Morpholino–RNA duplex exhibits robust, sustained, and safe steric-block antisense activity by intracerebroventricular and intrathecal injection

Domain: chemistry
Published: 2025-12-22
Editorial outcome: major_revision

Abstract
Intrathecal injection of steric-block antisense oligonucleotides (ASOs) provides therapeutic opportunities for targeting the central nervous system (CNS). Phosphorothioate backbone with 2′-O-methoxyethyl (MOE) is widely used in ASO drugs, with concerns about toxicity and potency. Phosphorodiamidate morpholino oligomer (PMO) is another chemistry with a favorable safety profile but less persistent effect due to its ineffective cellular uptake and poor binding affinity to serum proteins. Here, a cholesterol-conjugated PMO/RNA heteroduplex oligonucleotide (Chol-HDO[PMO]) outperformed conventional single-stranded ASOs, achieving up to a 16-fold increase in potency without detectable toxicity and maintaining stronger ASO activity throughout the observation period. Chol-HDO[PMO] showed enhanced antisense activity in both splice modulation and translation inhibition. Chol-HDO[PMO] successfully alleviated the CNS symptoms in a Duchenne muscular dystrophy mouse model. In contrast, Chol-HDO[MOE] failed to enhance tissue delivery or potency. The efficient tissue delivery relied on interactions with CSF lipoproteins, and binding affinity was determined by heteroduplex oligonucleotide chemistry. Collectively, Chol-HDO[PMO] may expand the therapeutic potential of steric-block ASOs targeting the CNS.

Introduction
Oligonucleotide therapies represent a promising platform for the treatment of various diseases, including neurological disorders, for which effective treatments are currently lacking. Over the past decade, two antisense oligonucleotides (ASOs) have been approved by the FDA for the treatment of neurological diseases via intrathecal (IT) injection: nusinersen for spinal muscular atrophy and tofersen for familial amyotrophic lateral sclerosis (ALS), respectively1. ASOs regulate gene expression via two mechanisms. Steric-block ASOs modulate RNA processes, such as splicing. For instance, nusinersen upregulates the expression of SMN2 protein by modulating SMN2 splicing without involving RNase H1. In contrast, gapmer ASOs like tofersen cleave bound RNA via the endogenous nuclease RNase H1. Several gapmer ASOs are currently undergoing clinical trials for the treatment of Alzheimer’s disease, Parkinson’s disease, ALS, and Huntington’s disease2. Similarly, siRNAs also cleave target RNA. Currently approved drugs such as vutrisiran and givosiran for transthyretin (TTR) amyloid polyneuropathy or acute hepatic porphyria, respectively, target genes expressed in the liver3. Clinical trials are underway for neurological disorders as well4. However, these siRNAs do not regulate splicing.

Several neurological diseases caused by monogenic mutations are associated with abnormal splicing5, leading to heightened expectations for steric-block ASOs to correct these splicing defects6,7. Steric-block ASOs also offer potential therapeutic approaches beyond RNA splicing modulation, including the regulation of translation, owing to their ability to manipulate various RNA-processing pathways8. However, challenges in the safety and efficacy of nucleic acids remain9.

To date, regardless of the route of administration, two modifications used in ASO have received FDA approval: phosphorothioate (PS) oligonucleotide, mainly used with 2′-O-methoxyethyl (MOE), and phosphorodiamidate morpholino oligomer (PMO)1. PS-MOE has been clinically used in both steric-block and gapmer ASO drugs, such as nusinersen and inotersen. Initially, all internucleotide linkages in these drugs were fully modified with PS10, but adverse events such as thrombocytopenia and renal toxicity have been reported1,11,12. Recent ASO drugs, such as tofersen and eplontersen, have been partially modified with PS. PS chemistry enhances cellular uptake and nuclease resistance, thereby improving the potency and duration of action11. However, it also causes non-specific protein binding, a major contributor to ASO toxicity13, leading to dosage limitations. In contrast, PMOs have been used in all four steric-block ASO drugs approved for the treatment of Duchenne muscular dystrophy (DMD) via systemic administration. ASOs with PMO chemistry exhibit favorable safety profiles, even at high dosages14,15. This is probably due to their uncharged backbone, which is characterized by low protein-binding affinity2. A comparison of the efficacy of SMN2 exon 7 inclusion demonstrated that single-stranded ASOs (ssASOs) with PMO and MOE (ssPMO and ssMOE) showed nearly equivalent effects following a single intracerebroventricular (ICV) administration. However, the low cellular uptake of PMO remains a challenge. PMO exhibits low tissue delivery, thereby requiring frequent administration and high dosages. This has hampered the clinical application of PMO, particularly in the central nervous system (CNS), because of the patient burden associated with repeated IT injections16. Therefore, a new platform that meets the requirement of potency, persistence, and safety is needed to enhance the therapeutic potential of steric-block ASOs for IT (ICV) administration.

Previously, we reported that systemic administration of lipid-conjugated gapmer ASO/RNA heteroduplex oligonucleotides (HDO[Gapmer]) achieved efficient gene silencing in various organs17–19. However, lipid-conjugated HDO[Gapmer] did not enhance CNS effects after IT or ICV administration compared to single-stranded Gapmer ASO (ssGapmer), and even attenuated the effect at high doses19. In this study, we aimed to develop an improved HDO platform by combining PMO with HDO technology to enhance effectiveness, persistence, and safety in the CNS following IT/ICV administration–features that were previously unattainable with conventional gapmer-based HDO technology.

Results
Chol-HDO[PMO] enhanced exon-skipping potency
Dystrophin, a cytoplasmic protein encoded by the DMD gene, contributes to cytoskeleton connections in the muscles and the brain20. Genetic loss of dystrophin causes Duchenne muscular dystrophy (DMD), which is characterized by motor dysfunction and CNS manifestations21. The mdx mouse, an animal model of DMD, carries a nonsense mutation in exon 23 of the Dmd gene, leading to a lack of dystrophin in the muscle and CNS tissues. The mice exhibit an exaggerated fear response as a CNS phenotype22. ssASOs targeting the splice-donor site induced functional dystrophin expression via exon 23 skipping in the mdx mouse22–24.

We used mdx mice to investigate HDO-mediated splice modulation in the CNS following cerebrospinal fluid (CSF) administration. First, 25-mers sPMO and 25-mers sMOE, which induce exon 23 skipping in the mouse Dmd gene, were synthesized22–24. Additionally, a 20-mer ssMOE was synthesized as 18–22 nucleotides are commonly used in PS-MOE chemistry3,25. Cholesterol-conjugated HDO (Chol-HDO) was generated by the hybridization of ssASO with its complementary RNA. The complementary RNA consisted of phosphodiester-linked RNAs flanked by three-nucleotide PS 2′-O-methyl RNAs, (OMe) with cholesterol conjugated at the 5′ end (Fig. 1a). We evaluated the formation and stability of the duplexes in CSF. Chol-HDOs comprising PMO and MOE chemistries (Chol-HDO[PMO] and Chol-HDO[MOE]) displayed a single band in nuclease-free water, confirming successful duplex formation (Supplementary Fig. 1a, b). Upon incubation in rat CSF, both duplexes remained intact for at least 1 h. At later time points, lower bands emerged whose mobilities matched those of no ligand HDOs, indicating degradation of the cholesterol linkage. Chol-HDO[PMO] degraded faster than Chol-HDO[MOE] at 24, 72, and 168 h, but both constructs were stable within the first 6 h (Supplementary Fig. 1a, b).

We then evaluated exon 23 skipping 1 week after the ICV injection of ssASOs or Chol-HDOs at 10 nmol into 6–7-week-old mdx mice. Interestingly, robust skipping activity was achieved with Chol-HDO[PMO] but not with Chol-HDO[MOE], regardless of its length (Fig. 1b).

We also assessed the potency of Chol-HDO[Gapmer] after ICV injection targeting mouse Malat1. Consistent with the previous report19, Chol-HDO[Gapmer] did not enhance the silencing activity (Fig. 1c and Supplementary Fig. 2). Thus, when administered via CSF, Chol-HDO activity varied depending on the chemistry of the antisense strand.

Efficient delivery by Chol-HDO[PMO]
To elucidate the tissue delivery of Chol-HDO[PMO], we quantified the accumulation of ssASO in tissue using a hybridization ELISA with a sequence-specific probe that detects 25-mer ssPMO and 25-mer ssMOE. At 24 h after ICV administration, a more than 5.7-fold increase in ssPMO was detected in CNS tissues following Chol-HDO[PMO] administration compared with ssPMO injection (Fig. 1d). In contrast, ssMOE accumulation in tissue was similar when Chol-HDO[MOE] or ssMOE was injected (Fig. 1e). We postulated that the ssMOE concentration in the tissue after Chol-HDO[MOE] injection was underestimated, as ssMOE may not have been released from the duplex structure, given that ELISA detects ssASO through probe hybridization. To test this possibility, we treated tissues from Chol-HDO[MOE]-injected mice with RNase A to degrade the Chol-HDO[MOE] structure and measured the ssMOE concentration. However, the ssMOE concentration after Chol-HDO[MOE] injection was comparable to that after ssMOE administration (Fig. 1e). Therefore, the tissue concentration of the antisense strand was enhanced with Chol-HDO[PMO] but not with Chol-HDO[MOE]. This observation was consistent with the finding that Chol-HDO[PMO] improved the potency, whereas Chol-HDO[MOE] did not (Fig. 1b). These results suggest that the chemistry of the antisense strand of the Chol-HDO structure played a critical role in tissue delivery and potency. In situ hybridization (ISH) using a ssPMO detection probe confirmed that the ssPMO signal was widely distributed across the CNS parenchyma 24 h after Chol-HDO[PMO] administration (Fig. 1f). Collectively, Chol-HDO[PMO], but not Chol-HDO[MOE], enhanced tissue delivery following ICV injection.

Structure–activity relationship of cholesterol-conjugated duplexes
Because the chemistry of the antisense strand markedly affected the potency of HDO, we further assessed the structure–activity relationship of the HDO platform. We first examined the effect of cholesterol conjugation position (Supplementary Fig. 3a). When cholesterol was conjugated to the 3′ end of the complementary strand, HDO[PMO] did not enhance potency (Supplementary Fig. 3b). This result aligns with our previous report showing that the silencing effect of Chol-HDO[Gapmer] was enhanced with cholesterol conjugation at the 5′ end, but not at the 3′ end, when systemically injected19. We next synthesized HDOs bearing various ligands at the 5′ end of the complementary strand. However, Chol-HDO[PMO] exhibited the most robust skipping effect (Supplementary Fig. 3b). We then attempted to evaluate cholesterol-conjugated ssPMO (Chol-ssPMO) and successfully synthesized the compound; however, it was not soluble in aqueous buffer26. As a result, Chol-ssPMO could not be injected into the CSF. Additionally, we also synthesized cholesterol-conjugated ssMOE (Chol-ssMOE) using a POd(TCA) linker, which is widely used for conjugating lipid ligands to PS oligos27–29. Chol-ssMOE was soluble in water and injected via ICV. However, it did not enhance potency compared with naked ssMOE and was less active than Chol-HDO[PMO] (Supplementary Fig. 3b).

We also synthesized complementary strands in which RNA in the gap region was replaced with DNA or OMe chemistry (Supplementary Fig. 3a). Chol-HDO[PMO/DNA] showed greater activity than ssPMO but lower activity than Chol-HDO[PMO] (Supplementary Fig. 3c). Chol-HDO[PMO/OMe] showed negligible skipping potency, likely because its structure was highly resistant to degradation by endogenous enzymes, thereby preventing ssPMO from accessing the target RNA. In our previous study, we measured the melting temperatures (Tm) of HDOs with different complementary strands26. In the current study, Chol-HDO[PMO/RNA] (Tm=84.5°C) showed greater potency than Chol-HDO[PMO/DNA] (Tm=73.7°C) and Chol-HDO[PMO/OMe] (Tm=86.7°C), indicating that Tm is not the primary determinant of potency.

We next evaluated Chol-HDO[PMO] containing a fully PS-modified complementary strand (Chol-HDO[PMO/full PS RNA]). The potency of Chol-HDO[PMO/full PS RNA] was lower than that of the original Chol-HDO[PMO/wing PS RNA] (Chol-HDO[PMO]) in the cerebellum, hippocampus, and cortex (Supplementary Fig. 3c). At 24 h after ICV injection, CNS tissue accumulation of ssPMO following Chol-HDO[PMO/full PS RNA] administration was comparable to or lower than that following Chol-HDO[PMO/wing PS RNA] (Supplementary Fig. 3d). To test whether the complementary strand interfered with ssPMO detection, we treated the samples with RNase A, but the ssPMO concentration after Chol-HDO[PMO/full PS RNA] injection remained similar or lower than that after Chol-HDO[PMO/wing PS RNA] injection (Supplementary Fig. 3d). These findings indicate that additional PS modification is not beneficial for CNS delivery. This result is consistent with the reduced potency of Chol-HDO[PMO/full PS RNA] compared with Chol-HDO[PMO/wing PS RNA] (Supplementary Fig. 3c).

Efficacy, distribution, and persistence of Chol-HDO[PMO] by ICV injection
Next, we compared the efficacy of Chol-HDO[PMO] with that of conventional ssASOs. We first tested 20-mer ssMOE and 25-mer ssMOE via ICV injection. The 20-mer ssMOE exhibited greater exon-skipping than the 25-mer ssMOE in the cortex (Supplementary Fig. 4). Subsequently, dose–response relationship was examined 1 week after ICV injection of 25-mer ssPMO, 20-mer ssMOE, and Chol-HDO[PMO]. Chol-HDO[PMO] outperformed both 25-mer ssPMO and 20-mer ssMOE, showing stronger exon-skipping activity across all CNS regions (Fig. 2a). Chol-HDO[PMO] exhibited enhanced efficacy by 5.3- to 16.1-fold compared with ssPMO. To calculate the ED50, the skipping rate achieved by Chol-HDO[PMO] at 40 nmol was defined as the maximum value.

One of the major challenges of ssASOs, especially ssPMO, in clinical use is the duration of action. Therefore, we conducted a time-course analysis of the exon-skipping activity following a single ICV injection of Chol-HDO[PMO] over a 9-week period. Consistent with a previous report30, we observed long-lasting activity of the exon 23 skipping induced by ssMOE, whereas ssPMO was less persistent (Fig. 2b). Remarkably, 9 weeks after a single dose, Chol-HDO[PMO] maintained robust exon-skipping activities that exceeded that of ssMOE in all CNS regions except the cortex, where efficacy was comparable (Fig. 2b). This observation is consistent with a previous report showing that once delivered to tissues, ssPMO exerts sustained effects over several weeks31. Thus, Chol-HDO[PMO] demonstrates robust and sustained exon-skipping activity across the CNS compared with conventional ssASOs.

Distribution and exon-skipping activity in rat CNS via IT bolus injection
The volume of CSF in rats is approximately 6–10-fold higher than that in mice32,33. Additionally, owing to their larger body size, rats can be administered IT injection, a clinically relevant administration route. Therefore, we investigated whether the activity of Chol-HDO[PMO] after IT injection in rats was compatible to that observed after ICV administration in mice.

We designed a novel 25-mer ssPMO that targeted the donor site of exon 23 in rat Dmd. Two weeks after the IT injection of Chol-HDO[PMO], robust skipping activity was observed in all examined the CNS regions (Fig. 2c). Previous studies of siRNA or ASO administered via IT injection reported that activity in the striatum, which lies upstream of the CSF flow, tended to be lower than that in other regions, such as the brainstem4,34. However, Chol-HDO[PMO] showed high activity in the striatum as well as other regions.

The skipping activity of Chol-HDO[PMO] reached ~30% in the lumbar spinal cord and ~20% in the thoracic region, levels that were comparable to those achieved with ssPMO (Fig. 2c). We speculated that the skipping activity plateaued in these regions, which are located closest to the injection site and were therefore more highly exposed to both ssPMO and Chol-HDO[PMO].

RNA drugs administered into the CSF may enter the systemic circulation and accumulate in the kidneys and liver, potentially causing organ-specific toxicity, such as acute kidney injury or hepatic failure13. However, only negligible skipping activity was detected in the kidney and liver after IT injection of Chol-HDO[PMO], implying low accumulation in these systemic organs (Fig. 2c).

Phenotypic rescue in mdx mice with Chol-HDO[PMO]
An isoform of dystrophin, Dp427, stabilizes the GABAA receptor in the neuronal membrane. Its deficiency, especially in the hippocampus and amygdala, contributes to the CNS phenotype of the mdx mouse, representing the CNS manifestation observed in patients with DMD21,22. To assess the therapeutic impact of Chol-HDO[PMO], we first confirmed that restored dystrophin expression was abundant in the hippocampus 2 weeks after the ICV administration of Chol-HDO[PMO] (Fig. 3a). To evaluate protein expression in the amygdala, we performed immunofluorescence using the DYS-1 antibody specific for the Dp427 isoform. In adult wild-type mice, Dp427 was abundant in the amygdala and partially colocalized with the GABAA receptor (Fig. 3b). In contrast, immunopositive signals for Dp427 were absent in mdx mice treated with PBS. Nine weeks after ICV injection of Chol-HDO[PMO], the immunochemical signal for Dp427 was markedly increased in the amygdala of mdx mice compared with ssPMO-treated mice (Fig. 3b, c). The restored dystrophin partially colocalized with the GABAA receptor, as observed in wild-type mice, suggesting that it served as a scaffold for the receptor.

Next, to evaluate the impact of Chol-HDO[PMO] on the phenotype of mdx mice, we performed an open-field test to assess the fear response induced by a brief restraint. PBS-treated mdx mice showed exaggerated fear responses, characterized by increased freezing time and decreased vertical activity, compared with wild-type mice (Fig. 3d–f). Following Chol-HDO[PMO] treatment, the abnormal fear response was alleviated: the mice showed reduced freezing, increased total distance traveled, and enhanced vertical activity (Fig. 3d–f). Taken together, Chol-HDO[PMO] restored functional dystrophin expression and ameliorated CNS symptoms in the DMD mouse model.

Chol-HDO[PMO] was effective in translation modulation
Steric-block ASOs can interfere with RNA-processing events beyond splicing. For instance, ASOs that bind to the primary open reading frame can downregulate target proteins by blocking ribosome translation initiation8. SOD1 mutations are associated with familial ALS35. Steric-block ASOs against the start codon in SOD1 mRNA reduce mutant SOD1 protein levels and prolongs the survival of transgenic mice36. To evaluate whether Chol-HDO[PMO] enhances steric-blocking activity beyond splice modulation, we tested its efficacy in translation inhibition.

We injected 20-mer ssMOE, 25-mer ssMOE, 25-mer ssPMO, and Chol-HDO[PMO] into 6–7-week-old human G93A mutant superoxide dismutase (SOD1) transgenic mice via ICV injection. The sequences of 25-mer ssMOE and 25-mer ssPMO were identical (Fig. 4a). Chol-HDO[PMO] was well tolerated even at the highest dose (40 nmol), whereas ssMOEs exhibited lethal toxicity in a dose-dependent manner (Fig. 4b). The 25-mer ssMOE resulted in a higher mortality rate than the 20-mer ssMOE. These observations indicate that Chol-HDO[PMO] exhibited a more favorable safety profile than ssASOs with PS-MOE chemistry.

Two weeks after injection, we evaluated human SOD1 protein levels in the cortex and spinal cord using western blotting. The protein levels decreased by approximately 50% in the Chol-HDO[PMO]-treated group, whereas no reduction was observed in the ssPMO-treated mice (Fig. 4c, d). qRT-PCR analysis revealed no decline in human SOD1 mRNA levels after Chol-HDO[PMO] injection (Supplementary Fig. 5). Thus, protein reduction was due to translation inhibition. In contrast, 20-mer ssMOE did not cause any apparent reduction in protein levels (Fig. 4e, f). Taken together, the Chol-HDO[PMO] technology was applicable not only to splice modulation but also to translation inhibition.

Safety evaluation of Chol-HDO[PMO]
As safety is a major concern in the development of therapeutic oligonucleotides, we evaluated the safety profile of Chol-HDO[PMO] from multiple perspectives (Fig. 5a). ssASOs administered into the CSF, particularly those with PS backbones, can cause acute neurotoxicity, including reduced locomotor activity37. To assess acute behavioral toxicity of Chol-HDO[PMO], we recorded the locomotor activity of mice 1 h after ICV injection using an open-field apparatus. For the mouse Dmd sequence, no significant change in immobility time was observed in mice treated with 40 nmol of Chol-HDO[PMO] or 20-mer ssMOE compared to the PBS-treated groups (Fig. 5b). However, reduced mobility was observed in mice treated with 40 nmol Chol-HDO[PMO/full PS RNA] and 10 nmol Chol-ssMOE, indicating neurotoxicity. For the human SOD1 sequence, no abnormal behavior was observed in mice after 40 nmol Chol-HDO[PMO]. In contrast, the same dose of 20-mer ssMOE induced reduced locomotor activity, as reflected by a marked increase in immobility time (Fig. 5c).

Next, the immunostimulatory effects of Chol-HDO[PMO] were evaluated. Some ASOs administered into the CSF induce immune responses in the CNS that can persist for up to 2 months after injection38. We evaluated proinflammatory cytokines, including Aif1, Gfap, and Tnfα, in the mouse cortex 1 week after administration by quantitative RT-PCR. However, mice treated with Chol-HDO[PMO] targeting Dmd or SOD1 did not exhibit increased levels of these proinflammatory cytokines (Fig. 5d, e).

A previous study reported that ssASOs can induce anorexia and weight loss, which are known as late-phase toxicities39. Therefore, we evaluated weight changes in mice over 4 weeks after ICV injection of 40 nmol Chol-HDO[PMO]. No significant difference in body weight was observed compared with the PBS-treated groups (Fig. 5f). Collectively, Chol-HDO[PMO] had a favorable safety profile concerning acute-phase toxicity, immunostimulatory effects, and late-phase toxicity.

HDO chemistry affected the affinity for the CSF endogenous lipoproteins and contributed to receptor-mediated delivery
Finally, we aimed to elucidate the mechanism underlying the effective tissue delivery of Chol-HDO[PMO]. Several studies have shown that the chemical structures of oligonucleotides affect their interactions with serum proteins, thereby influencing their biodistribution during systemic administration40. Therefore, we hypothesized that the chemistry of Chol-HDOs influences their interactions with CSF proteins, which in turn facilitate efficient delivery when injected into the CSF.

To test this hypothesis, we first examined rat CSF proteins that were bound to ssPMO or Chol-HDO[PMO] using native polyacrylamide gel electrophoresis. As ssPMO is uncharged, we conjugated Alexa 647 at the 5′ end to enable electrophoretic migration of ssPMO. Alexa 647-PMO showed no additional bands after mixing with rat CSF, indicating no detectable binding to CSF proteins. In contrast, Chol-HDO[Alexa 647-PMO], containing Alexa-PMO, showed shifted bands (Fig. 6a), suggesting that it formed complexes with specific CSF proteins.

Next, we compared the protein-binding profiles of Chol-HDO[PMO] and Chol-HDO[MOE]. To exclude possible interference of the fluorescent dye with protein binding40, Chol-HDOs without the dye were mixed with rat CSF and visualized using GelRed. As expected, different migration patterns were observed for Chol-HDO[PMO] and Chol-HDO[MOE] (Fig. 6b). We focused on the band indicated by “*,” which was present in the mixture with Chol-HDO[PMO] but absent with Chol-HDO[MOE]. Neither ssMOE nor cholesterol-conjugated complementary RNA strand showed the shifted band marked “*” (Supplementary Fig. 6a). Mass spectrometric analysis identified apolipoprotein E (ApoE) and transferrin as components of the protein band marked “*” (Fig. 6c and Supplementary Fig. 6b).

ApoE is a major component of lipoproteins in the CSF. Although the molecular weight of ApoE is 37 kDa, the apparent molecular weight of the band marked by “*” exceeded 180 kDa. We therefore inferred that this band represented lipoprotein particles containing ApoE bound to Chol-HDO[PMO]. To confirm this, we isolated lipoprotein particles from rat CSF through ultracentrifugation followed by size-exclusion, and conducted a binding assay using native gel electrophoresis. The mixture containing Chol-HDO[PMO] and the rat CSF lipoprotein fraction showed a clear shifted band (Fig. 6d), whereas no shift was observed for the mixture containing Chol-HDO[MOE]. This result was consistent with the observation shown in Fig. 6b. Chol-HDO[PMO] bound to human CSF lipoproteins as well (Fig. 6e). These results indicate that Chol-HDO[PMO] formed a complex with CSF lipoproteins. Chol-HDO[PMO] was further confirmed to bind human ApoE protein using native gel electrophoresis (Supplementary Fig. 6c), whereas no binding was observed with purified transferrin (Supplementary Fig. 6d). These findings further support the specificity of the interaction between Chol-HDO[PMO] and ApoE.

Therefore, we hypothesized that Chol-HDO[PMO] enhances CNS tissue delivery by exploiting endogenous CSF lipoproteins as vectors through a receptor-mediated mechanism. Lipid transport in the CSF is mediated by lipoproteins of the low-density lipoprotein receptor (LDLR) family41. Therefore, we treated mdx mice with receptor-associated protein (RAP), an antagonist of the LDLR family42, before and after ICV administration of Chol-HDO[PMO] to block the interaction between CSF lipoproteins and the LDLR family (Fig. 6f). As expected, Chol-HDO[PMO] activity in the hippocampus was markedly reduced in RAP-treated mice (Fig. 6g). These findings indicate that the delivery of Chol-HDO[PMO] depends on lipoprotein-LDLR family interactions. We confirmed that no ligand HDO[PMO] did not bind to rat or human CSF lipoproteins, indicating that Chol-HDO[PMO] interacts with lipoproteins through its cholesterol moiety (Supplementary Fig. 7). Collectively, Chol-HDO[PMO], but not Chol-HDO[MOE], exhibited efficient binding affinity to endogenous CSF lipoproteins, which contribute to tissue delivery.

Methods
Animals
All animal studies were conducted in accordance with the protocols approved by the Institutional Animal Care and Use Committee at Tokyo Medical and Dental University (#A2023-144A). C57BL/10ScSn wild-type male mice and C57BL/10ScSn-Dmdmdx/J male mice (mdx mice) were obtained from CLEA Japan (Tokyo, Japan). Human SOD1 transgenic male mice (B6.Cg-Tg(SOD1-G93A)1Gur/J) and Sprague–Dawley wild-type male rats were purchased from Oriental Yeast (Tokyo, Japan). The animals were maintained on a 12-h light/12-h dark cycle with ad libitum access to water and food. The ambient temperature was kept at 22–24°C, and the humidity level was maintained at 40–60%. The following primers were used for genotyping of SOD1 Tg mice: 3′-CATCAGCCCTAATCCATCTGA-5′ (forward) and 3′-CGCGACTAACAATCAAAGTGA-5′ (reverse).

Animals were euthanized through cervical dislocation at the end of experiments, and tissues were collected for molecular and biochemical analyses.

Antisense oligonucleotides and duplex generation
Morpholino antisense oligonucleotides and complementary strands were purchased from Gene Tools (Philomath, OR, USA) and Gene Design (Osaka, Japan), respectively. The oligonucleotide sequences are listed in Supplementary Table 1. To produce HDO, single-stranded ASOs were hybridized with an equimolar complementary strand, heated at 95°C in nuclease-free water for 5 min, and then cooled at 37°C for 1 h. To validate duplex formation, HDOs were analyzed on 16% polyacrylamide gels. To assess stability, Chol-HDO[PMO] and Chol-HDO[MOE] were incubated with rat CSF at 37°C for 0, 1, 6, 24, 72, and 168 h. At each time point, SDS (0.025%) was added to disrupt protein–oligonucleotide interactions. Samples were then frozen in liquid nitrogen, stored at −80°C, and analyzed by electrophoresis.

ICV and IT injection
Six- to seven-week-old male mdx and hSOD1 Tg mice were anesthetized with 3% isoflurane and placed in a stereotaxic instrument (Narishige Instruments, Tokyo, Japan). A small hole was drilled into the skull above the left hemisphere at the injection site (1.0 mm lateral, 0.3 mm posterior to the bregma). A 25-µL NeuroSyringe with a 33-gauge needle (Hamilton, Nevada, USA) was lowered to a depth of 3.0 mm, and 10 µL of PBS or oligonucleotide solution was injected over 3 min. After ICV dosing, the mice were placed in their home cages to recover from anesthesia. In the LDLR family inhibition study, recombinant mouse LDLR-associated protein (RAP; R&D Systems, Minneapolis, USA) was diluted in water to a final concentration of 50 µM. A 5 µL RAP solution was injected into the left lateral ventricle 30 min before and 150 min after Chol-HDO[PMO] administration.

Five-week-old Sprague–Dawley rats were anesthetized with 4% isoflurane, and the spinal column was exposed to identify the injection site. A total of 30 μL of nuclease-free water containing ssPMO or Chol-HDO[PMO] was intrathecally injected via lumbar puncture into the dorsal region between the L4/5 vertebral space, followed by suturing.

The mdx and SOD1 transgenic mice used in this study weighed 19–21 g; therefore, a 10 nmol dose of ssPMO corresponded to approximately 476–526 nmol/kg. The rats weighed 180–200 g, and correspondingly, a 60 nmol dose equates to approximately 300–333 nmol/kg.

RT-PCR study
Total RNA was extracted from each tissue using ISOGEN II (NIPPON GENE, Tokyo, Japan), according to the manufacturer’s instructions. For Dmd exon skipping analysis, 900 ng of total RNA was subjected to one-step RT-PCR using a commercial kit (Qiagen, Venlo, Netherlands). The following primers were used: mouse Dmd, 3′- ATCCAGCAGTCAGAAAGCAAA-5′ (forward) and 3′- CAGCCATCCATTTCTGTAAGG-5′ (reverse); rat Dmd, 3′- TACCACCAATGCGCTATCAA-5′ (forward) and 3′- CCATCCATTTCTGCAAGGTT-5′ (reverse). Cycling conditions were as follows: 50°C for 30 min for 1 cycle; 95°C for 15 min for 1 cycle; 94°C 1 min, 60°C for 1 min, 72°C for 1 min for 35 cycles; and 72°C for 7 min for 1 cycle. The predicted sizes of the full-length and delta-23 transcripts were 334 and 121 bp, respectively. Each PCR product was evaluated by a bioanalyzer (Agilent), and the exon-skipping rate was calculated as the molarity of skipped product divided by the molarity of the total product, as expressed as a percentage.

The dose–response curves were fitted using a four-parameter logistic model. The ED50 values corresponded to the dose of ssASO or Chol-HDO[PMO] required to achieve 50% of the maximum exon 23 skipping in each CNS region.

Hybridization ELISA
Twenty-four hours after ICV injection of 10 nmol oligonucleotides, the cerebellum, hippocampus, and cortex were collected following PBS perfusion. The ASO concentration in each sample was measured using hybridization ELISA, as previously described52. Briefly, the tissues were homogenized in RIPA buffer supplemented with 2 mg/mL Proteinase K, followed by incubation at 55°C overnight. The lysate was then centrifuged at 16,200 rpm for 15 min at 25°C, and the supernatant was aliquoted and stored at −80°C. The DNA probe complementary to the ASO sequence was dually labeled with digoxigenin at the 5′ end and biotin at the 3′ end (3′-CCGGTTTGGAGCCGAATGGACTTTA-5′; GeneDesign, Japan). After hybridization, the biotin moiety to the probe was captured by avidin on Pierce NeutrAvidin-Coated 96-Well Plates, Black (Thermo Fisher Scientific, Waltham, MA, USA), and unhybridized probes were digested with micrococcal nuclease (New England Biolabs, Massachusetts, USA). Alkaline phosphatase-conjugated anti-digoxigenin antibody (1:5000; #11093274910, Roche, Switzerland) was added to each well, followed by AttoPhos AP fluorescent substrate (Promega, Tokyo, Japan). Fluorescence intensity was measured using an Infinite M1000 PRO microplate reader (Tecan, Männerdorf, Switzerland).

In situ hybridization
Mouse brains were collected 24 h after ICV injection of 10 nmol single-stranded PMO or Chol-HDO[PMO]. Brain sections were stained using the miRNAscope HD (RED) assay kit (Advanced Cell Diagnostics, Westminster, USA), following the manufacturer’s protocol. Briefly, fresh mouse brains were embedded in an OCT compound and then sliced into sections at a thickness of 10 µm. After mounting onto Superfrost Plus slides (Thermo Fisher Scientific), the sections were dried at −20°C for 2 h and fixed in 4% paraformaldehyde for 1 h. The sections were dehydrated in 50% ethanol for 5 min, 70% ethanol for 5 min, and twice in 100% ethanol for 5 min at room temperature. Hydrogen peroxide was added to each section, followed by incubation for 10 min at 25°C. The sections were washed twice with distilled water for 1 min. The sections were treated with Protease IV at room temperature for 30 min and then washed with PBS for 1 min. The ssPMO-specific probe used in this study was designed by Advanced Cell Diagnostics, Inc. (ACD) based on the target ssASO sequence information we provided. The general probe architecture followed ACD’s RNAscope design53—comprising a 14-base tail sequence, a spacer, and a target-specific region complementary to ssPMO. We previously used the same vendor-provided ssPMO-targeting probe in our earlier work26. As in that study, a pair of target probes bearing distinct tail sequences hybridized contiguously to the target ssASO; the two tails then serve as a scaffold for subsequent signal amplification. The exact probe sequences and chemical modifications are proprietary and not disclosed by the manufacturer. For the present experiments, we used a PMO probe (SR-ASO-PMO-S1, #1088271-S1; Advanced Cell Diagnostics, Westminster, UK); slides were incubated with the probe for 2 h at 40°C. The target probe signal was amplified using the miRNAscope kit Amp 1-6 before being detected using Red-A and Red-B (1:60). The slides were counterstained with hematoxylin for 2 min and mounted with EcoMount (EM897L, Biocare Medical, California, USA). Images were captured using a SLIDEVIEW VS200 (Olympus, Tokyo, Japan) at 20× magnification.

Western blotting
For mouse dystrophin analysis, tissues were homogenized in 4 M urea buffer (125 mM Tris-HCl, pH 6.4, 10% glycerol, 4% SDS, 4 M urea, 10% beta-mercaptoethanol, and 0.005% bromophenol blue, Complete Ultra protease inhibitor tablet (Roche, Switzerland)). Protein aliquots were precipitated using Compat-Able Protein Assay Preparation Reagent Sets (Thermo Fisher Scientific, Massachusetts, USA) for quantification. Protein concentration was determined using BCA assay (Thermo Fisher Scientific, Massachusetts, USA). Subsequently, 30 µg of total protein was loaded onto the NuPAGE Tris-Acetate Precast Gel (Thermo Fisher Scientific, Massachusetts, USA). For human SOD1 analysis, proteins were extracted using RIPA buffer (1% NP-40, 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, Complete Ultra protease inhibitor tablet) supplemented with 1% SDS, and 5 µg of total protein was loaded onto 15% acrylamide gel.

After electrophoresis, the proteins were electrophoretically transferred onto a PVDF membrane (Bio-Rad Laboratories, California, USA). The membrane was blocked with 5% non-fat milk in 0.05% TBS-T, followed by overnight incubation with primary antibodies at 4°C. DYS-1 (1:50, mouse monoclonal; Leica Biosystems, Wetzlar, Germany) and anti-human SOD1 (1:5000, sheep polyclonal; Merck, Darmstadt, Germany) were used as primary antibodies. The membrane was washed three times with TBS-T and incubated at room temperature for 1 h with horseradish peroxidase-conjugated goat anti-mouse and anti-sheep IgG (1:10,000) (Thermo Fisher Scientific, MA, USA). For signal detection, SuperSignal West Dura Extended Duration Substrate (Thermo Fisher, Massachusetts, USA) was used. Chemiluminescence was detected using a ChemiDoc Imaging System, and protein band intensity was quantified using Image Lab (ver. 6.0.1, Bio-Rad Laboratories, California, USA).

Immunofluorescence
The brains were harvested by perfusion with cold PBS. The tissue was embedded in the OCT compound, and 16-µm-thick coronal sections were prepared for immunostaining. After blocking with 10% normal goat serum in PBS-T for 1 h, the sections were incubated with Mouse-to-Mouse Blocking Reagent (ScyTek Laboratories, Logan, UT, USA) for 30 min. The sections were then washed and incubated with primary antibodies (mouse monoclonal anti-dystrophin NCL-DYS1, diluted 1:5, Leica Biosystems, Wetzlar, Germany; polyclonal anti-GABA Aα2 receptor, diluted 1:400, Alomone Lab, Israel), followed by secondary antibodies conjugated to Alexa 488 or Alexa 568 (1:200; Jackson Immunoresearch, Suffolk, UK) for 1 h at room temperature. Images were captured using an A1R confocal laser-scanning microscope (Nikon, Tokyo, Japan).

Restrain-induced fear response
Behavioral tests were performed between 8:00 am and 1:00 pm. Each mouse was restrained by gently pinching its neck between the thumb and forefinger, holding the tail between the fourth and fifth fingers, and tilting the animal to expose the abdomen. After 10 s, the mouse was released into an observation box illuminated at 20 lx and was video-monitored for 10 min using Image OF software (O’Hara & Co. Ltd., Tokyo, Japan). The total distance traveled, vertical activity (leaning and rearing), and immobile time were measured. Freezing was defined as the absence of any movement except for respiration. This was quantified as the time during which the mouse moved by less than 0.5 cm per second. Vertical activity was assessed by an investigator blinded to the treatment.

Toxicity evaluation
To evaluate acute toxicity, mice were video-monitored for 10 min to measure immobile time 1 h after ICV injection. To quantify proinflammatory cytokine levels after treatment, the mouse cortex was collected 1 week after dosing. Following RNA extraction and cDNA synthesis, qRT-PCR was performed.

CSF collection, lipoprotein fractionation, and polyacrylamide native gel electrophoresis
Sprague-Dawley rats were anesthetized with 4% isoflurane inhalation and perfused with cold PBS. The rats were then placed in the prone position on the operating table, and the muscle tissue was removed to expose the cisterna magna (CM). An insulin syringe was inserted into the CM, and approximately 120 µL of CSF was collected from each rat by gentle aspiration. Human CSF from healthy donors was purchased from KAC (Kyoto, Japan). The CSF was stored at −80°C for subsequent analysis. Purified human and mouse transferrin were obtained from Bio-Rad (California, USA) and Jackson ImmunoResearch (Pennsylvania, USA), respectively.

Lipoproteins were isolated from rat and human CSF by ultracentrifugation according to a previously described serum lipoprotein isolation method, with minor modifications54. One volume of CSF was mixed with an equal volume of NaBr solution (density = 1.478 g/mL) and centrifuged at 266,000 × g for 7.5 h at 16°C. The upper half of the solution was collected and filtered using an Amicon Ultra 50kDa centrifugal filter (Merck Millipore, Massachusetts, USA).

Oligonucleotides (200 pmol) were mixed with 8 µL of nuclease-free water, CSF, CSF lipoprotein fraction, or transferrin solution (30 µg/mL) and incubated for 1 h at 37°C. Subsequently, each mixture was loaded onto 5–20% gradient polyacrylamide gel (ATTO, Tokyo, Japan) containing 2 µL of 30% sucrose solution as a loading agent and electrophoresed at 120 V for 110 min at 4°C after preloading at 200 V for 60 min. Bands were stained and visualized using GelRed (Biotium, California, USA) and scanned using a ChemiDoc Imaging System (Bio-Rad, Hercules, CA, USA).

Mass spectrometry (MS)
The parameters of MS are summarized in Supplementary Table 2. Chol-HDOs were incubated in rat CSF and separated using native gel electrophoresis, followed by visualization with GelRed. Gel pieces were washed with water and then dehydrated with acetonitrile. The samples were incubated with 10 mM DTT and 100 mM NH4HCO3 at 60°C for 1 h. After blocking with 50 mM iodoacetamide at room temperature for 45 min, the samples were digested with 1 pmol sequencing grade modified trypsin (Promega, Madison, WI, USA). The peptides were analyzed using a mass spectrometer (LTQ Orbitrap Velos, Thermo Scientific) coupled with a nano-LC (EASY-nLC II, Thermo Scientific). Capillary Ex-Nano Mono cap C18 Trap column (0.075 mm i.d. × 50 mm, GL Sciences) and Capillary Ex-Nano Mono cap C18 Nano-flow Analytical column (0.075 mm i.d. × 150 mm, GL Sciences) were used. For LC conditions, the following gradient was used (A, 0.1% formic acid/H2O; B, 70% ACN, 0.1% formic acid in H2O): 0 min 0% B; 0–2 min 0–29% B; 2–42 min 29–85% B; 42–47 min 85–100% B, 47–52 min 100% B, 300 nl/min. A top 10 data-dependent acquisition MS method was used.

Statistical analysis
Data were analyzed using GraphPad Prism version 10.6.1 (GraphPad Software, San Diego, CA, USA) and presented as means ± standard error of the mean (SEM). The n value refers to the number of animals.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Yanagidaira et al. present data using the Yokota lab’s HDO approach to deliver PMOs, a neutral and very difficult oligonucleotide to deliver. The study is well performed with most of the appropriate controls. The data is highly compelling and will garner a strong interest from the RNA oligonucleotide therapeutics field. Figure 2b is amazing in the duration of effect, which is entirely consistent with both the lack of an endogenous mechanism to degrade PMOs and more PMO’s being delivered into the cytosol and nucleus of cells. I have several questions, comments and concerns, as follows:\nMajor Concerns:\n#1) Fig 1b-f. While the naked PMO is one appropriate control, we really need to see the Chol-PMO compared to Chol-HDO[PMO] throughout the key experiments in this study. Unfortunately, it appears that molecule is insoluble (Juri. Nat. Communication. in press). We would need to see this study for a proper review here.\n#2) Fig 1a. Does a full PS backbone on the RNA in HDO change any properties of HDO[PMO)] (activity, distribution, etc)?\n#3) Extended Fig 2c. The 2’-OMe strand also has a much higher Tm than native 2’-OH RNA sense strand. Does Tm affect HDO[PMO] activity?\n#4) Fig 2. The authors need to investigate the Chol-ssMOE oligos too. That is the appropriate control vs. Chol-HDO[PMO].\n#5) Fig. 6. To address what part of the molecule is interacting with ApoE, the authors need to repeat 6 with Chol-HDO[PMO] vs. HDO[PMO] (no cholesterol).\nMinor Concerns:\n#6) Needs some minor English grammar editing", "reviewer_2": "This is the first demonstration of using cholesterol-conjugated RNA to deliver PMO to CNS. It is a novel approach and could have significant impact for treating CNS diseases using the PMO ASO platform. The facts presented here are well-substantiated by evidence. However, there are some inaccurate statements made and some foundational information is lacking.\nLine 27. Spell out the full name for PMO, phosphorodiamidate morpholino oligomer\nLine 27-28. The statement “Morpholino (PMO) … but a less persistent effect” is misleading. If PMO gets into cells, it has sustained effect for many weeks (see PMC2671676). PMO is very stable and can’t be degraded by any known enzymes. The issue with PMO’s duration of effect lies with its poor cellular uptake and serum protein binding, but PMOs modified with a cell-penetrating peptide or with an antibody have been shown to have long-lasting and persistent efficacy. Consider changing the sentence to “… but a less persistent effect due to its ineffective cellular uptake and poor binding affinity to serum proteins”.\nLine 74. Is this referring to plasma half-life? References 16 and 17 don’t contain pharmacokinetic data to indicate that PMO has a short half-life.\nLine 125-126, No PK study was done in the manuscript so the sentence about PK behavior of two constructs are not correct. This was an assay of of tissue concentration at one point in time, not pharmacokinetics which refers to studies of concentration profiles over time (hence “kinetics”).\nLine 306-308. Plasma PK was not done in the study, so this statement is not true. “CSF concentration” would be a reasonable term to describe what was assessed.\nCharacterization of ASO duplexes: there is no experimental validation that the duplexes used in the studies have been formed, and there is no information on their stability in solution or over time. No methods are given for checking the identity of the analytes.\nIdentification of apolipoprotein E as the main binding partner of the chol-HDO[PMO]: the method and data by mass spec analysis of the protein is missing. A direct binding assay between apolipoprotein E and chol-HDO[PMO] is needed to validate the claim. Does chol-HDO[PMO] bind to human apolipoprotein E? This is not difficult to check with commercially available ApoE protein.\nMouse dosing: nanomole doses are cited, but there is no mouse weight given--actual dosage (nmole/kg) is not given, nor is variability in mouse weight shown.\nFigure 1f: stain color for ASO-specific probe is not given, making interpretation of the image difficult.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nScott Bittner", "reviewer_4": "Nagata et al have carried out very interesting experiments that advance the field of nucleic acid therapeutics. The authors describe a unique method to leverage the safety of the PMO chemistry while enhancing delivery and potency by forming a heteroduplex with a complementary RNA strand stabilized at the ends with minimal phosphorothioate inter-nucleoside linkages. Thus, their HDO constructs have the potential to decrease nucleic acid chemistry based toxicity while maintaining or increasing potency of nucleic acid technology to enhance splicing or exon skipping particularly in multiple areas of the brain which is unprecedented.\nThe work is quite detailed and meets the standards of the field. Moreover the authors have investigated the importance of RNA vs DNA complementary strands. They have also determined whether length of ASO or chemistry of the ASO affects potency and delivery and provided sufficient, clear data to demonstrate the superiority of their HDO constructs. In addition they have tested whether the cholesterol linkage used is similar or superior to other lipid linkage used for CNS targeting of nucleic acids. Their methods are detailed and should be sufficient for anyone schooled in the art of nucleic acid technology to reproduce or evaluate their data. I would recommend the authors to spell check the document as well as reconcile Fig.1a with the description provided in the text for the 5' vs 3' cholesterol linkage.\nOverall, this is an exciting and seminal work in the field of nucleic acid therapeutics and i was pleased to review the manuscript."}, "author_response": "Response to Referees Letter :\nWe sincerely appreciate the thoughtful suggestions and insights.\nThe manuscript has been rechecked and the necessary changes have been made in accordance with the reviewers’ suggestions. In the responses below, the revised or newly added parts of the manuscript are indicated in red for clarity.\nIn addition to addressing the reviewers’ comments, we have made several voluntary improvements to the manuscript, which are summarized at the end of this response letter.\nWe hope these improvements further increase the manuscript’s clarity and rigor.\nResponses to the Reviewers’ comments\nReviewer #1:\nYanagidaira et al. present data using the Yokota lab’s HDO approach to deliver PMOs, a neutral and very difficult oligonucleotide to deliver. The study is well performed with most of the appropriate controls. The data is highly compelling and will garner a strong interest from the RNA oligonucleotide therapeutics field. Figure 2b is amazing in the duration of effect, which is entirely consistent with both the lack of an endogenous mechanism to degrade PMOs and more PMO’s being delivered into the cytosol and nucleus of cells. I have several questions, comments and concerns, as follows:\nComment 1: Fig 1b-f. While the naked PMO is one appropriate control, we really need to see the Chol-PMO compared to Chol-HDO[PMO] throughout the key experiments in this study. Unfortunately, it appears that molecule is insoluble (Juri. Nat. Communication. in press). We would need to see this study for a proper review here.\nResponse: Thank you for your remarks regarding the cholesterol-conjugated PMO (Chol-ssPMO).\nWe acknowledge that Chol-ssPMO would be an important control in this study. However, we were unable to formulate Chol-ssPMO under aqueous conditions suitable for in vivo administration, due to its poor solubility. Therefore, a direct comparison with Chol-HDO[PMO] in animal experiments was technically unfeasible in the current study, as noted in the open peer-review file of the reference article (Hasegawa, et al. 2024.). Briefly, Chol-ssPMO was synthesized from Cyclooctyne-PMO and Cholesteryl-TEG azide in 100% ethanol. MALDI-TOF MS analysis showed a peak at approximately m/z 9630, consistent with the expected mass of Cholesteryl-TEG-PMO. The reaction mixture was then dissolved in 50% acetonitrile and analyzed by RP-HPLC, which revealed two peaks: one at 15 minutes (Cyclooctyne-PMO) and one at 23 minutes (Cholesteryl-TEG-PMO) (Figure L1A and B). However, this 23-minute peak was not detected in the aqueous supernatant, indicating that Chol-ssPMO was poorly soluble in water. (Figure L1C).\nFigure L1 (This data was quoted and modified from our previous work (Hasegawa, et al. 2024.))\nWe speculate that this poor solubility is due to the uncharged nature of the PMO backbone. In contrast, Chol-HDO[PMO] exhibited high water solubility, likely due to the negatively charged complementary RNA strand.\nWe have revised the corresponding sentence in the Results section as follows:\nFrom\nWe also attempted to conjugate cholesterol directly to ssPMO; however, the resulting compound did not dissolve in water or PBS.\nTo\nWe then attempted to test cholesterol-conjugated ssPMO (Chol-ssPMO) and successfully synthesized the compound; however, it was not soluble in aqueous solution26. As a result, Chol-ssPMO could not be injected into the CSF. (Page 8, Lines 155–158)\nNew Reference in the manuscript\n26. Hasegawa J., Nagata T., Ihara K., et al. Heteroduplex oligonucleotide technology boosts oligonucleotide splice switching activity of morpholino oligomers in a Duchenne muscular dystrophy mouse model. Nat Commun. 2024:1-15.\nComment 2: Fig 1a. Does a full PS backbone on the RNA in HDO change any properties of HDO[PMO)] (activity, distribution, etc)?\nResponse: We appreciate the reviewer's insightful question. To address this, we generated Chol-HDO[PMO] with a fully phosphorothioated complementary RNA strand (Chol-HDO[PMO/full PS RNA] and we evaluated its activity, tissue distribution, and toxicity.\nFirst, we observed that the exon-skipping activity of Chol-HDO[PMO/full PS RNA] was reduced compared to Chol-HDO[PMO/wing PS RNA] (the original compound) in the cerebellum, hippocampus, and cortex one week after injection (New Supplementary Figure 3c).\nTo assess distribution, we measured ssPMO levels in CNS tissues 24 hours after ICV injection using hybridization ELISA. The ssPMO accumulation was comparable to or lower than that of Chol-HDO[PMO/wing PS RNA] (New Supplementary Figure 3d). We also conducted RNase A treatment to degrade the complementary strand, allowing ssPMO to bind the ELISA probe. Still, the detected ssPMO concentration remained equal to or lower than Chol-HDO[PMO/wing PS RNA], suggesting that additional PS modification does not enhance delivery. This was consistent with the observation that Chol-HDO[PMO/full PS RNA] was less active than Chol-HDO[PMO/wing PS RNA] (New Supplementary Figure 3c).\nIn terms of toxicity, mice injected with 40 nmol of Chol-HDO[PMO/full PS RNA] exhibited acute neurotoxicity, observed as reduced locomotor activity one hour post-injection (New Figure 5b). Since Chol-HDO[PMO/wing PS RNA] did not show such effects, the observed toxicity is likely due to the increased PS content.\nWe have included these new findings and a schematic in the manuscript. Additionally, we have added the following sentences:\nResults section (Page 8, Lines 171–181)\nWe next tested Chol-HDO[PMO] with fully PS-modified complementary strand (Chol-HDO[PMO/full PS RNA]). The potency of Chol-HDO[PMO/full PS RNA] was lower than that of Chol-HDO[PMO/wing PS RNA] (the original compound, Chol-HDO[PMO]) in the cerebellum, hippocampus, and cortex (Supplementary Figure 3c). The CNS tissue accumulation of ssPMO 24 h after ICV injection of Chol-HDO[PMO/wing PS RNA] was comparable to or lower than that of Chol-HDO[PMO/wing PS RNA] (Supplementary Figure 3d). To test whether the complementary strand interfered with ssPMO detection, we performed RNase A treatment; however, the concentration of Chol-HDO[PMO/wing PS RNA] remained similar or lower to Chol-HDO[PMO/wing PS RNA]. (Supplementary Figure 3d). These findings indicate that additional PS modification is not beneficial for CNS delivery. This result is consistent with the reduced potency of Chol-HDO[PMO/full PS RNA] compared to Chol-HDO[PMO/wing PS RNA] (Supplementary Figure 3c).\nResults section (Page 12, Lines 275–276)\nHowever, reduced mobility was observed in mice treated with 40 nmol of Chol-HDO[PMO/full PS RNA] and 10 nmol Chol-ssMOE, indicating neurotoxicity.\nDiscussion section (Page 17, Lines 370–379)\nMoreover, additional PS in Chol-HDO[PMO] reduced tissue delivery and caused neurotoxicity (Supplementary Figure 3c, d; Fig. 5b). These findings suggest that the efficacy and safety of Chol-HDO[PMO] could be further enhanced by replacing PS with alternative chemical modifications that confer nuclease resistance.\nWe assume that the heteroduplex structure must be degraded by endogenous enzymes for ssPMO to bind target RNA. This is supported by the observation that Chol-HDO[PMO/full OMe], highly resistant to nuclease cleavage, exhibited no exon skipping (Supplementary Figure 3c). Moreover, 10-fold higher ssPMO was detected after RNase A treatment in CNS tissues (Supplementary Figure 3d), suggesting a substantial portion of PMO remains duplexed in vivo. Designing duplexes that efficiently release ssPMO may enhance efficacy.\nComment 3: Extended Fig 2c. The 2’-OMe strand also has a much higher Tm than native 2’-OH RNA sense strand. Does Tm affect HDO[PMO] activity?\nResponse: Thank you for this valuable comment. In our previous study, we evaluated the melting temperatures (Tm) of various HDOs with different complementary strands (Hasegawa, et al. 2024.) (Table L1). As noted, Chol-HDO[PMO/full OMe] exhibited a higher Tm than chol-HDO[PMO/RNA]. In the current ICV study, Chol-HDO[PMO/RNA] (Tm = 84.5°C) showed greater potency than chol-HDO[PMO/DNA] (Tm = 73.7°C) and Chol-HDO[PMO/OMe] (Tm = 86.7°C). These results suggest that Tm alone does not determine HDO potency.\nWe added the following sentence:\nResults section (Page 8, Line 166– Line170)\nIn our previous study, we measured the melting temperatures (Tm) of HDOs with different complementary strands26. In the current ICV study, Chol-HDO[PMO/RNA] (Tm = 84.5°C) showed greater potency than Chol-HDO[PMO/DNA] (Tm = 73.7°C) and Chol-HDO[PMO/OMe] (Tm = 86.7°C), indicating that Tm is not the primary determinant of potency.\nNew Reference in the manuscript\n26. Hasegawa J., Nagata T., Ihara K., et al. Heteroduplex oligonucleotide technology boosts oligonucleotide splice switching activity of morpholino oligomers in a Duchenne muscular dystrophy mouse model. Nat Commun. 2024:1-15.\nComment 4: Fig 2. The authors need to investigate the Chol-ssMOE oligos too. That is, the appropriate control vs. Chol-HDO[PMO].\nResponse: We appreciate your feedback. We synthesized a 20 mer cholesterol-conjugated ssMOE (Chol-ssMOE) using the cleavable PO d(TCA) linker, commonly used for lipid-conjugated PS-MOE ASOs. The chemical structure is shown below.\nChol-ssMOE was water-soluble and injectable via ICV. However, it showed no significant increase in activity over naked ssMOE (p = 0.11, Supplementary Figure 3b), and was less potent than Chol-HDO[PMO]. Moreover, Chol-ssMOE induced acute neurotoxicity even at a relatively low dose (10 nmol) (New Figure 5b). These results suggest that Chol-HDO[PMO] is superior in both efficacy and safety.\nThe new schema and results have been included in the article. Furthermore, based on the new results, we added the following sentence to the manuscript:\nResults section (Page 8, Lines 158–161)\nAdditionally, we synthesized cholesterol-conjugated ssMOE using a PO d(TCA) linker, which is widely used for attaching lipid ligands to PS oligos27, 28, 29. Chol-ssMOE was soluble in water and injected via ICV. However, it did not improve potency over naked ssMOE and was less active than Chol-HDO[PMO] (Supplementary Figure 3b).\nResults section (Page 12, Lines 275–276)\nHowever, reduced mobility was observed in mice treated with 40 nmol of Chol-HDO[PMO/full PS RNA] and 10 nmol Chol-ssMOE, indicating neurotoxicity.\nNew References in the manuscript:\n27. Prakash TP, Mullick AE, Lee RG, et al. Fatty acid conjugation enhances potency of antisense oligonucleotides in muscle. Nucleic Acids Res. 2020;47(12):6029-6044. doi:10.1093/nar/gkz354\n28. Wang S, Allen N, Prakash TP, Liang X, Crooke ST. Lipid Conjugates Enhance Endosomal Release of Antisense Oligonucleotides Into Cells. Nucleic Acid Ther. June 2019. doi:10.1089/nat.2019.0794\n29. Østergaard ME, Jackson M, Low A, et al. Conjugation of hydrophobic moieties enhances potency of antisense oligonucleotides in the muscle of rodents and non-human primates. Nucleic Acids Res. 2019;47(12):6045-6058. doi:10.1093/nar/gkz360\nMoreover, the observation that chol-ssMOE did not enhance activity despite having a similar overall charge to chol-HDO[PMO] suggests that enhanced activity is not simply attributed to lower nucleic acid charge, but rather that the charge-to-molecular weight ratio is a critical factor. Therefore, we have revised the corresponding part of the Discussion Section as follows.\nDiscussion section (Page 18, Lines 412)\nFrom\nIn contrast, Chol-HDO[PMO], with a less negative charge than Chol-HDO[MOE] or Chol-HDO[Gapmer], can bind to CSF lipoproteins via hydrophobic interactions, even at low concentrations.\nTo\nIn contrast, Chol-HDO[PMO], with a lower charge-to-molecular weight ratio than Chol-HDO[MOE] or Chol-HDO[Gapmer], can bind to CSF lipoproteins via hydrophobic interactions, even at low concentrations.\nComment 5: Fig. 6. To address what part of the molecule is interacting with ApoE, the authors need to repeat 6 with Chol-HDO[PMO] vs. HDO[PMO] (no cholesterol).\nResponse: Thank you for this important suggestion. We prepared ligandless HDO[PMO] (without cholesterol) and performed a native gel assay. Ligandless HDO[PMO] did not bind to purified rat or human CSF lipoproteins, whereas Chol-HDO[PMO] did (New Supplementary Figure 7). This demonstrates that cholesterol, not the heteroduplex structure, is responsible for lipoprotein binding. This result also supports the observation that ligandless HDO[PMO] lacks skipping activity (Supplementary Figure 3b).\nThis result has been included as New Supplementary Figure 7 and we added the following sentence to the manuscript:\nResults section (Page 15, Lines 334–336)\nWe confirmed that ligandless HDO[PMO] did not bind to rat or human CSF lipoproteins, indicating that Chol-HDO[PMO] interacts with lipoproteins via cholesterol (Supplementary Figure 7).\nComment 6: Minor Concerns: Needs some minor English grammar editing\nResponse: Thank you for the suggestion. We carefully reviewed the entire manuscript for grammatical accuracy and also submitted it to a professional English editing service for thorough proofreading.\nReviewer #2:\nThis is the first demonstration of using cholesterol-conjugated RNA to deliver PMO to CNS. It is a novel approach and could have significant impact for treating CNS diseases using the PMO ASO platform. The facts presented here are well-substantiated by evidence. However, there are some inaccurate statements made and some foundational information is lacking.\nComment 1: Line 27. Spell out the full name for PMO, phosphorodiamidate morpholino oligomer\nComment 2: Line 27-28. The statement “Morpholino (PMO) … but a less persistent effect” is misleading. If PMO gets into cells, it has sustained effect for many weeks (see PMC2671676). PMO is very stable and can’t be degraded by any known enzymes. The issue with PMO’s duration of effect lies with its poor cellular uptake and serum protein binding, but PMOs modified with a cell-penetrating peptide or with an antibody have been shown to have long-lasting and persistent efficacy. Consider changing the sentence to “… but a less persistent effect due to its ineffective cellular uptake and poor binding affinity to serum proteins”.\nResponse: Thank you for your comments. We have revised the sentence as follows:\nAbstract (Page 3, Lines 36–38)\nFrom\nMorpholino (PMO) is another chemistry with a favorable safety profile but a less persistent effect.\nTo\nPhosphorodiamidate morpholino oligomer (PMO) is another chemistry with a favorable safety profile but less persistent effect due to its ineffective cellular uptake and poor binding affinity to serum proteins.\nAlso, we have added a sentence in the Results section to clarify the consistency between our results and the reference you kindly suggested, which has been added as a new citation in the revised manuscript.\nResults section (Page 9, Lines 198–199)\nThis is consistent with a previous report showing that once delivered to tissues, PMO exert sustained effects over several weeks31.\nNew Reference in the manuscript\n31. Jearawiriyapaisarn N, Moulton HM, Buckley B, et al. Sustained dystrophin expression induced by peptide-conjugated morpholino oligomers in the muscles of mdx mice. Mol Ther. 2008;16(9):1624-1629. doi:10.1038/mt.2008.12\nComment 3: Line 74. Is this referring to plasma half-life? References 16,17 don’t contain pharmacokinetic data to indicate that PMO has a short half-life.\nResponse: Thank you for the clarification. We agree that the references do not include pharmacokinetic data. Therefore, we have removed the reference to half-life and revised the sentence:\nFrom\nPMO exhibits low tissue delivery and a short half-life16,17, thereby requiring frequent administration and high dosages.\nTo\nPMO exhibits low tissue delivery and a short half-life16,17, thereby requiring frequent administration and high dosages. (Page 5, Lines 81–82)\nComment 4: Line 125-126, No PK study was done in the manuscript so the sentence about PK behavior of two constructs are not correct. This was an assay of of tissue concentration at one point in time, not pharmacokinetics which refers to studies of concentration profiles over time (hence “kinetics”).\nResponse: We agree with the reviewer. We have revised the sentences:\nFrom\nTo elucidate the pharmacokinetics of Chol-HDO[PMO], we quantified the tissue accumulation of ssASO by hybridization ELISA using a sequence-specific probe that detects 25mer-ssPMO and 25mer-ssMOE.\nTo\nTo elucidate the tissue delivery of Chol-HDO[PMO], we quantified the tissue accumulation of ssASO by hybridization ELISA using a sequence-specific probe that detects 25 mer ssPMO and 25 mer ssMOE. (Page 7, Lines 128)\nFrom\nTherefore, the pharmacokinetics of antisense strand was improved by Chol-HDO[PMO] but not Chol-HDO[MOE].\nTo\nTherefore, the tissue concentration of antisense strand was increased by Chol-HDO[PMO] but not Chol-HDO[MOE]. (Page 7, Lines 138–139)\nFrom\nHere, Chol-HDO[PMO] demonstrated vast and sustained steric block activity across the CNS with improved pharmacokinetics following IT and ICV injection.\nTo\nHere, Chol-HDO[PMO] demonstrated vast and sustained steric block activity across the CNS with improved delivery following IT and ICV injection. (Page 16, Lines 343)\nComment 5: Line 306-308. Plasma PK was not done in the study, so this statement is not true. “CSF concentration” would be a reasonable term to describe what was assessed.\nResponse: We appreciate your feedback. We apologize for the confusion. Since we measured the ASO strand concentration in the CNS parenchyma in Figure 1d and e, we have changed the sentence as follows:\nFrom\nThe pharmacokinetics of Chol-HDO following ICV administration varied remarkably depending on the antisense strand chemistry, which determined the binding capability of CSF lipoproteins.\nTo\nThe CNS accumulation of Chol-HDO following ICV administration varied remarkably depending on the antisense strand chemistry, which determined the binding capability of CSF lipoproteins. (Page 16, Lines 348)\nComment 6: Characterization of ASO duplexes: there is no experimental validation that the duplexes used in the studies have been formed, and there is no information on their stability in solution or over time. No methods are given for checking the identity of the analytes.\nResponse: Thank you for this important comment. We agree that clear experimental validation of duplex formation and stability is essential.\nTo confirm duplex formation, we performed polyacrylamide gel electrophoresis after annealing the antisense strand with the cholesterol-conjugated complementary strand. Both Chol-HDO[PMO] and Chol-HDO[MOE] showed a single band corresponding to the expected duplex size (~40 bp), confirming successful heteroduplex formation (New Supplementary Figure 1a, b).\nTo evaluate the stability of the duplexes, we incubated Chol-HDO[PMO] and Chol-HDO[MOE] in rat CSF at 37 °C for up to 7 days. At each time point, SDS (0.025%) was added to dissociate potential protein–oligonucleotide complexes and ensure accurate electrophoretic mobility. The samples were then analyzed by gel electrophoresis. At 0 and 1 hour, both Chol-HDOs maintained a single band, indicating structural integrity. However, at later time points (24, 72, and 168 h), lower bands appeared with mobilities matching those of ligandless HDOs, suggesting that the linkage between cholesterol and the oligonucleotide was progressively degraded in CSF. Notably, degradation of Chol-HDO[PMO] occurred more rapidly than Chol-HDO[MOE], but both retained substantial stability up to 6 hours. These results demonstrate that the duplexes were successfully formed and that their stability is adequate for initial CNS delivery. The identity of the analytes and their time-dependent structural changes were verified by native PAGE analysis\nWe have added these data to the manuscript and expanded the relevant sections in the Results and Methods, as follows:\nResults section (Page 6, Lines 111–117)\nWe evaluated the formation and stability of the duplexes in CSF. Chol-HDO[PMO] and Chol-HDO[MOE] displayed a single band in nuclease-free water, confirming successful duplex formation (Supplementary Figure 1 a, b). Upon incubation in rat CSF, both duplexes remained intact for at least 1 hour. At later time points, lower bands emerged whose mobilities matched those of ligandless HDOs, indicating degradation of the cholesterol linkage. Chol-HDO[PMO] degraded faster than Chol-HDO[MOE] at 24, 72, and 168 h, but both constructs were stable within the first 6 h (Supplementary Figure 1 a, b).\nMaterials and Methods section (Page 20, Lines 427–431)\nTo validate duplex formation, HDOs were analyzed on 16% polyacrylamide gels. To assess stability, Chol-HDO[PMO] and Chol-HDO[MOE] were incubated with rat CSF at 37 °C for 0, 1, 6, 24, 72, and 168 h. At each time point, SDS (0.025%) was added to disrupt protein–oligonucleotide interactions. Samples were then frozen in liquid nitrogen, stored at –80 °C, and analyzed by electrophoresis.\nComment 7_1: Identification of apolipoprotein E as the main binding partner of the Chol-HDO[PMO]: the method and data by mass spec analysis of the protein is missing.\nResponse: Thank you for this insightful comment. To address the concern, we have now included detailed methods for the mass spectrometric identification of proteins bound to Chol-HDO[PMO] in the revised manuscript. Specifically, protein bands extracted from native gels of rat CSF samples incubated with Chol-HDO[PMO] were subjected to in-gel digestion followed by LC-MS/MS analysis.\nWe have added the sentence as follows:\nMaterials and Methods section (Page 26, Lines 582–594)\nMass spectrometry (MS)\nThe parameters of MS are summarized in Table S2. Chol-HDOs were incubated in rat CSF and separated using native gel electrophoresis, followed by visualization with GelRed. Gel pieces were washed with water and then dehydrated with acetonitrile. The samples were incubated with 10 mM DTT and 100 mM NH HCO at 60 °C for 1 h. After blocking with 50 mM iodoacetamide at room temperature for 45 min, the samples were digested with 1 pmol sequencing grade modified trypsin (Promega, Madison, WI, USA). The peptides were analyzed using a mass spectrometer (LTQ Orbitrap velos, Thermo Scientific) coupled with a nano-LC(EASY-nLC II, Thermo Scientific). Capillary Ex-Nano Mono cap C18 Trap column (0.075mm i.d. × 50 mm, GL sciences) and Capillary Ex-Nano Mono cap C18 Nano-flow Analytical column (0.075 mm i.d. × 150 mm, GL sciences) were used. For LC conditions, the following gradient was used (A, 0.1% formic acid/H2O; B, 70% ACN, 0.1% formic acid in H2O): 0 min 0% B; 0–2 min 0-29% B; 2–42 min 29-85% B; 42–47 min 85-100% B, 47–52 min 100% B, 300 nl/min. A top ten data-dependent acquisition MS method was used.\nResults section (Page 14, Lines 312–314)\nMass spectrometric analysis identified apolipoprotein E (ApoE) and transferrin as components of the protein band marked “*” (Fig. 6c and Supplementary Figure 6b).\nWe have included the mass spectrometry parameters as New Supplementary Table 2 and the detected peptides mapped on the ApoE protein sequence as New Supplementary Figure 6b.\nComment 7_2: A direct binding assay between apolipoprotein E and Chol-HDO[PMO] is needed to validate the claim. Does Chol-HDO[PMO] bind to human apolipoprotein E? This is not difficult to check with commercially available ApoE protein.\nResponse: Thank you for highlighting this fundamental point. To address this, we conducted a direct binding assay using commercially available human apolipoprotein E (ApoE) and Chol-HDO[PMO]. Native gel electrophoresis revealed a shifted band upon mixing the two, indicating complex formation (New Supplementary Figure 6c).\nInterestingly, the shifted band appeared at a molecular weight exceeding 180 kDa, despite ApoE being approximately 34 kDa. This result is consistent with previous reports that ApoE can form lipid-associated multimers with a molecular mass around 250 kDa (Raussens et al., J. Biol. Chem. 2000, Pubmed:10986285). We therefore interpret the observed shift as binding of Chol-HDO[PMO] to an ApoE multimer.\nWe have added this result as Supplementary Figure 6c and revised the manuscript accordingly with the following sentence:\nResults section (Page 14, Lines 323–326)\nChol-HDO[PMO] was also confirmed to bind human ApoE protein using native gel electrophoresis (Supplementary Figure 6c), whereas no binding was observed with purified transferrin (Supplementary Figure 6d). These findings further support the specificity of the interaction between Chol-HDO[PMO] and ApoE.\nComment 8: Mouse dosing: nanomole doses are cited, but there is no mouse weight given--actual dosage (nmole/kg) is not given, nor is variability in mouse weight shown.\nResponse: Thank you for pointing out this important issue. In this study, the mdx and SOD1 transgenic mice weighed 19–21 grams, corresponding to an approximate dosage of 476–526 nmol/kg for a 10 nmol per mouse injection. The rats weighed 180–200 grams, resulting in a dosage of approximately 300–316 nmol/kg for a 60 nmol injection per rat. To clarify the actual dosing relative to body weight, we have added this information to the Materials and Methods section.\nMaterials and Methods section (Page 21, Lines 460–462)\nThe mdx and SOD1 transgenic mice used in this study weighed 19–21 g. Therefore, a 10 nmol dose of PMO corresponds to approximately 476–526 nmol/kg. The rats weighed 180–200 g, and thus a 60 nmol dose equates to approximately 300–333 nmol/kg.\nComment 9: Figure 1f: stain color for ASO-specific probe is not given, making interpretation of the image difficult.\nResponse: Thank you for the reviewer’s helpful suggestion. To improve clarity, we have added the stain color information to Figure 1f and updated the figure legend accordingly.\nRevised Figure 1f legend (Page 37, Line 805):\nBiodistribution of ssPMO was visualized by in situ hybridization. Unfixed fresh frozen sections were stained with an ssPMO-specific probe and counterstained with hematoxylin 24 hours after dosing with 10 nmol of either ssPMO (upper) or Chol-HDO[PMO] (lower). Red: ssPMO; Blue: Hematoxylin. Bar = 20 µm. All graphs represent mean ± s.e.m.\nReviewer #3:\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nScott Bittner\nComment: We would like to sincerely thank Dr. Scott Bittner for his contribution to the peer review process as a co-reviewer. We greatly appreciate the constructive feedback provided through this collaborative review effort, which helped us to improve the quality of our manuscript.\nReviewer #4:\nNagata et al have carried out very interesting experiments that advance the field of nucleic acid therapeutics. The authors describe a unique method to leverage the safety of the PMO chemistry while enhancing delivery and potency by forming a heteroduplex with a complementary RNA strand stabilized at the ends with minimal phosphorothioate inter-nucleoside linkages. Thus, their HDO constructs have the potential to decrease nucleic acid chemistry based toxicity while maintaining or increasing potency of nucleic acid technology to enhance splicing or exon skipping particularly in multiple areas of the brain which is unprecedented.\nThe work is quite detailed and meets the standards of the field. Moreover the authors have investigated the importance of RNA vs DNA complementary strands. They have also determined whether length of ASO or chemistry of the ASO affects potency and delivery and provided sufficient, clear data to demonstrate the superiority of their HDO constructs. In addition they have tested whether the cholesterol linkage used is similar or superior to other lipid linkage used for CNS targeting of nucleic acids. Their methods are detailed and should be sufficient for anyone schooled in the art of nucleic acid technology to reproduce or evaluate their data.\nComment1:\nI would recommend the authors to spell check the document as well as reconcile Fig.1a with the description provided in the text for the 5' vs 3' cholesterol linkage.\nOverall, this is an exciting and seminal work in the field of nucleic acid therapeutics and i was pleased to review the manuscript.\nResponse: We would like to express our gratitude to the reviewer’s comment. We corrected Figure 1a as the reviewer kindly pointed out. We also checked the spelling throughout the manuscript and have arranged for proofreading of the manuscript.\nWe have also corrected the grammatical and typing errors that the reviewer kindly pointed out.\nFrom\nIn consistent with the previous report19, Chol-HDO[Gapmer] did not boost the silencing activity (Fig. 1c and Supplementary Fig. 1). Hence, when administered via CSF, Chol-HDO activity varied depending on the chemistry of the antisense strand.\nTo\nConsistent with the previous report19, Chol-HDO[Gapmer] did not boost the silencing activity (Fig. 1c and Supplementary Fig. 1). Hence, when administered via CSF, Chol-HDO activity varied depending on the chemistry of the antisense strand. (Page 7, Line 123)\nFrom\nInterestingly, Chol-HDO[PMO/OMe deplex] showed negligible skipping potency, probably because its structure was highly resistant to degradation by endogenous enzymes; therefore, ssPMO was unable to access the target RNA.\nTo\nChol-HDO[PMO/OMe deplex] showed negligible skipping potency, probably because its structure was highly resistant to degradation by endogenous enzymes; therefore, ssPMO was unable to access the target RNA. (Page 8, Line 165)\nFrom\nAfter Chol-HDO[PMO] treatment, the abnormal fear response was alliveated: the mice showed reduced freezing, increased traveled distances, and enhanced vertical activity (Fig. 3d-f).\nTo\nAfter Chol-HDO[PMO] treatment, the abnormal fear response was alleviated: the mice showed reduced freezing, increased traveled distances, and enhanced vertical activity (Fig. 3d-f). (Page 11, Line 240)\nIn addition to addressing the reviewers’ comments, we made the following improvements to enhance the overall quality of the manuscript.\n1. We have renamed all “Extended Figures” as “Supplementary Figures” throughout the manuscript and figure legends, in accordance with the journal’s guidelines.\n2. We annotated all graphs with precise p-values to facilitate interpretation of statistical significance.\n3. Figure 3b was replaced with a higher-resolution image acquired from a different region of the same sample, solely to improve visual clarity. The replacement depicts the same experimental condition and is representative of the same findings. This change does not affect the interpretation or conclusions of the study.\n4. In the course of revision, we identified that the fluorescence intensity values for puncta in Figures 3c were originally calculated as the sum of mean fluorescence intensities (MFI) without accounting for puncta size. To more accurately reflect the total signal within images of a fixed size, we reanalyzed the images by calculating the sum of area x MFI for each dystrophin punctum in each image, thereby obtaining the total integrated fluorescence intensity. The updated values are shown in the revised graphs, and the figure legends have been modified accordingly. This change does not alter the statistical significance or conclusions of the study.\n5. Inaccuracies in the numerical data of Figures 1d and 2a have been corrected.\nIn the original Figure 1d, 24 h after ICV injection, the concentration of Chol-HDO[PMO] relative to PMO was shown as 14.2-fold in the cerebellum, 11.9-fold in the hippocampus, and 6.6-fold in the cortex. After correcting the data, the actual fold changes are 5.7-fold, 6.7-fold, and 5.7-fold, respectively. This correction does not affect the study’s conclusions, as the precise fold-change values were not critical to the interpretation. We have revised the corresponding sentence as follows:\nResults section (Page 7, Line 130)\nFrom\nAfter 24 h following ICV administration, a 6.6-to 14-fold increase in ssPMO was detected in CNS tissues with Chol-HDO[PMO] administration compared to ssPMO injection (Fig. 1d).\nTo\nAfter 24 h following ICV administration, an increase of more than 5.7-fold in ssPMO was detected in CNS tissues with Chol-HDO[PMO] administration compared to ssPMO injection (Fig. 1d).\nIn the table of the original Figure 2a, the fold changes in ED50 of skipping activity by Chol-HDO[PMO] relative to PMO, measured 1 week after ICV injection, were shown as 8.7 in the brainstem, 11.2 in the striatum, 6.7 in the cortex, and 5.7 in the spinal cord. After correction, these values are 9.9, 15.4, 6.8, and 6.5, respectively. This correction does not affect the study’s conclusions, and no changes have been made to the manuscript as a result.\n6. In Figure 1e, the axis limit was adjusted to improve visual clarity."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have gone and above and beyond to address all of my previous concerns. I have no additional concerns. This is an extremely well done and exciting study that will be well recognized by the field.", "reviewer_2": "The authors have addressed my previous concerns satisfactorily. I have only a few minor points that should be addressed in the revision:\n1. Materials and Methods: The description of the “ssPMO-specific probe” in line 803: is missing. Please provide details of its nature and composition.\n2. Abstract: The statement “Here, a cholesterol-conjugated PMO/RNA heteroduplex (Chol-HDO[PMO]) outperformed 39 single-stranded ASOs with conventional chemistries, achieving up to 16-fold higher potency, safety, and longer activity across the CNS.” appears overstated. It is not clear how “16-fold higher safety” and “longer activity” were measured. The authors should revise this statement to more accurately reflect the available data.", "reviewer_4": "The manuscript is noteworthy and important to the field. I support the responses and revisions from the authors, and recommend publication of the manuscript"}, "author_response": "Response to Referees Letter:\nWe would like to express our sincere gratitude to the reviewer for the constructive suggestions, which have significantly improved our manuscript.\nThe manuscript has been changed in accordance with the reviewers’ comments. In the responses below, the revised or newly added parts of the manuscript are indicated in red for clarity.\nWe have voluntarily revised Figure 1a, 4a, 5a, and 6g to improve clarity. These revisions are purely illustrative and do not involve any scientific changes. In addition, minor grammatical corrections were made.\nWe hope these improvements further enhance the manuscript’s clarity and rigor.\nResponses to the Reviewers’ comments\nReviewer #1:\nThe authors have gone and above and beyond to address all of my previous concerns. I have no additional concerns. This is an extremely well done and exciting study that will be well recognized by the field.\nResponse:\nWe sincerely thank the reviewer for the constructive and encouraging comment.\nReviewer #2:\nThe authors have addressed my previous concerns satisfactorily. I have only a few minor points that should be addressed in the revision:\nComment 1. Materials and Methods: The description of the “ssPMO-specific probe” in line 803: is missing. Please provide details of its nature and composition.\nResponse:\nWe appreciate your valuable feedback. We have contacted Advanced Cell Diagnostics, Inc., the supplier of the probe. The company provided a reference paper describing the general design strategy of their probes; however, the specific sequence and chemical modifications remain confidential. Based on the information in this information, we have added a description of the probe’s characteristics and composition, along with its catalog number.\nMethods section (Page 22, Line 511–519)\nThe ssPMO-specific probe used in this study was designed by Advanced Cell Diagnostics, Inc. (ACD) based on the target ssASO sequence information we provided. The general probe architecture followed ACD’s RNAscope design53—comprising a 14-base tail sequence, a spacer, and a target-specific region complementary to ssPMO. We previously used the same vendor-provided ssPMO-targeting probe in our earlier work26. As in that study, a pair of target probes bearing distinct tail sequences hybridized contiguously to the target ssASO; the two tails then serve as a scaffold for subsequent signal amplification. The exact probe sequences and chemical modifications are proprietary and not disclosed by the manufacturer. For the present experiments, we used a PMO probe (SR-ASO-PMO-S1, #1088271-S1; Advanced Cell Diagnostics, Westminster, UK); slides were incubated with the probe for 2 h at 40 °C.\nNew reference in the manuscript\n53. Wang F, Flanagan J, Su N, et al. RNAscope: A novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. J Mol Diagnostics. 2012;14(1):22-29. doi:10.1016/j.jmoldx.2011.08.002\nComment 2. Abstract: The statement “Here, a cholesterol-conjugated PMO/RNA heteroduplex (Chol-HDO[PMO]) outperformed 39 single-stranded ASOs with conventional chemistries, achieving up to 16-fold higher potency, safety, and longer activity across the CNS.” appears overstated. It is not clear how “16-fold higher safety” and “longer activity” were measured. The authors should revise this statement to more accurately reflect the available data.\nResponse:\nThank you for raising this important point. We have revised the sentence as follows:\nFrom\nHere, a cholesterol-conjugated PMO/RNA heteroduplex (Chol-HDO[PMO]) outperformed single-stranded ASOs with conventional chemistries, achieving up to 16-fold higher potency, safety, and longer activity across the CNS.\nTo (Page 3, Line 39)\nHere, a cholesterol-conjugated PMO/RNA heteroduplex (Chol-HDO[PMO]) outperformed conventional single-stranded ASOs, achieving up to a 16-fold increase in potency without detectable toxicity and maintaining stronger ASO activity throughout the observation period.\nReviewer #4:\nThe manuscript is noteworthy and important to the field. I support the responses and revisions from the authors, and recommend publication of the manuscript\nResponse:\nWe sincerely thank the reviewer for the thorough evaluation and helpful feedback, which have helped us refine both the content and presentation of our study."}]

Decision letter