Unnatural Amino Acid‐Based Ionic Liquid Enables Oral Treatment of Nonsense Mutation Disease in Mice

Abstract This investigation addresses the challenge of suboptimal unnatural amino acid (UAA) utilization in the site‐specific suppression of nonsense mutations through genetic code expansion, which is crucial for protein restoration and precise property tailoring. A facile and economical oral liquid formulation is developed by converting UAAs into ionic liquids, significantly enhancing their bioavailability and tissue accumulation. Empirical data reveal a 10‐fold increase in bioavailability and up to a 13‐fold rise in focal tissue accumulation, alongside marked improvements in UAA incorporation efficiency. A 4‐week oral administration in mdx mice, a model for Duchenne muscular dystrophy (DMD), demonstrates the formulation's unprecedented therapeutic potential, with up to 40% dystrophin expression restoration and 75% recovery of normal fiber functions, surpassing existing treatments and exhibiting substantial long‐term safety. This study presents a potent oral dosage form that dramatically improves UAA incorporation into target proteins in vivo, offering a significant advance in the treatment of nonsense mutation‐mediated disorders and holding considerable promise for clinical translation.

ion source (AB SCIEX, USA).Infrared (IR) spectroscopic analysis was carried out using a FITR spectrometer (Thermo Fisher, USA) to elucidate the molecular vibrations and chemical bonds.
Furthermore, 1 H NMR spectroscopy was performed on an AVANCE III 400MHz NMR spectrometer (Bruker, Switzerland) to confirm the chemical structure and purity of the UAA-based ILs.
Additionally, the aqueous solubility of the UAAs (NAEK, Anap, and pAcF) and the corresponding UAA-based ILs was determined by measuring the mass of each compound that reached saturation in 100 g of deionized water (ddH2O).This metric provided a quantitative assessment of the solubility enhancements achieved through IL formation.

Cell Culture and Transfection
HEK293T cells (ATCC CRL-11268) were cultured in Dulbecco's Modified Eagle Medium (DMEM, Corning) supplemented with 10% fetal bovine serum (FBS, Gibco) and maintained at 37°C in a humidified atmosphere containing 5% CO2.For the readthrough assay, pCMV-EGFP vectors harboring a site-specific TAA nonsense mutation at position 39 were co-transfected with PylRS-tRNA Pyl , OMeYRS-tRNA Tyr , or AnapRS-tRNA Leu plasmids into HEK293T cells.The transfection was facilitated using Megatran 2.0 reagent (Origene) following the manufacturer's protocol.Six hours post-transfection, the cell culture medium was replaced with fresh DMEM supplemented with NAEK (ChNAEK) /pAcF (ChpAcF) (1mM) or Anap (ChAnap) (100 μM).The cells were then incubated for an additional 48 hours to allow for sufficient expression and readthrough of the mutated GFP.Post-incubation, GFP fluorescence was assessed using a fluorescence microscope (Nikon) to qualitatively visualize the efficacy of the nonsense mutation suppression by the UAA-based ILs.Each treatment group was conducted in triplicate to ensure the reliability and reproducibility of the results.

Cell Viability Assay and Cell Growth Curve
To evaluate the impact of UAA-based ionic liquids (ILs) on cellular viability and proliferation, a series of preliminary preparations and dilutions were conducted.Initially, precise quantities of UAA-based ILs were weighed and subsequently dissolved in water to create three distinct 50 mM aqueous solutions of the UAA-based ILs.These solutions served as stock concentrations from which further dilutions were made.Subsequently, the culture medium for 293T cells was supplemented with varying concentrations of the UAA-based ILs.A gradient of dilutions was prepared to encompass a comprehensive range of concentrations, including 0 mM (control), 0.5 mM, 1.5 mM, 2 mM, 4 mM, 6 mM, and 8 mM.These varying concentrations were meticulously prepared to assess the cellular response across a spectrum of IL exposure levels.The prepared media containing the specified concentrations of UAA-based ILs were then applied to 293T cell cultures.Following treatment, cell viability assays and growth curve analyses will be conducted at designated time points to quantitatively assess the cytotoxicity and proliferative effects of the UAA-based ILs.

Cell Planking
Cells from 10cm dishes were digested into single cells using 1 mL of 0.25% Trypsin-EDTA and resuspended, and the cell count was determined using a cell counter.The cells were then seeded into a six-well plate at a density of 3×10 5 cells per well and incubated overnight at 37°C.Upon reaching about 70% confluence, transfection was performed.Cells were treated with medium containing different concentrations of UAA-based ILs, observed for 48 hours for cell status and viability.After 48 hours, cells were digested, collected, and counted using a cell counter to create a fitting curve of cell number and UAA-based ILs concentration.

Cell Growth Curve
Two experimental groups were established, one with UAA aqueous solution and the other with an equal molar amount of UAA-based ILs added to 293T cells.Three replicate wells were cultured for each group.Cells from each group were collected and counted at 0 h, 6 h, 24 h, 48 h, 56 h, and 72 h to plot cell growth curves under different conditions.

Cellular Internalization of UAAs
293T cells were cultured in six-well plates at 4×10 5 /well.Six groups were established, and three replicate wells of cells were used for each group.The medium was replaced with fresh DMEM containing NAEK/pAcF (1 mM), Anap (100 μM), Ch-NAEK/Ch-pAcF (1 mM), or Ch-Anap (100 μM).Cells were incubated for an additional 6 and 12 hours.Cells from each group were collected, washed with PBS three times, then resuspended in 100 μL dH2O for 5 minutes, and centrifuged at 12000 g for 10 minutes.The UAA concentration in the supernatant from each group was quantified using high-performance liquid chromatography-mass spectrometry (HPLC-MS) (Waters ACQUITY UPLC H-Class, Waters Xevo TQ-S).

Flow Cytometry and FACS
To quantify the proportion of GFP-positive HEK293T cells indicative of successful readthrough of the targeted nonsense mutation, flow cytometry and fluorescence-activated cell sorting (FACS) were employed 48 hours post-medium change.GFP-positive HEK293T cells were collected 48 hours after medium change, digested into single cells using trypsin/EDTA, and washed three times with PBS.The cells were then transferred into 1.5 mL ep tubes containing 500 μL PBS and analyzed on a BD FACSAria (BD Biosciences) with appropriate filter settings (488nm-FITC coherent sapphire laser for GFP excitation).Data were analyzed using FlowJo software.

qRT-PCR Analysis
To quantitatively evaluate the gene expression levels in the tibialis anterior muscle tissues of both transgenic and wild-type (WT) mice, a rigorous qRT-PCR analysis was conducted.Total RNA from transgenic or WT mice's tibialis anterior muscle was extracted using the Quick-RNA MicroPrep kit (Zymo Research), reverse-transcribed, and then subjected to quantitative PCR with the LightCycler® System (Roche).Target gene transcripts were normalized to internal GAPDH control, and relative gene expression fold was calculated using the 2−ΔΔCt method.qRT-PCR primer details are provided in Table S2.

Genotype Identification of Transgenic Mice by Tail DNA PCR Analysis
To ascertain the genotypes of the three distinct transgenic mouse lines, a comprehensive PCR analysis of tail DNA was conducted.Tail samples from young generation mice were collected into 1.5 mL centrifuge tubes on ice, and 500 μL of cracking solution (containing 50 μg protease) was added.The subsequent DNA extraction was meticulously performed following the standard protocol provided by the genomic DNA extraction kit (TIANGEN), ensuring the purity and quality of the isolated DNA.Post-extraction, the concentration of the obtained DNA was accurately measured to determine its suitability for PCR amplification.A precise quantity of 200 ng of DNA from each sample was then used as the template for the PCR analysis.The PCR was conducted using specific primer sequences designed to target the unique genetic modifications of each transgenic mouse line.These primer sequences, integral to the accurate identification of the transgenic genotypes, are comprehensively listed in Supplementary Table S3.

Bioavailability and Tissue Distribution of UAAs from UAA Solutions/UAA-based ILs
In evaluating the bioavailability and tissue distribution of unnatural amino acids (UAAs) and their respective ionic liquid (IL) derivatives, wild-type C57BL/6 mice were orally administered with UAAs or equimolar doses of UAA-based ILs.Serum samples were collected at specified intervals (0 h to 22 h post-administration) for UAA quantification.The protein in the serum was precipitated using acetonitrile, and the UAA-containing supernatants were collected for analysis at different time points.Additionally, various tissues were harvested 9 hours post-administration for UAA quantification.High-performance liquid chromatography-mass spectrometry (HPLC-MS) (Waters ACQUITY UPLC H-Class, Waters Xevo TQ-S) was employed for UAA concentration analysis as per established protocols.Pharmacokinetic parameters were calculated using the DAS (Data Analysis System) software package (Version 2.0, BioGuider Co., Shanghai, China) according to a one-compartment model.

Western Blotting Analysis
Post one, two, or four weeks of oral administration of ChNAEK or NAEK, tissue samples from the mice were homogenized, and cellular debris was removed via centrifugation at 4°C.Proteins from different groups were extracted using lysis buffer for 10 minutes and then quantified using a BCA assay (Thermo).A total of 50 µg of protein from each sample was boiled with loading buffer, separated on 4-12% Nu-PAGE (Invitrogen), and transferred onto a polyvinylidene difluoride membrane overnight.The membrane was blocked with 5% (v/v) non-fat milk in TBST (50 mM Tris-HCl, 150mM NaCl, and 0.02% Tween-20, pH 7.5) at room temperature for 1 hour and then incubated with rabbit or mouse polyclonal antibodies overnight at 4°C.Antibodies used included anti-dystrophin (1:500, ab7164, Abcam), anti-vinculin (1:3,000, ab91459, Abcam), anti-GFP (1:3,000, 66002-1-Ig, Proteintech), and anti-mCherry (ab183628) diluted in TBST containing 5% (v/v) defatted milk.Membranes were washed three times with TBST, incubated with horseradish peroxidase-conjugated goat anti-rabbit/mouse IgG (1:3,000) at room temperature for 1 hour, developed using an enhanced chemiluminescent detection kit (Millipore), and visualized using an automated chemiluminescent image analysis system (Tanon).The integrated optical density (IOD) of western blot bands was semi-quantified using the ImagePro Plus software package.

AAV Construction and Administration
The AAV-pylRS-tRNA Pyl UUA construct comprised four tRNA copies under a U6 promoter and a full-length PylRS with a Myc tag under the CMV promoter at the C-terminal.As a control, the AAV-CMV-mScarlet-3-Myc flag excluded the PylRS-tRNA.Both were packaged into the AAV2/9 serotype, with the AAV-PylRS-tRNA vector having a titre of 4.10×10 13 viral genomes (vg)/mL.Male mdx mice, aged 6-8 weeks, were anesthetized for the topical administration of 50 μL each of AAV-PylRS-tRNAPyl and control AAV into their right and left tibialis anterior muscles, respectively.Daily oral administration of either NAEK (30 mg) or an equimolar dose of ChNAEK followed.At 1, 2, and 4 weeks post-administration, some mice were euthanized for tissue and blood analyses.The body weight and survival rates were monitored and recorded.

Immunofluorescence Staining
Tibialis anterior muscles from various groups were fixed in 4% paraformaldehyde (PFA) at room temperature for an hour, then immersed in 30% (w/v) sucrose until fully submerged before embedding and freezing in Optimal Cutting Temperature (OCT) compound.Using a cryostat at -20 °C, serial 12-μm sections were obtained from the embedded tissue.These sections were blocked with 5% normal donkey serum in PBST for 30 min and incubated with an anti-dystrophin antibody (1:500, ab15277, Abcam) diluted in blocking buffer overnight at 4 °C.Post-incubation, sections were treated with secondary goat anti-rabbit IgG Alexa Fluor 488 (1:400, A-11037, LifeTech) for an hour at room temperature, stained with 0.5 µg mL -1 Hoechst, and mounted.Photographs of the stained sections were taken under a Nikon Ti-S microscope, and Dystrophin-positive cells were quantified to assess restoration efficiency.

Histological Analysis
For histopathological examination, tibialis anterior muscle, liver, and stomach tissues were harvested from various groups of mice and fixed in 4% paraformaldehyde (PFA) for 48 hours.The tissues were then subjected to sequential dehydration through ethanol gradients (70%, 95%, and 100%), cleared in xylene, and finally embedded in paraffin.Sections of 10-μm thickness were prepared and stained with Hematoxylin and Eosin (H&E) to assess tissue architecture and any pathological changes.The stained sections were scanned using a NanoZoomer Slice scanner, and the histological morphology was comparatively analyzed across the different mouse cohorts.

Grip Strength Test
The grip strength meter (Saiangsi) was used to measure the muscle strength of all four limbs in each group.Randomly picked mice from each group (wild-type C57BL/6, mdx, and mdx administered with NAEK or ChNAEK for 1, 2, and 4 weeks) were weighed and induced to grab the shelf connected to the meter by lifting them by the tail.The peak force in grams was recorded, with each mouse undergoing 4-6 trials and the average force noted.All measurements were performed blindly without knowledge of the group information.

Body Weight and Survival Rate
The body weight of each mouse was monitored daily throughout the experimental period.
The survival rates of wild-type C57BL/6 mice orally administered with NAEK aqueous solution or ChNAEK formulation were observed, with untreated mdx mice serving as controls.This longitudinal tracking provided insights into the general health and potential systemic effects of the treatments.
Table S3.The primer sequences for the three kinds of transgenic mice tail DNA PCR analysis.

Figure
Figure S2 The Mass spectra of ChNAEK.The peak at 466 is due to the clusting of 2Choline 1NAEK.Clusters of the [(AB)nA] + form consisting of cation A and anion B are reportedly one of the typical features of FAB-MS spectroscopy of ion beams at room temperature.

Figure S5 .
Figure S5.Effect of UAA-based ILs and UAAs on read-through rate of GFP 39TAA with the corresponding suppression system by fluorescence flow analysis (n=3).The y axes represent the number of the cells, and the x axes shows the GFP fluorescence intensity (in arbitrary units).P3 represents the GFP restoration-positive cell percentage.

Figure S7 .
Figure S7.Verification of the AnapRS/tRNACUA transgenic mice (A) and OMeYRS/tRNAUUA transgenic mice (B) by PCR analysis of mouse tail DNA.(A) Primer 1 targeting the sequence at 500 bp and Primer 2 targeting the sequence at 300 bp were used (n=3).The seven progenies were from the F1 generation.The results showed that Progeny 6 and 7 were positive.(B) Primer 1 targeting the sequence at 2223 bp and Primer 2 targeting the sequence at 2518 bp were used (n=3).

Figure S8 .
Figure S8.The western blot analysis of report protein in mice muscle to evaluate the restoration efficiency produced by the incorporation of UAAs.(A) The M-cherry 154TAA restoration of the transgenic mice harbored with OMeYRS-(U6-tRNAUUA)4-M-cherry 154TAA gene after intramuscular injection ChpAcF (the same molar dose of 20mg or 30 mg pAcF) and 20 mg pAcF aqueous suspention every day for 1 week.When the same dose of pAcF (20 mg) reached the local tissue, the mCherry expression of ChpAcF was significantly higher than that of the suspension group (n=2).(B) The GFP 39TAG restoration of the transgenic mice harbored withAnapRS-(H1-tRNACUA)8-GFP 39TAG gene after intramuscular injection ChAnap (the same molar dose of 30 mg Anap) or 30 mg Anap aqueous suspention every day for 1 week (n=2).When the same dose of Anap (30 mg) reached the focal muscle tissue, the GFP expression of ChAnap was significantly higher than that of the suspension group.The bigger dose of UAA, the higher reporter protein restoration.

Figure S9 .
Figure S9.The western blot analysis of the report protein restoration in focal tissues of the transgenic mice after one-week oral treatment.(A) The M-cherry 154TAA restoration in heart, brain, muscle and stomach tissue of the transgenic mice treated with same molar dose of ChpAcF or pAcF aqueous suspension (30mg).(B) The GFP 39TAG restoration in heart, brain, muscle and stomach tissue of the transgenic mice treated with same molar dose of ChAnap or Anap aqueoussuspension (20 mg).The results showed that the expression of M-cherry (pAcF groups) or GFP (Anap groups) in these focal tissues in UAA-based ILs groups were significantly higher than that of free UAA aqueous suspension groups, respectively, which were consistent with the trend of UAAs accumulation in tissues after oral administration.

Figure S10 .
Figure S10.The efficiency of pAcF incorporation evaluated in muscle of the OMeYRS-tRNA-M-cherry 154TAA transgenic mice after oral administration by western blot analysis.

Figure S11 .
Figure S11.The western blot analysis of dystrophin restoration from the muscle of mdx mice after one-week injection treatment.(A) Same molar dose of ChNAEK or NAEK aqueous solution (20 mg) was injection intramuscularly every two days (n=2).(B) Same molar dose of ChNAEK or NAEK aqueous solution (30 mg) was injection intraperitoneally every two days (n=2).

Figure S12 .
Figure S12.Survival rates of four different mice groups within 30 days (n=4).The results were statistical analyzed using GraphPad Prism Software (Version 8.0, GraphPad Software, San Diego, CA) and presented as the mean ± S.D.