene from the NCBI database with accession number AB019551.1). was performed using Lipofectamine 2000 when cells were 50~70% confluent. In the control group, only Lipofectamine 2000 was used for transfection. Before harvesting, transfected cells were cultured in the same growth medium for 24 h.
2.4. Western Blotting
Western blotting was used to assess the expression of α-Gal A and β-actin in HEK293 cells. Briefly, an RIPA buffer was used to extract the total protein from these cells, after which protein levels were quantified with a BCA kit. Total proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were then incubated with appropriate primary and secondary antibodies, after which protein bands were detected with the G: Box Chemi XRQ Imaging System.
2.5. Animal Model Experiments
GLA gene knockout (KO) mice were used as Fabry disease model animals. These mice were generated in the Shanghai Model Organisms Center (Shanghai, China) using CRISPR/Cas9 technology. Genotyping was performed using DNA from collected tail samples to confirm the loss of the whole precursor-Gal gene encoding sequence (1400 bp). All mice used in the study were housed and bred in the Shanghai Model Organisms Center (Shanghai, China). During all experiments, mice had free access to clean food and water in a facility with a 12-h light/dark cycle. Animal experiments were authorized by the Animal Care and Use Committee of Shanghai Nanfang Model Biotechnology Co., Ltd (Shanghai, China) (2021-0012). Mice were randomly divided into three groups: a control group (PBS), a low-dose mRNA group (1 mg/kg), and a high-dose mRNA group (3 mg/kg). The mRNA encapsulated by LNP was injected into the mouse body via tail vein injection. The plasma, heart, liver, and kidneys of mice were collected at different times for testing α-Gal A activity and Lyso-Gb3 levels.
Male mice (wild-type) were housed in the Shanghai Model Organisms Center (Shanghai, China). The firefly luciferase mRNA (NCBI accession number M15077.1) encapsulated in LNP (1 mg/kg) was injected into the mouse body via tail vein injection. Fluorescence was observed at different locations and time points (0, 2, 6, 12, 24, 48, and 72 h) using a small animal imager (Clinx, IVScope 8000, Shanghai, China).
2.6. α-Gal A Activity Assay
A-Gal A activity assay was performed according to published instructions [
10]. Briefly, lysates or plasma from mice were mixed with 2-Methylum-belliferyl-a-D-galactopyranoside and 50 mM N-acetylgalactosamine in 100 mM sodium citrate buffer. The reaction mixture was incubated at 37 °C for 2 h. The reaction was stopped by adding 200 μL of stop solution. Fluorescence detection was then performed at the respective excitation and emission wavelengths of 360 nm and 445 nm. α-Gal A activity was expressed as nmol/h/mL plasma or nmol/h/mg total protein.
2.7. Immunohistochemistry
Immunohistochemical (IHC) staining was performed using paraffin-embedded sections of murine heart, liver, and kidney tissues. Slides were deparaffinized, hydrated, and antigen-retrieved, and then treated with primary polyclonal anti-mouse α-Gal A (1:500, Abcam, ab168341) overnight at 4 °C. Samples were then incubated with a secondary antibody (1:1000, Abcam, ab6785) for 1 h at room temperature, after which DAB staining was used to visualize the IHC reaction. Sections were imaged with a digital camera (AxioCam, Carl Zeiss, Inc., Thornwood, NY, USA).
2.8. Lyso-Gb3 Analyses
The Lyso-Gb3 level in plasma, heart, liver, and kidney samples was measured using a liquid chromatography–tandem mass spectrometry (LC-MS/MS) approach. Briefly, homogenized samples were mixed with methanol and extracted with chloroform/methanol (2:1). The mixed samples were centrifuged at 12,000× g for 15 min, and 20 μL of extracted supernatant was injected into the LC-MS/MS system and separated using an acetonitrile-formic acid-methanol gradient on an XSelect Peptide HSS T3 C18 column. Using analyte peak integration, Lyso-Gb3 levels were quantified with reference to prepared standard curves. The MS conditions were set as follows: ESI mode was positive; the spray voltage was 5.5 kV; the sheath was of 40 AU; the auxiliary gas was 12 AU; the capillary temperature was 320 °C; and the collision energy was 33 eV. The following parallel reaction monitoring (PRM) transitions were monitored, with a dwell time of 40 ms: 786.4 > 282.2 (Lyso-Gb3). The mobile phase A consisted of 5% ACN, 95% water, and 0.2% FA, and the mobile phase B consisted of ACN and 0.2% FA. The flow rate was set at 0.3 mL/min. The two mobile phases formed the following gradient: 0~2 min (50%, B), 2~8 min (90%, B), and 8.1~10 min (5%, B).
2.9. Metabonomic Analyses
To precipitate plasma proteins, 50 μL of plasma was mixed with 200 μL of methanol, vortexed for 20 s, and left on ice for 30 min. The mixed samples were centrifuged at 15,000× g for 20 min, and the supernatant was then analyzed using an HPLC-QE Orbitrap-MS/MS approach.
The HPLC system used for this study consisted of an HSS T3 column coupled to an Orbitrap MS instrument (Thermo Fisher Scientific, Waltham, MA, USA). The mobile phase for this study consisted of 0.1% formic acid in water (A) and acetonitrile (B), and the gradient elution settings were as follows: 0 min, 2% B; 1 min, 2% B; 18 min, 100% B; 22 min, 100% B; 22.1 min, 2% B; 25 min, 2% B. A flow rate of 0.3 mL/min was used with an injection volume of 5 μL. During LC/MS experiments, the QE mass spectrometer was used to acquire MS/MS spectra on an information-dependent acquisition (IDA) basis. Electrospray ionization source settings were as follows: Aux gas flow rate = 16 Arb; full ms resolution = 70,000; collision energy = 25 eV in the NCE model; MS/MS resolution = 17,500; and spray voltage = −3.0 kV or 3.6 kV.
Metabolite quantification was performed by using the ABF Converter software (
http://www.reifycs.com/AbfConverter/, accessed on 1 December 2023) to convert raw data into “Analysis Base File” (ABF) files. For data processing, the MSDIAL 2.2.62 software was used for peak detection, deconvolution, and alignment. SIMCA (version 14.1) was used to import the obtained data. Principal component analysis (PCA) and orthogonal partial least squares discrimination analysis (OPLS-DA) analyses were performed on the SIMCA data. Mass values of differential metabolites were retrieved from the Human Metabolome Database (HMDB). MetabolAnalyst 5.0 was used to analyze differential metabolites in clusters and pathway analyses.
2.10. Statistical Analysis
Statistical analyses were performed using SAS 9.2 for Windows (SAS Institute Inc., Cary, NC, USA). All data were presented as means with standard deviations and analyzed using one-way ANOVAs and Duncan’s multiple range test when more than two groups were compared, while the student’s t-test was utilized when only two groups were compared. Statistical significance was defined as p < 0.05.
4. Discussion
Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the
GLA gene encoding α-Gal A. Glycosphingolipids, particularly Gb3 and Lyso-Gb3, accumulate in the context of α-Gal A deficiency [
2]. After the administration of α-Gal mRNA, the activity of α-Gal A significantly increased, which resulted in a reduction in the Lyso-Gb3 level in the plasma, heart, liver, and kidney of the Fabry disease model mice. The levels of Lyso-Gb3 remained low in the Fabry disease mice at 120 h post-administration, highlighting a prolonged α-Gal mRNA therapeutic window. Compared to the study of Zhu et al. [
10], the duration time of our present study is much shorter. The main reason is that mRNA encapsulated by LNP can be metabolized by the body in a relatively short period of time, and as our goal is to explore the effects of messenger ribonucleic acid drugs on the body’s metabolic pathways, we decided to evaluate the metabolome of treated mice within a shorter time frame after the administration of the RNA drug. However, it will be of interest to further investigate metabolomic changes over a longer period, which will shed light on the duration and long-term effects of the mRNA on the metabolome of the entire body.
Lipid accumulation in the liver induces hepatic infiltration by immune cells and consequent inflammation [
14]. Given that α-Gal A activity was detected in the liver of Fabry disease model mice following the intravenous injection of α-Gal A mRNA, the activity of liver transaminases was quantified to assess hepatic injury. Compared with control mice, α-Gal A mRNA treatment resulted in a significant decrease in the AST-to-ALT ratio in the murine liver. Fabry disease is characterized by a chronic inflammatory response, of which the accumulation of Lyso-Gb3 and Gb3, lactosylceramide, glucosylceramide, and other substrates causes an inflammatory response in the liver and other organs. Our current study demonstrated that even low-dose α-Gal A mRNA administration was sufficient to significantly reduce the level of NF-κB in the mouse liver, suggesting that the mRNA can efficiently abrogate liver inflammation in Fabry disease model mice.
HPLC-MS/MS-based metabolomic analysis revealed marked changes in the plasma metabolite profiles of Fabry disease mice following α-Gal A mRNA administration. In addition to the changes in Lyso-Gb3 levels, 19 metabolites exhibited significant changes in abundance following the application of α-Gal A mRNA. Isoprostanes are generated following the peroxidation of arachidonic acid and arachidonic esters of phospholipids in the context of inflammation. In the treatment groups, significant changes in arachidonic acid and phosphatidylinositol levels were noted, demonstrating that such treatment was sufficient to regulate inflammation governed by the phosphatidylinositol signaling system and arachidonic acid metabolism. Interestingly, there is also literature indicating that elevated levels of arachidonic acid and 5-HETE promote inflammatory responses. The increase in arachidonic acid level can promote the elevation of prostaglandin G2, which further raises the level of prostaglandin E2, thereby strengthening the occurrence and development of inflammation [
15]. The reason for the above situation needs to be investigated further. However, a possible reason may be due to the presence of a large amount of lipids in the LNP components.
Metabolites that are significantly enriched in the glycolysis/gluconeogenesis pathways, including alpha-D-glucose, alpha-D-Glucose 1,6-bisphosphate, and fructose 1,6-bisphosphat,e were found to decrease greatly in α-Gal A mRNA-treated mice. Although these energy metabolism-related compounds are generally considered to be suppressed in Fabry model cells [
16], it was also demonstrated that the energy metabolism was shifted toward glycolysis from fatty acid β-oxidation in Fabry model cells, suggesting that the deficiency of the
GLA gene can result in enhanced glycolysis. Similarly, metabolites that are related to amino acid metabolism were reduced in the treatment group. Amino acid metabolism is interwound with carbohydrate and fatty acid metabolisms. Hence, the interference of one metabolic pathway will likely directly or indirectly affect others. We speculate that the recovery of lipid metabolism in the treatment group led to the balancing of the other metabolic pathways, which exhibited a suppressive effect. Such an unexpected profile highlights the necessity of evaluating the consequences of mRNA administration in the in vivo system, as the metabolic processes in the body may differ from those in the cellular model, and different metabolic pathways need to cross-talk to each other and maintain the homeostasis of the full system.
The malfunction of lipid metabolism is a major phenomenon in Fabry disease. However, only two related metabolites, i.e., globotriaosylsphingosine and phosphatidylglyceride 22, were found to be altered in our present metabolomic analysis. Phosphatidylglyceride 22 is not only a component of biomembranes but is also involved in the formation of bile and membrane surfactants and participates in the recognition and signal transduction processes. Its level was found to rapidly accumulate in the bodies of Fabry patients [
17]. The level of phospholipid was also increased in our treatment group, which is different from previous research results. It is likely caused by the presence of cholesterol in the LNP that encapsulates mRNA. Additionally, the lack of major changes in lipid metabolism observed in our current study may be due to the relatively short duration of the experiment. Fabry disease is a lifelong illness, and a significant change in the profile of lipid-related metabolites may require a longer treatment period and possibly multiple injections. A future long-term drug administration study will be needed to clarify this issue.
Compared with the control group, we found that the levels of phosphatidylinositol 16, 17, and 18 were significantly increased in the treatment group. Phosphatidylinositol is composed of 1,2-diacylglycerol phosphate and inositol. Phosphatidylinositol plays a crucial role in cell morphology, metabolic regulation, signal transduction, and various physiological functions [
17]. In terms of signal transduction, phosphatidylinositol transfer protein and phosphatidylinositol are important upstream regulatory factors involved in the regulation of the Hippo signaling pathway [
18] and the phosphatidylinositol 3-kinase (PI3K) signaling pathway. Studies have shown that the PI3K signaling pathway plays an important role in regulating cellular lipid metabolism [
19]. The significant increase in phosphatidylinositol in the treatment group is likely due to the activation of the PI3K signaling pathway, which further regulates lipid metabolism and related pathways. Further study is warranted to clarify whether the PI3K signaling pathway is activated in mRNA-treated mice and involved in the regulation of lipid metabolism.
Studies have shown that polyunsaturated fatty acids are sensitive to oxidation [
20], and low-density lipoprotein oxidation is a critical step in the development of atherosclerosis [
21]. Fabry disease patients harbor increased lipid, protein oxidation, and inflammation levels, together with decreased levels of antioxidant defenses such as heme oxygenase 1 [
22]. Moreover, endothelial dysfunction was reported in Fabry disease patients [
23]. Oxidative stress was initially assumed in Fabry disease based on elevated markers of inflammation and oxidative damage. Shen et al. observed a positive association between Gb3 levels and increased production of reactive oxygen species in cultured vascular endothelial cells [
24]. Furthermore, it was observed that endothelial cells exposed to plasma obtained from individuals with Fabry disease exhibited a significantly elevated production of reactive oxygen species in comparison to those treated with plasma derived from healthy individuals. The greatly suppressed level of polyunsaturated fatty acid in the mRNA-treated mice hence suggested that the therapeutic agent not only recovered the function of α-Gal A, but may also improve the redox environment in vivo. However, how such a change affects the oxidative status of Fabry disease mice remains unclear.
LNP is a novel drug delivery system that can safely and effectively deliver nucleic acids, such as DNA or RNA, to target cells. Compared with other nano-drug delivery systems, LNP has a higher nucleic acid encapsulation rate and transfection capability. In the meantime, LNP has lower cytotoxicity and immunogenicity compared to viral vectors. However, there have been reports indicating that cationic lipids have potential toxicity and immunogenicity [
25]. Further, the storage conditions of LNP are relatively harsh, and the stability is poor. LNP is generally composed of ionizable lipids, phospholipids, cholesterol, and polyethylene glycol (PEG) lipids. Studies have shown that LNP is mainly distributed in the liver (81%), and the expression level can reach up to 96% through intravenous injection into mice [
26]. We injected luciferase LNP into mice via intravenous injection to study its tissue distribution. The result showed that the luciferase LNP continued to be expressed for 48 h, and the majority of luciferases are distributed in the liver, with a small portion also found in the spleen, kidneys, and heart. In fact, it has been demonstrated that by changing the proportion of LNP, the LNP-mRNA can be accurately transported to organs such as the lungs, spleen, and liver of mice [
27]. On the other hand, due to the presence of lipids in LNP, injection into the body may affect the metabolism of endogenous lipids. Hence, long-term and more systematic investigations such as monthly dosing cycles, application of different dosages, and long-term metabolomics studies of urine in addition to plasma will need to be carried out to understand and evaluate the LNP-mRNA system more thoroughly in in vivo Fabry’s disease systems.
In summary, a significant impact occurs in the glycolysis/gluconeogenesis pathways, amino acid metabolism, and arachidonic acid metabolism of mice after administration of α-Gal A mRNA (
Figure 6). Our results highlighted the efficacy of α-Gal A mRNA treatment, emphasizing its potential as a therapeutic intervention for the management of Fabry disease. Moreover, metabolomics analytical techniques were applied to investigate the metabolome alterations in Fabry disease model mice after mRNA nucleic acid injection, providing the basis for a more comprehensive evaluation of the efficacy and safety of this kind of novel treatment in the future.