Journal: Molecular Therapy. Nucleic Acids

Article Title: Unraveling and controlling late-onset neurotoxicity of antisense oligonucleotides through strategic chemical modifications

doi: 10.1016/j.omtn.2025.102692

Figure Lengend Snippet: 2′-OMe modification at gap position 2 mitigates late-onset neurotoxicity of i.c.v.-injected ASO1 (A–C) Neuro-2a cells were transfected with 50 nM ASO1. (A) Representative microscopic images of cells 24 and 72 h after transfection. (B) Cell number 24 and 72 h post-transfection. (C) LDH release in medium 24 and 72 h post-transfection. Data in (B) and (C) are presented as mean ± SEM, as a percentage relative to vehicle (Lipofectamine 2000 with PBS) ( n = 4 biological replicates per group). ns, not significant ( p > 0.05). ## p ≤ 0.01, ### p ≤ 0.001, and #### p ≤ 0.0001; data were analyzed using Student’s two-tailed t tests with vehicle. (D) Schematic of the in vitro experiment and assessment workflow. (E) Schematic of the in vitro experiment introducing 2′-OMe RNA at each gap position (gap walking) in ASO1. (F) Neuro-2a cells were transfected with 4 nM ASO1 (knockdown activity assessment) or 50 nM ASO1 (toxicity assessment), with or without 2′-OMe modification at each gap position. KD, knockdown. Blue letters indicate LNA, whereas blue “C” indicates LNA with 5-methylcytosine. Black letters indicate DNA, and red letters indicate 2′-OMe-modified DNA. ΔT m indicates the T m of the sequence minus the T m of the parent ASO. Target mRNA levels, cell number, LDH release, Cdkn1a mRNA levels, and Il-6 mRNA levels 72 h post-transfection are shown as the mean percentage relative to vehicle ( n = 2 biological replicates per group for Il-6 mRNA of ASO1 with 2′-OMe at gap position 4; n = 4 for others). The highlighted sequences with 2′-OMe modifications show an increase in cell number and a decrease in LDH release compared to the parent ASOs. See also . (G and H) Seven-week-old female ICR mice ( n = 3 per group for the vehicle and n = 4 for all other groups) were injected intracerebroventricularly with 38.4 nmol of ASO1 and ASO1 modified with 2′-OMe at gap position 1 or 2. Toxicity was assessed on the indicated days, and mice were sacrificed 10 days postinjection. (H) Tolerability scores were measured at the indicated time points, and data are presented as mean ± SEM. ## p ≤ 0.01 and ### p ≤ 0.001; data were analyzed using Student’s two-tailed t tests with vehicle. ∗ p ≤ 0.05 and ∗∗ p ≤ 0.01; data were analyzed using one-way ANOVA, followed by Tukey’s post hoc tests.

Article Snippet: The primers and probes for mouse Mapt (Mm00521988_m1), Cdkn1a (Mm01303209_m1), Il-6 (Mm00446190_m1), Hdac2 (Mm00515108_m1), Casp3 (Mm01195085_m1), Casp7 (Mm00432322_m1), human SNCA (Hs01103383_m1), CDKN1A (Hs00355782_m1), rat Hprt1 (Rn01527840_m1), and Cdkn1a (Rn00589996_m1) were purchased from Thermo Fisher Scientific.

Techniques: Modification, Injection, Transfection, Two Tailed Test, In Vitro, Knockdown, Activity Assay, Sequencing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Unraveling and controlling late-onset neurotoxicity of antisense oligonucleotides through strategic chemical modifications

doi: 10.1016/j.omtn.2025.102692

Figure Lengend Snippet: 2′-OMe at gap position 2 increases late-onset neurotoxicity of i.c.v.-injected ASO2 (A–C) BE(2)-M17 cells were transfected with 20 nM ASO2, with or without 2′-OMe modification at gap position 2 Cell number (A), LDH release in medium (B), and CDKN1A mRNA levels (C) are shown as the mean percentage relative to vehicle ( n = 4 biological replicates per group). (D) Neuro-2a cells were transfected with 2 nM ASO2, with or without 2′-OMe modification at gap position 2. Target Hdac2 mRNA levels 72 h post-transfection are shown as the mean percentage relative to vehicle ( n = 4 biological replicates per group). (E–H) Seven-week-old female C57BL/6 mice ( n = 4 per group) were injected intracerebroventricularly with 19.0 nmol of ASO2, with or without 2′-OMe modification at gap position 2. Toxicity was assessed on the indicated days, and mice were sacrificed 7 days postinjection. Tolerability scores (F) and maximum speed in open-field tests (G) were documented 6 days postinjection. Body weight was measured at 3 and 6 days postinjection (H). Data in (A)–(D) and (F)–(H) are presented as mean ± SEM. # p ≤ 0.05, ### p ≤ 0.001, and #### p ≤ 0.0001; data were analyzed using Student’s two-tailed t tests with vehicle. ∗ p ≤ 0.05 and ∗∗∗∗ p ≤ 0.0001; data were analyzed using one-way ANOVA, followed by Tukey’s post hoc tests.

Article Snippet: The primers and probes for mouse Mapt (Mm00521988_m1), Cdkn1a (Mm01303209_m1), Il-6 (Mm00446190_m1), Hdac2 (Mm00515108_m1), Casp3 (Mm01195085_m1), Casp7 (Mm00432322_m1), human SNCA (Hs01103383_m1), CDKN1A (Hs00355782_m1), rat Hprt1 (Rn01527840_m1), and Cdkn1a (Rn00589996_m1) were purchased from Thermo Fisher Scientific.

Techniques: Injection, Transfection, Modification, Two Tailed Test

Journal: Molecular Therapy. Nucleic Acids

Article Title: Unraveling and controlling late-onset neurotoxicity of antisense oligonucleotides through strategic chemical modifications

doi: 10.1016/j.omtn.2025.102692

Figure Lengend Snippet: 5′-CP modification in gap mitigates neurotoxicity of both ASO1 and ASO2 (A) Schematic of introducing 5′-CP DNA at each gap position in ASO1. (B) Neuro-2a cells were transfected with 4 or 50 nM ASO1, with or without 5′-CP at each gap position. Green letters indicate 5′-CP-modified DNA. n = 3 biological replicates per group for Cdkn1a and Il-6 mRNA of ASO1 with 5′-CP gap positions 4 and 10; n = 4 for others. See also . (C–E) Seven-week-old female ICR mice were injected intracerebroventricularly with 38.4 nmol of ASO1, with or without 5′-CP position 3 ( n = 3 for ASO1 with 5′-CP position 3; n = 4 for others) and sacrificed 21 days postinjection. Tolerability scores at the indicated times (D) and left hippocampal Mapt mRNA levels at 21 days postinjection (E) were measured. (F–J) Seven-week-old female C57BL/6 mice ( n = 4) were injected intracerebroventricularly with 19.0 nmol of ASO2, with or without 5′-CP position 3, and sacrificed 11 days postinjection. (G) Tolerability scores were recorded. (H) Representative track plots 10 days postinjection. (I and J) Target Hdac2 and Cdkn1a mRNA levels in the left hippocampus were measured 8–11 days postinjection, with round, triangle, and rhombus symbols indicating measurements at days 11, 9, and 8, respectively. (K) Seven-week-old female C57BL/6 mice ( n = 3–4 per group) were injected intracerebroventricularly with 9.49 nmol of ASO2, with or without 5′-CP position 3, labeled with Alexa Fluor 647 on the 3′ end. ASO delivery was assessed by measuring fluorescence intensity across brain regions 24 h and 11 days postinjection. (L and M) Nine-week-old male Slc:SD rats ( n = 4) were injected intrathecally via spinal canal catheters with 190 nmol of ASO2, with or without 5′-CP position 3, and sacrificed 14 days postinjection. (M) Tolerability scores were documented postinjection. Data in (D), (E), (G), (I), (J), (K), and (M) are presented as mean ± SEM. ## p ≤ 0.01 and #### p ≤ 0.0001; data were analyzed using Student’s two-tailed t tests with vehicle. ns, not significant ( p > 0.05). ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, and ∗∗∗∗ p ≤ 0.0001; data were analyzed using one-way ANOVA, followed by Tukey’s post hoc tests.

Article Snippet: The primers and probes for mouse Mapt (Mm00521988_m1), Cdkn1a (Mm01303209_m1), Il-6 (Mm00446190_m1), Hdac2 (Mm00515108_m1), Casp3 (Mm01195085_m1), Casp7 (Mm00432322_m1), human SNCA (Hs01103383_m1), CDKN1A (Hs00355782_m1), rat Hprt1 (Rn01527840_m1), and Cdkn1a (Rn00589996_m1) were purchased from Thermo Fisher Scientific.

Techniques: Modification, Transfection, Injection, Labeling, Fluorescence, Two Tailed Test

Journal: Molecular Therapy. Nucleic Acids

Article Title: Unraveling and controlling late-onset neurotoxicity of antisense oligonucleotides through strategic chemical modifications

doi: 10.1016/j.omtn.2025.102692

Figure Lengend Snippet: In vitro and in vivo neurotoxicity evaluation of PS-ASOs with PO replacement (A) Structures of a phosphodiester (PO) bond and a phosphorothioate (PS) bond. (B) BE(2)-M17 cells were transfected with 20 nM ASO2 with or without replacement of PS bond 5′-adjacent to a DNA monomer at gap positions 1, 3, or 9 with a PO bond (PS-1(−), PS-3(−), or PS-9(−)). Cell number and LDH release 72 h post-transfection are shown as the mean percentage relative to vehicle ( n = 4 biological replicates per group). Underlined letters indicate substitution of a PS bond with a PO bond at the 5′-adjacent site of the DNA monomer. See also . (C–G) Seven-week-old female C57BL/6 mice were injected intracerebroventricularly with 19.0 nmol of ASO2 or ASO2 with PS-1(−), PS-3(−), or PS-9(−) ( n = 4 per group for vehicle and the parent ASO2; n = 5 per group for the others). Toxicity was assessed on the indicated days, and mice were sacrificed 10 days postinjection. Tolerability scores (D) and maximum speed in open-field tests (E) were documented postinjection. Target Hdac2 and Cdkn1a mRNA levels in the left hippocampus were measured 6–10 days postinjection (F and G). White round and triangle symbols represent measurements taken on days 10 and 9 postinjection, respectively. Red triangle and square symbols represent samples taken postmortem on days 9 and 6 postinjection, respectively. For (F) and (G), n = 5 per group for ASO2 PS-3(−); n = 4 per group for others. Data in (D)–(G) are presented as mean ± SEM. # p ≤ 0.05, ## p ≤ 0.01, ### p ≤ 0.001, and #### p ≤ 0.0001; data were analyzed using Student’s two-tailed t tests with vehicle. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, and ∗∗∗ p ≤ 0.001; data were analyzed using one-way ANOVA, followed by Tukey’s post hoc tests.

Article Snippet: The primers and probes for mouse Mapt (Mm00521988_m1), Cdkn1a (Mm01303209_m1), Il-6 (Mm00446190_m1), Hdac2 (Mm00515108_m1), Casp3 (Mm01195085_m1), Casp7 (Mm00432322_m1), human SNCA (Hs01103383_m1), CDKN1A (Hs00355782_m1), rat Hprt1 (Rn01527840_m1), and Cdkn1a (Rn00589996_m1) were purchased from Thermo Fisher Scientific.

Techniques: In Vitro, In Vivo, Transfection, Injection, Two Tailed Test

Journal: Molecular Therapy. Nucleic Acids

Article Title: Unraveling and controlling late-onset neurotoxicity of antisense oligonucleotides through strategic chemical modifications

doi: 10.1016/j.omtn.2025.102692

Figure Lengend Snippet: 5′-CP modification with PO replacement mitigates late-onset neurotoxicity of i.c.v.-injected PS-ASOs without reducing knockdown activity (A) Schematic of introducing DNA monomers with 5′-CP modification and PO replacement of the 5′-adjacent PS bond at each ASO1 gap position. (B) Neuro-2a cells were transfected with 4 or 50 nM ASO1, with or without replacement of 5′-CP with PO replacement. n = 3 biological replicates per group for cell number of vehicle and ASO1; n = 4 for others. N.A., not applicable due to insufficient RNA. See also . (C–E) Seven-week-old female ICR mice ( n = 4) were injected intracerebroventricularly with 38.4 nmol of ASO1 and ASO1 with 5′-CP position 3 PS-3(−) and sacrificed 21 days postinjection. Tolerability scores (D) and target Mapt mRNA levels in the left hippocampus and lumbar cord (E) were measured. (F) 5′-CP positions 1,3, or 9 with or without PO replacement in ASO2. (G) Neuro-2a cells were transfected with 2 nM ASOs and BE(2)-M17 cells for 20 nM ASOs: ASO2; ASO2 2′-OMe position 2; and ASO2 5′-CP position 1, 3, or 9 with or without PO replacement ( n = 4 biological replicates per group). See also . (H–K) Seven-week-old female C57BL/6 mice ( n = 4) were injected intracerebroventricularly with 19.0 nmol ASO2, with 5′-CP positions 1, 3, or 9, with or without PO replacement, and sacrificed 14 days postinjection. (I) Tolerability scores were documented. Target Hdac2 (J) and Cdkn1a (K) mRNA levels in the left hippocampus were measured 10–14 days postinjection. Symbols indicate collection days: rounds (day 14), rhombus (day 10), and red triangles (day 13, postmortem). Data in (D), (E), and (I)–(K) are presented as mean ± SEM. # p ≤ 0.05, ## p ≤ 0.01, ### p ≤ 0.001, and #### p ≤ 0.0001; data were analyzed using Student’s two-tailed t tests with vehicle. ns, not significant ( p > 0.05). ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, and ∗∗∗ p ≤ 0.001; data were analyzed using one-way ANOVA, followed by Tukey’s post hoc tests.

Article Snippet: The primers and probes for mouse Mapt (Mm00521988_m1), Cdkn1a (Mm01303209_m1), Il-6 (Mm00446190_m1), Hdac2 (Mm00515108_m1), Casp3 (Mm01195085_m1), Casp7 (Mm00432322_m1), human SNCA (Hs01103383_m1), CDKN1A (Hs00355782_m1), rat Hprt1 (Rn01527840_m1), and Cdkn1a (Rn00589996_m1) were purchased from Thermo Fisher Scientific.

Techniques: Modification, Injection, Knockdown, Activity Assay, Transfection, Two Tailed Test

Journal: Molecular Therapy. Nucleic Acids

Article Title: Unraveling and controlling late-onset neurotoxicity of antisense oligonucleotides through strategic chemical modifications

doi: 10.1016/j.omtn.2025.102692

Figure Lengend Snippet: 2′-OMe modification at gap position 2 mitigates late-onset neurotoxicity of i.c.v.-injected ASO1 (A–C) Neuro-2a cells were transfected with 50 nM ASO1. (A) Representative microscopic images of cells 24 and 72 h after transfection. (B) Cell number 24 and 72 h post-transfection. (C) LDH release in medium 24 and 72 h post-transfection. Data in (B) and (C) are presented as mean ± SEM, as a percentage relative to vehicle (Lipofectamine 2000 with PBS) ( n = 4 biological replicates per group). ns, not significant ( p > 0.05). ## p ≤ 0.01, ### p ≤ 0.001, and #### p ≤ 0.0001; data were analyzed using Student’s two-tailed t tests with vehicle. (D) Schematic of the in vitro experiment and assessment workflow. (E) Schematic of the in vitro experiment introducing 2′-OMe RNA at each gap position (gap walking) in ASO1. (F) Neuro-2a cells were transfected with 4 nM ASO1 (knockdown activity assessment) or 50 nM ASO1 (toxicity assessment), with or without 2′-OMe modification at each gap position. KD, knockdown. Blue letters indicate LNA, whereas blue “C” indicates LNA with 5-methylcytosine. Black letters indicate DNA, and red letters indicate 2′-OMe-modified DNA. ΔT m indicates the T m of the sequence minus the T m of the parent ASO. Target mRNA levels, cell number, LDH release, Cdkn1a mRNA levels, and Il-6 mRNA levels 72 h post-transfection are shown as the mean percentage relative to vehicle ( n = 2 biological replicates per group for Il-6 mRNA of ASO1 with 2′-OMe at gap position 4; n = 4 for others). The highlighted sequences with 2′-OMe modifications show an increase in cell number and a decrease in LDH release compared to the parent ASOs. See also . (G and H) Seven-week-old female ICR mice ( n = 3 per group for the vehicle and n = 4 for all other groups) were injected intracerebroventricularly with 38.4 nmol of ASO1 and ASO1 modified with 2′-OMe at gap position 1 or 2. Toxicity was assessed on the indicated days, and mice were sacrificed 10 days postinjection. (H) Tolerability scores were measured at the indicated time points, and data are presented as mean ± SEM. ## p ≤ 0.01 and ### p ≤ 0.001; data were analyzed using Student’s two-tailed t tests with vehicle. ∗ p ≤ 0.05 and ∗∗ p ≤ 0.01; data were analyzed using one-way ANOVA, followed by Tukey’s post hoc tests.

Article Snippet: The primers and probes for mouse Mapt (Mm00521988_m1), Cdkn1a (Mm01303209_m1), Il-6 (Mm00446190_m1), Hdac2 (Mm00515108_m1), Casp3 (Mm01195085_m1), Casp7 (Mm00432322_m1), human SNCA (Hs01103383_m1), CDKN1A (Hs00355782_m1), rat Hprt1 (Rn01527840_m1), and Cdkn1a (Rn00589996_m1) were purchased from Thermo Fisher Scientific.

Techniques: Modification, Injection, Transfection, Two Tailed Test, In Vitro, Knockdown, Activity Assay, Sequencing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Unraveling and controlling late-onset neurotoxicity of antisense oligonucleotides through strategic chemical modifications

doi: 10.1016/j.omtn.2025.102692

Figure Lengend Snippet: 2′-OMe at gap position 2 increases late-onset neurotoxicity of i.c.v.-injected ASO2 (A–C) BE(2)-M17 cells were transfected with 20 nM ASO2, with or without 2′-OMe modification at gap position 2 Cell number (A), LDH release in medium (B), and CDKN1A mRNA levels (C) are shown as the mean percentage relative to vehicle ( n = 4 biological replicates per group). (D) Neuro-2a cells were transfected with 2 nM ASO2, with or without 2′-OMe modification at gap position 2. Target Hdac2 mRNA levels 72 h post-transfection are shown as the mean percentage relative to vehicle ( n = 4 biological replicates per group). (E–H) Seven-week-old female C57BL/6 mice ( n = 4 per group) were injected intracerebroventricularly with 19.0 nmol of ASO2, with or without 2′-OMe modification at gap position 2. Toxicity was assessed on the indicated days, and mice were sacrificed 7 days postinjection. Tolerability scores (F) and maximum speed in open-field tests (G) were documented 6 days postinjection. Body weight was measured at 3 and 6 days postinjection (H). Data in (A)–(D) and (F)–(H) are presented as mean ± SEM. # p ≤ 0.05, ### p ≤ 0.001, and #### p ≤ 0.0001; data were analyzed using Student’s two-tailed t tests with vehicle. ∗ p ≤ 0.05 and ∗∗∗∗ p ≤ 0.0001; data were analyzed using one-way ANOVA, followed by Tukey’s post hoc tests.

Article Snippet: The primers and probes for mouse Mapt (Mm00521988_m1), Cdkn1a (Mm01303209_m1), Il-6 (Mm00446190_m1), Hdac2 (Mm00515108_m1), Casp3 (Mm01195085_m1), Casp7 (Mm00432322_m1), human SNCA (Hs01103383_m1), CDKN1A (Hs00355782_m1), rat Hprt1 (Rn01527840_m1), and Cdkn1a (Rn00589996_m1) were purchased from Thermo Fisher Scientific.

Techniques: Injection, Transfection, Modification, Two Tailed Test

Journal: Molecular Therapy. Nucleic Acids

Article Title: Unraveling and controlling late-onset neurotoxicity of antisense oligonucleotides through strategic chemical modifications

doi: 10.1016/j.omtn.2025.102692

Figure Lengend Snippet: 5′-CP modification in gap mitigates neurotoxicity of both ASO1 and ASO2 (A) Schematic of introducing 5′-CP DNA at each gap position in ASO1. (B) Neuro-2a cells were transfected with 4 or 50 nM ASO1, with or without 5′-CP at each gap position. Green letters indicate 5′-CP-modified DNA. n = 3 biological replicates per group for Cdkn1a and Il-6 mRNA of ASO1 with 5′-CP gap positions 4 and 10; n = 4 for others. See also . (C–E) Seven-week-old female ICR mice were injected intracerebroventricularly with 38.4 nmol of ASO1, with or without 5′-CP position 3 ( n = 3 for ASO1 with 5′-CP position 3; n = 4 for others) and sacrificed 21 days postinjection. Tolerability scores at the indicated times (D) and left hippocampal Mapt mRNA levels at 21 days postinjection (E) were measured. (F–J) Seven-week-old female C57BL/6 mice ( n = 4) were injected intracerebroventricularly with 19.0 nmol of ASO2, with or without 5′-CP position 3, and sacrificed 11 days postinjection. (G) Tolerability scores were recorded. (H) Representative track plots 10 days postinjection. (I and J) Target Hdac2 and Cdkn1a mRNA levels in the left hippocampus were measured 8–11 days postinjection, with round, triangle, and rhombus symbols indicating measurements at days 11, 9, and 8, respectively. (K) Seven-week-old female C57BL/6 mice ( n = 3–4 per group) were injected intracerebroventricularly with 9.49 nmol of ASO2, with or without 5′-CP position 3, labeled with Alexa Fluor 647 on the 3′ end. ASO delivery was assessed by measuring fluorescence intensity across brain regions 24 h and 11 days postinjection. (L and M) Nine-week-old male Slc:SD rats ( n = 4) were injected intrathecally via spinal canal catheters with 190 nmol of ASO2, with or without 5′-CP position 3, and sacrificed 14 days postinjection. (M) Tolerability scores were documented postinjection. Data in (D), (E), (G), (I), (J), (K), and (M) are presented as mean ± SEM. ## p ≤ 0.01 and #### p ≤ 0.0001; data were analyzed using Student’s two-tailed t tests with vehicle. ns, not significant ( p > 0.05). ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, and ∗∗∗∗ p ≤ 0.0001; data were analyzed using one-way ANOVA, followed by Tukey’s post hoc tests.

Article Snippet: The primers and probes for mouse Mapt (Mm00521988_m1), Cdkn1a (Mm01303209_m1), Il-6 (Mm00446190_m1), Hdac2 (Mm00515108_m1), Casp3 (Mm01195085_m1), Casp7 (Mm00432322_m1), human SNCA (Hs01103383_m1), CDKN1A (Hs00355782_m1), rat Hprt1 (Rn01527840_m1), and Cdkn1a (Rn00589996_m1) were purchased from Thermo Fisher Scientific.

Techniques: Modification, Transfection, Injection, Labeling, Fluorescence, Two Tailed Test

Journal: Molecular Therapy. Nucleic Acids

Article Title: Unraveling and controlling late-onset neurotoxicity of antisense oligonucleotides through strategic chemical modifications

doi: 10.1016/j.omtn.2025.102692

Figure Lengend Snippet: In vitro and in vivo neurotoxicity evaluation of PS-ASOs with PO replacement (A) Structures of a phosphodiester (PO) bond and a phosphorothioate (PS) bond. (B) BE(2)-M17 cells were transfected with 20 nM ASO2 with or without replacement of PS bond 5′-adjacent to a DNA monomer at gap positions 1, 3, or 9 with a PO bond (PS-1(−), PS-3(−), or PS-9(−)). Cell number and LDH release 72 h post-transfection are shown as the mean percentage relative to vehicle ( n = 4 biological replicates per group). Underlined letters indicate substitution of a PS bond with a PO bond at the 5′-adjacent site of the DNA monomer. See also . (C–G) Seven-week-old female C57BL/6 mice were injected intracerebroventricularly with 19.0 nmol of ASO2 or ASO2 with PS-1(−), PS-3(−), or PS-9(−) ( n = 4 per group for vehicle and the parent ASO2; n = 5 per group for the others). Toxicity was assessed on the indicated days, and mice were sacrificed 10 days postinjection. Tolerability scores (D) and maximum speed in open-field tests (E) were documented postinjection. Target Hdac2 and Cdkn1a mRNA levels in the left hippocampus were measured 6–10 days postinjection (F and G). White round and triangle symbols represent measurements taken on days 10 and 9 postinjection, respectively. Red triangle and square symbols represent samples taken postmortem on days 9 and 6 postinjection, respectively. For (F) and (G), n = 5 per group for ASO2 PS-3(−); n = 4 per group for others. Data in (D)–(G) are presented as mean ± SEM. # p ≤ 0.05, ## p ≤ 0.01, ### p ≤ 0.001, and #### p ≤ 0.0001; data were analyzed using Student’s two-tailed t tests with vehicle. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, and ∗∗∗ p ≤ 0.001; data were analyzed using one-way ANOVA, followed by Tukey’s post hoc tests.

Article Snippet: The primers and probes for mouse Mapt (Mm00521988_m1), Cdkn1a (Mm01303209_m1), Il-6 (Mm00446190_m1), Hdac2 (Mm00515108_m1), Casp3 (Mm01195085_m1), Casp7 (Mm00432322_m1), human SNCA (Hs01103383_m1), CDKN1A (Hs00355782_m1), rat Hprt1 (Rn01527840_m1), and Cdkn1a (Rn00589996_m1) were purchased from Thermo Fisher Scientific.

Techniques: In Vitro, In Vivo, Transfection, Injection, Two Tailed Test

Journal: Molecular Therapy. Nucleic Acids

Article Title: Unraveling and controlling late-onset neurotoxicity of antisense oligonucleotides through strategic chemical modifications

doi: 10.1016/j.omtn.2025.102692

Figure Lengend Snippet: 5′-CP modification with PO replacement mitigates late-onset neurotoxicity of i.c.v.-injected PS-ASOs without reducing knockdown activity (A) Schematic of introducing DNA monomers with 5′-CP modification and PO replacement of the 5′-adjacent PS bond at each ASO1 gap position. (B) Neuro-2a cells were transfected with 4 or 50 nM ASO1, with or without replacement of 5′-CP with PO replacement. n = 3 biological replicates per group for cell number of vehicle and ASO1; n = 4 for others. N.A., not applicable due to insufficient RNA. See also . (C–E) Seven-week-old female ICR mice ( n = 4) were injected intracerebroventricularly with 38.4 nmol of ASO1 and ASO1 with 5′-CP position 3 PS-3(−) and sacrificed 21 days postinjection. Tolerability scores (D) and target Mapt mRNA levels in the left hippocampus and lumbar cord (E) were measured. (F) 5′-CP positions 1,3, or 9 with or without PO replacement in ASO2. (G) Neuro-2a cells were transfected with 2 nM ASOs and BE(2)-M17 cells for 20 nM ASOs: ASO2; ASO2 2′-OMe position 2; and ASO2 5′-CP position 1, 3, or 9 with or without PO replacement ( n = 4 biological replicates per group). See also . (H–K) Seven-week-old female C57BL/6 mice ( n = 4) were injected intracerebroventricularly with 19.0 nmol ASO2, with 5′-CP positions 1, 3, or 9, with or without PO replacement, and sacrificed 14 days postinjection. (I) Tolerability scores were documented. Target Hdac2 (J) and Cdkn1a (K) mRNA levels in the left hippocampus were measured 10–14 days postinjection. Symbols indicate collection days: rounds (day 14), rhombus (day 10), and red triangles (day 13, postmortem). Data in (D), (E), and (I)–(K) are presented as mean ± SEM. # p ≤ 0.05, ## p ≤ 0.01, ### p ≤ 0.001, and #### p ≤ 0.0001; data were analyzed using Student’s two-tailed t tests with vehicle. ns, not significant ( p > 0.05). ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, and ∗∗∗ p ≤ 0.001; data were analyzed using one-way ANOVA, followed by Tukey’s post hoc tests.

Article Snippet: The primers and probes for mouse Mapt (Mm00521988_m1), Cdkn1a (Mm01303209_m1), Il-6 (Mm00446190_m1), Hdac2 (Mm00515108_m1), Casp3 (Mm01195085_m1), Casp7 (Mm00432322_m1), human SNCA (Hs01103383_m1), CDKN1A (Hs00355782_m1), rat Hprt1 (Rn01527840_m1), and Cdkn1a (Rn00589996_m1) were purchased from Thermo Fisher Scientific.

Techniques: Modification, Injection, Knockdown, Activity Assay, Transfection, Two Tailed Test