Subcutaneous DG9-PMO administration enhances survival and improves motor function and muscle strength. To examine whether DG9 peptide can increase the efficacy of PMO, we conjugated it to an 18-mer PMO with a sequence identical to nusinersen. The DG9-PMO, unconjugated PMO, and MOE were injected into SMA model mice [Cg-Tg(SMN2)2HungSmn1tm1Hung/J; abbreviated as Smn–/– SMN2Tg/Tg; commonly called the Taiwanese model]. We also used an arginine-rich peptide (R6G) that is currently being explored in clinical trials for Duchenne muscular dystrophy (DMD) and used in preclinical studies for Hutchinson-Gilford progeria syndrome (37, 38). The benchmark control peptide R6G was conjugated to the same PMO sequence used in our study (R6G-PMO). We used an SMA mouse model that exhibits a severe phenotype and has a median survival of 8 days (39, 40). We subcutaneously injected mice with the AOs or saline (nontreated, NT) administered at postnatal day 0 (PD0) at 2 doses: 40 mg/kg or 80 mg/kg. In our preliminary experiments, we also tested lower doses of 10 and 20 mg/kg (data not shown). We chose higher doses of 40 and 80 mg/kg to demonstrate the safety profile and dose-dependent effect of AOs. The survival was recorded until a humane endpoint was reached. For the 40 mg/kg doses, the median survival was 12 days for unconjugated PMO, 15.5 days for MOE, and 17 days for R6G-PMO, which was increased to 58 days for DG9-PMO treatment (Figure 1A and Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.160516DS1). The median survival increased to 57 days for unconjugated PMO, 64 days for MOE, and 121 days for DG9-PMO at the higher dose of 80 mg/kg (Figure 1A). DG9-PMO significantly and more effectively increased the life span of SMA mice than unmodified PMO and R6G-PMO. In spite of no statistical difference between DG9-PMO and MOE groups, the median survival of DG9-PMO still was higher than MOE, as only 1 mouse was alive until PD200 following a 40 mg/kg injection of MOE.

Subcutaneous administration of DG9-PMO at PD0 extends survival and improves motor function in severe SMA mice. (A) Survival curves of heterozygous mice (Smn+/– SMN2Tg/–) (Hets), nontreated (NT) mice, unconjugated PMO (PMO), DG9-PMO, and MOE injected at PD0 at a dose of either 40 or 80 mg/kg. For 40 mg/kg studies, n = 15 (Hets), n = 22 (NT), n = 49 (unconjugated PMO), n = 14 (DG9-PMO), n = 29 (MOE). For 80 mg/kg studies, n = 15 (Hets), n = 22 (NT), n = 7 (unconjugated PMO), n = 6 (DG9-PMO), n = 4 (MOE) (*P < 0.05, **P < 0.01, ****P ≤ 0.0001, log-rank Mantel-Cox test). (B) Weight of mice at PD7 administered with either 40 or 80 mg/kg doses. Each dot (symbol) indicates a neonatal pup. (C) Hind limb suspension assay (HLS). Mice were treated with 40 mg/kg AOs at PD0. Score is based on the position of the hind limbs when suspended from a tube (n = 12–20 mice per group). (D) Righting reflex test. Mice were treated with 40 mg/kg AOs at PD0. The ability of mice to right themselves on their paws was measured every alternate day PD2 to PD20 (left) (n = 12–20 mice per group). The mean righting reflex time at PD6 and PD8 was also indicated (right: box-and-whiskers plots). Box edges, 25th and 75th percentiles; central line, median; whiskers, range. (E) Forelimb grip strength measured in adult mice at PD30 and PD60 from 40 mg/kg treatment groups normalized to the body weight. In B, D (box-and-whisker plots), and E, 1-way ANOVA followed by post hoc Tukey’s test was performed. Single symbols represent P < 0.05, double symbols represent P < 0.01, and triple symbols represent P < 0.005. In C and D (left graph), 2-way ANOVA followed by Holm-Šídák multiple comparison was performed. Single symbols represent P < 0.03; double symbols represent P < 0.002; triple symbols represent P < 0.0002. *NT, #PMO, @DG9-PMO, &MOE. Error bars: SEM.

At both doses, DG9-PMO–treated mice were significantly heavier than the NT and unconjugated PMO–treated littermates at PD7, with no significant difference when compared to the age-matched Hets that were used as healthy controls (Figure 1B). The overall appearance of DG9-PMO–treated mice looked similar to the Hets, while the unconjugated PMO–treated neonates exhibited a weak phenotype, similar to the NT mice (Supplemental Figure 1B). DG9-PMO–treated neonates also weighed significantly more than R6G-PMO–treated neonates at a dose of 40 mg/kg (Supplemental Figure 1C).

To evaluate the effects of systemic DG9-PMO treatment on muscle strength and the function of motor neurons, we used several functional tests and compared motor function in the treated and NT mice at various time points. We performed the hind limb suspension (HLS) assay and the righting reflex test during the early weeks of life. In the HLS assay, NT mice had a decreasing HLS score as they could not extend their hind limbs when suspended by the tail after PD6 (Figure 1C). These mice also exhibited a reduced latency on the tube. On the other hand, DG9-PMO–treated neonates exhibited hind limb strength comparable to the Hets at PD12, with a significantly higher score and greater latency on the tube than the unconjugated PMO–treated and NT mice (Figure 1C and Supplemental Figure 1D), indicating improvement in muscle strength. Consistent with the HLS assay, DG9-PMO mice took a significantly shorter time to right themselves on their paws between PD6 and PD10 when compared with the other groups in the righting reflex test, suggesting improved muscle strength and coordination (Figure 1D), and greater latency on the tube than the unconjugated PMO–treated and NT mice (Figure 1C and Supplemental Figure 1E). It should be noted that the results beyond PD12 are biased, as only the healthiest unconjugated PMO–, MOE-, and R6G-PMO–treated mice survived to these time points.

This improvement was further bolstered by the forelimb grip strength assessment at PD30 in the DG9-PMO–treated mice (Figure 1E and Supplemental Figure 2A). DG9-PMO–treated mice exhibited a forelimb grip force comparable to the Hets and significantly higher than the unconjugated PMO– and MOE-treated mice when injected with 40 mg/kg AOs (Figure 1E). At PD60, the performance demonstrated by the Hets, MOE-treated mice, and DG9-PMO–treated mice was similar (Figure 1E). Unconjugated PMO–treated mice did not survive until PD60 and were excluded from this portion of the study. We evaluated the relationship between the forelimb data and the sex at PD30. Surprisingly, we observed no significant difference between males across the groups but saw significant differences in the females (Supplemental Figure 2B). We also performed a rotarod test PD30 for the 40 mg/kg groups to assess their overall muscle coordination and balance (41). We observed that although the unconjugated PMO–treated mice had reduced forelimb grip strength, they were able to balance themselves on the rotating beam similar to DG9-PMO–treated mice owing to their smaller size and weight (Supplemental Figure 2C). Since we had only 2 MOE mice in this experiment, they were not included in the statistics. Taken together, based on conclusive findings from these functional tests, systemic injection of DG9-PMO improves motor function and muscle strength in both neonatal and adult SMA mice.

Subcutaneous DG9-PMO administration increases SMN2 expression dose-dependently. To evaluate the change of FL-SMN2 expression relative to Δ7 SMN2 transcripts, we used real-time quantitative PCR (RT-qPCR) at PD7; the mice were treated with DG9-PMO at PD0. In both doses, DG9-PMO treatment led to a 4- to 30-fold higher FL-SMN2 expression than NT control in both peripheral and CNS tissues (Figure 2A and Supplemental Figure 3A). It also led to a ~5-fold increase in FL-SMN2 expression when compared with unconjugated PMO, MOE, and R6G-PMO treatments in the majority of the tissues (Figure 2A, Supplemental Figure 3A, and Supplemental Figure 1F). These data demonstrate that DG9-PMO can induce FL-SMN2 expression more efficiently than the unmodified AOs. These findings were validated at the protein level using Western blotting, where DG9-PMO treatment increased SMN protein levels in both the peripheral and CNS tissues (Figure 2B and Supplemental Figure 3B). SMN levels were higher in CNS tissues in the DG9-PMO–treated than MOE-treated mice, though no statistical significance was seen. We also found that subcutaneous administration of DG9-PMO at 40 mg/kg led to sustained levels of SMN expression at PD30 (Supplemental Figure 4). SMN levels were similar in DG9-PMO and MOE in the tissues collected from mice at PD30.

Subcutaneous administration of DG9-PMO at PD0 increases SMN expression. (A) Relative expression levels of full-length SMN2 (FL-SMN2) compared with deleted SMN2 transcripts (Δ7 SMN2) measured by quantitative PCR. (B) Representative images from Western blotting and the quantification of SMN protein levels, relative to β-tubulin. The Hets were used as a control with the relative SMN expression set to 1. A total of 40 mg/kg AOs were injected on PD0. Tissues were collected at PD7. One-way ANOVA followed by post hoc Tukey’s test was performed. Single symbols represent P < 0.05, double symbols represent P < 0.01, and triple symbols represent P < 0.005. *NT, #PMO, @DG9-PMO, &MOE. Error bars: SEM.

DG9-PMO treatment improves breathing function in neonatal SMA mice. The majority of patients with SMA suffer from compromised breathing functions and rely on an external source of breathing support. To evaluate the effects of DG9-PMO treatment on breathing function, we performed whole-body plethysmography recordings at PD7 under normoxic (21% O2) and hypoxic (11% O2) conditions (Figure 3A). The Hets did not present a respiratory phenotype under normoxic conditions. The NT mice on the other hand had slow, irregular breathing denoted by a higher coefficient of variation of frequency (CV, a measure of relative variability) and marked apneas (absence of airflow/pressure changes for a period equivalent to or greater than 2 complete respiratory cycles) (Figure 3, B and F). In contrast, under the same normoxic conditions, the majority of the DG9-PMO–treated (n = 11) and MOE-treated (n = 10) mice did not exhibit any respiratory phenotype and were similar to the Hets (Figure 3, B–F). Half of the unconjugated PMO–treated mice (n = 14) exhibited parameters as seen in NT mice, while the other half were similar to the Hets and demonstrated an increase in fR (number of breaths per minute), VE, and VT (amount of air flowing in or out of the lungs during each respiratory cycle) when compared with the NT control (Figure 3, B and D).

DG9-PMO treatment improves breathing function at PD7 in SMA mice. (A) Representative traces of whole-body plethysmograph recording from PD7 pups in normoxia (left column) and hypoxia (11% O2, right column) (n = 6 each). (B) Respiratory frequency (fR). (C) Tidal volume (VT) relative to the mean of heterozygotes (100%) in normoxia. (D) Minute ventilation (VE) relative to the mean of heterozygotes (100%) in normoxia. (E) Coefficient of variation of frequency (CV). (F) Total apnea duration (seconds in 1 minute). For those data (fR, VT, VE, and CV) in B–E that passed the normality test (Shapiro-Wilk) and equal variance test (Brown-Forsythe), parametric statistics were used with 2-way repeated measures ANOVA, followed by Holm-Šídák method. If the data did not pass the normality test, nonparametric statistics were used. (G) Correlation between respiratory frequency and body weight. Respiratory frequency was plotted against the body weight in heterozygotes (n = 10) with a correlation coefficient of 0.303 (P = 0.394). The homozygotes (n = 43) had a correlation coefficient of 0.791 (P < 0.001). A total of 40 mg/kg AOs were injected on PD0. Comparison of the difference in normoxia, or hypoxia, was conducted with Kruskal-Wallis 1-way ANOVA on ranks, followed by Dunn’s method. The difference between hypoxia and normoxia was conducted with a signed-rank test. P < 0.05 is taken as a statistically significant difference; single symbols represent P < 0.05, double symbols represent P < 0.01, and triple symbols represent P < 0.001 compared between groups indicated (*NT, #PMO); $ < 0.05, $$ < 0.01, $$$ < 0.001 compared with normoxia.

When switched to a hypoxic environment, the Hets, DG9-PMO–treated mice, and MOE-treated mice exhibited an increase in the respiratory parameters fR, VE, and VT relative to normoxia (Figure 3, B–D). Even though hypoxia reduced the number of apneas, breathing was still slow and irregular as seen by the high CV in NT mice (Figure 3E). Most of the unconjugated PMO–treated neonates exhibited an increase in the respiratory parameters relative to normoxia with no effects on apnea when compared to NT mice but still had slow, weak, and irregular breathing when compared with the Hets (Figure 3, B–D). We also examined the correlation of the severity of respiratory phenotype (decrease in fR or VE) with the decrease in body weight by using the Pearson product moment correlation t test (Figure 3G). We observed no correlation between fR and body weight in the Hets but found a strong correlation in the NT and treated mice (grouped as homozygotes). With a lower CV and a marked improvement in respiratory parameters, DG9-PMO treatment ameliorated the breathing dysfunction seen in SMA mice.

DG9-PMO treatment improves muscle pathology and neuromuscular junction characteristics. Atrophic musculature is a classical characteristic feature of SMA with diminished skeletal muscle fiber size. To determine the treatment effects on muscle pathology, we assessed the physiology and architecture of the myofibers of 2 affected muscle groups, the quadriceps and intercostal muscles, as well as the sparingly affected diaphragm at PD7 and PD30 (42, 43). We quantified the cross-sectional area (CSA), the minimal Feret’s diameter, and centrally nucleated fibers, a hallmark of pathological muscle degeneration/regeneration cycle, for at least 500 myofibers per muscle for each treatment using hematoxylin and eosin (H&E) staining at PD7 and PD30 (Figure 4, A and B, and Supplemental Figures 5 and 6). At PD7, the Feret’s diameter and CSA of the myofibers were significantly larger in all 3 muscle groups following DG9-PMO treatment when compared with the NT control (Figure 4B and Supplemental Figure 5). In the quadriceps muscle, DG9-PMO–treated myofiber was significantly larger than that of unconjugated PMO and MOE. In the diaphragm and intercostal muscle, all 3 treatment groups exhibited a similar myofiber size (Figure 4B and Supplemental Figure 5). In addition, DG9-PMO treatment led to a significant decrease in the percentage of centrally nucleated fibers (Figure 4C). The effect of DG9-PMO persisted at PD30 (Supplemental Figure 6). Unconjugated PMO– and MOE-treated mice had significantly smaller myofibers (Supplemental Figure 6) and a higher percentage of central nuclei (data not shown) compared with DG9-PMO in all 3 tissue types. These data demonstrate that DG9-PMOs improve muscle pathology in SMA mice.

Systemic administration of DG9-PMO improves muscle pathology in SMA mice. (A) Representative images from H&E staining of the quadriceps muscle (top row), diaphragm (middle row), and intercostal muscle (bottom row) at PD7 in the heterozygous, NT control and treated groups. Original magnification, 40×. (B) Frequency distribution (top) and the quantification (bottom) of the minimal Feret’s diameter (μm) of individual myofibers. Box edges, 25th and 75th percentiles; central line, median; whiskers, range (n = 3–7 per group). We measured 1,292–1,653 fibers for the quadriceps, 917–1,746 fibers for the diaphragm and 642–1,127 for the intercostal muscle. (C) Centrally nucleated fibers quantified from the H&E images (%). A total of 40 mg/kg AOs were injected at PD0. One-way ANOVA followed by post hoc Tukey’s test. Single symbols represent P < 0.05, double symbols represent P < 0.01, and triple symbols represent P < 0.005. *NT, #PMO, @DG9-PMO, &MOE. Error bars: SEM.

The SMA mouse model typically begins to exhibit neuropathological deficits around PD4–PD5, with denervated and collapsed neuromuscular junctions (NMJs) as the disease progresses (44). We assessed the NMJ architecture of the quadriceps and the intercostal muscles at PD30 to understand the phenotypic rescue following injections at PD0 (Figure 5A). DG9-PMO treatment restored the integrity of the NMJ, increased the endplate size, reduced denervation, and exhibited innervation patterns similar to the Hets (Figure 5, A–C). The peripheral synapses in DG9-PMO–treated muscles exhibited more than 60% full innervation, while unconjugated PMO and MOE treatments had close to 40%–50% innervation (Figure 5B). Unconjugated PMO–treated mice exhibited smaller endplates and immature vesicles (termed as collapsed), especially in the intercostal muscle (Figure 5C). We also assessed the expression of atrogenes Atrogin-1 and muscle ring finger-1 (MuRF-1) that are routinely upregulated in the denervated muscle (Supplemental Figure 7) (45). Although there was no statistical significance, we observed upregulation of both atrogenes in the unconjugated PMO–treated mice and upregulation of only Atrogin-1 in MOE-treated mice in the quadriceps muscle at approximately PD30. The expression was comparable between Hets and DG9-PMO–treated mice. We also evaluated the expressions of muscle specific kinase (MuSK) and acetylcholine receptor alpha (AChRα), which play a crucial role in NMJ formation and maintenance (46–50). The expression was comparable across treatment groups for MuSK, while there was a reduced expression of AChRα in unconjugated PMO mice, albeit not statistically significant (Supplemental Figure 7). Taken together, these data indicate that DG9-PMO treatment ameliorates the phenotype of the NMJs in SMA mice. The findings corroborate our hypothesis that DG9-PMO treatment rescues the crosstalk between the muscle and the neurons by improving muscle pathology and the characteristics of NMJs.

DG9-PMO treatment leads to improvement in the NMJs. (A) Representative confocal images of the NMJ staining in quadriceps and intercostal muscles collected at PD30. Scale bar: 100 μm. Postsynaptic endplates were stained using a-bungarotoxin (red, a-BTX) while neurofilament (2H3) and synaptic vesicles (SV2) were indicative of neurons (green). Denervated endplates can be identified as a-BTX endplates without overlapping synaptophysin-stained axons, while partially denervated endplates are identified as less than 50% occupancy of the presynaptic nerve terminals in an endplate. White arrowheads: full innervation. Yellow arrows: partial innervation. Blue arrows: denervation. White arrows: collapsed NMJs. (B) Innervation characteristics: full innervation, partial innervation, denervation, collapsed NMJs were quantified from at least 300–500 NMJs per group (n = 3–7) and plotted as percentages of total NMJs analyzed. (C) The number of collapsed vesicles (flat, not pretzel shaped) was quantified (n = 4–6 per group). Error bars: SEM. 40 mg/kg AOs were injected at PD0. In B, 2-way ANOVA followed by Holm-Šídák multiple comparison was used. In C, 1-way ANOVA followed by post hoc Tukey’s test. #PMO, &MOE. Single symbols represent P < 0.05, and double symbols represent P < 0.01.

DG9 enhances PMO uptake in both systemic and CNS tissues. To determine the effects of DG9 peptide in increasing PMO uptake, we evaluated the biodistribution of unconjugated PMO and DG9-PMO in the peripheral and CNS tissues. We used a well-established hybridization-based ELISA designed specifically to detect the PMO to compare the concentration of DG9-PMO and unconjugated PMO in the tissues of treated PD7 mice (51). Conjugation of DG9 to the PMO led to a significantly higher uptake in most tissues except for the liver, with an average AO detection of 12,202 pM (quadriceps muscle, n = 5), 2 × 108 pM (liver, n = 5), 8,359 pM (brain, n = 5), 13,907 pM (spinal cord, n = 4), 8,886 pM (heart, n = 4), and 2 × 107 pM (kidney, n = 6) (Figure 6A). Both unconjugated PMO and DG9-PMO displayed higher levels in the liver and kidney, as PMOs are metabolically stable and resistant to nucleases.

DG9 increases uptake of PMO in target tissues following subcutaneous administration at PD0. (A) Concentrations of PMO (pM) were measured by ELISA using the avidin-biotin affinity system and compared between DG9-PMO and unconjugated PMO treatments at 40 mg/kg doses in the quadriceps, liver, heart, kidney, brain, and spinal cord (n = 3–7 per group). *P < 0.05, **P < 0.01; unpaired 2-tailed Student’s t test. Error bars: SEM. (B) Representative IHC images from PD7 heart, quadriceps muscle, brain, and spinal cord following fluorescently tagged DG9-PMO subcutaneous administration at PD0. Green: fluorescein-DG9-PMO. Magenta: DAPI. DG9-PMO without fluorescent tag was used for the negative control. White arrows indicate DG9-PMO overlapped with nuclei (DAPI). n = 3. Scale bar: 50 μm.

PMO uptake is mediated by the caveolin-dependent pathway in myotubes (52). Some CPP-PMOs get trapped in endosomes, limiting the efficiency of splicing correction (53). To reveal the intracellular localization of DG9-PMO, we attached a fluorescent tag to the DG9-PMO. We subcutaneously injected the fluorescent DG9-PMOs into SMA mice at PD0 (40 mg/kg) and performed immunohistochemistry (IHC) to examine the localization in frozen tissue sections collected at PD7. We found some DG9-PMOs localized in nuclei of cells in the hearts and quadriceps muscle and to a lesser extent in the CNS tissues (Figure 6B). This experiment shows that DG9 promotes the uptake of PMO in both the peripheral and CNS tissues following a single subcutaneous administration, thereby globally increasing SMN levels and ameliorating the SMA phenotype.

DG9-PMO reaches the CNS in a mild SMA model. To target the motor neurons in the CNS via systemic administration, AOs need to cross the BBB in patients with SMA. For this study, we used a milder SMA model (F0) (Smn–/– SMN2+/+) with 4 SMN2 copies obtained from Jackson Laboratory (JAX 005058) that are viable, are fertile, and have shortened, thick tails since severe SMA model mice have a median survival of only 8 days, making it difficult to evaluate the treatment efficacy when the BBB is mature. The mild model typically exhibits necrosis of the tail and ears around 8–12 weeks of age and represents type III SMA (39). To determine the ability of DG9-PMO to reach the CNS, we subcutaneously injected 40 mg/kg or 80 mg/kg fluorescently tagged DG9-PMO into F0 mice at PD0 or PD5. Fluorescently tagged DG9-PMO was detected inside the nuclei of the cells in both peripheral and CNS tissues at both PD7 and PD13 (Figure 7A and Supplemental Figure 8A), indicating that DG9-PMOs can reach the CNS tissues when the BBB is mature in SMA mice. We also performed an ELISA to examine the biodistribution of unconjugated PMO and DG9-PMO at PD7. Despite subcutaneous administration at PD5, DG9 significantly increased the uptake of PMO in the CNS and peripheral tissues at PD7 (Figure 7B). We analyzed the FL-SMN2 levels and observed a significant increase in the FL-SMN2 expression in both the CNS and peripheral tissues of the treated mice at PD7 (40 mg/kg) and PD13 (80 mg/kg) (Supplemental Figure 8B and Figure 7C). These findings demonstrate that the DG9-PMO can ensure widespread distribution of the AOs to both the peripheral and CNS tissues.

DG9-PMO reaches the CNS tissues and increases FL-SMN2 expression in a mild SMA model. (A) Representative IHC images at PD7 from the quadriceps muscle, heart, brain, and spinal cord, following fluorescently tagged DG9-PMO (green) subcutaneous administration (40 mg/kg) at PD5 in the milder SMA model (F0 mice, Smn–/– SMN2+/+). Magenta: DAPI. n = 3 per group. White arrows indicate DG9-PMO overlapped with nuclei (DAPI). Scale bar: 50 μm. (B) Concentrations of PMO (pM) at PD7 were detected by ELISA using the avidin-biotin affinity system and compared between DG9-PMO and unconjugated PMO treatments at 40 mg/kg injected at PD5 in the F0 mice (n = 3–6 per group). Statistics performed using unpaired 2-tailed Student’s t test. **P < 0.01, ***P < 0.001. (C) Relative expression levels of full-length SMN2 (FL-SMN2) compared to deleted SMN2 transcripts (Δ7 SMN2) in the quadriceps muscle, liver, heart, spleen, brain, and spinal cord. Saline or DG9-PMO at 80 mg/kg doses was injected at PD5 subcutaneously (n = 3–4 per group). The tissues were collected at PD13. In C, 1-way ANOVA followed by post hoc Tukey’s test was performed. *P < 0.05. Error bars: SEM.

DG9-PMO treatment rescues the SMA phenotype without apparent toxicity. Peptides can typically pose as antigens, leading to immune reactions. Therefore, we examined the susceptibility of DG9-PMO to cause immune activation at an early neonatal stage, by looking at CD68+ cells, indicative of circulating and tissue macrophages, in the quadriceps muscle sections at PD7 (Supplemental Figure 9, A and B). The unconjugated PMO– and MOE-treated muscle had a significantly higher number of CD68+ macrophages when compared with the Hets, while NT and DG9-PMO mice had no significant difference (Supplemental Figure 9, A and B). The apparent reduction in circulating macrophages following DG9-PMO treatment is likely due to amelioration of the atrophic musculature, which would compensate for any elevation seen from the treatment itself.

To further elucidate the possibility of long-term toxicity, we collected serum from the Hets and AO-treated mice at PD30–PD35. A toxicological evaluation was performed on the levels of alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate aminotransferase (AST), creatine kinase (CK), creatinine, total bilirubin, total protein, albumin, globulin, and gamma-glutamyl transferase (GGT). All examined indicators were comparable between the groups, suggesting no apparent toxic effects (Supplemental Figure 9C). We also performed a qualitative histological analysis of the liver and kidney and found no signs of observable toxicity (Supplemental Figure 9D). We also analyzed levels of urinary kidney injury marker-1 (KIM-1) following DG9-PMO treatment at both 40 and 80 mg/kg. We observed no apparent increase in this marker compared to the Hets at PD30 (Supplemental Figure 9E). These findings emphasize that DG9-PMO is associated with no apparent toxicity or immune dysfunction in mice.