Compounds and methods for treating human subjects

Abstract

Described herein are compounds and methods that can be used to treat diseases or conditions by increasing functional MeCP2 proteins.

Description

[0001] cross-application

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 498,404, filed April 26, 2023, which is incorporated herein by reference in its entirety. Background Technology

[0003] MeCP2 protein deficiency or activity has been observed in subjects with Rett syndrome, a rare and severe neurodevelopmental disorder that primarily affects women. In over 95% of cases, Rett syndrome is associated with MECP2 mutations. These mutations are almost always de novo, with T158M being the most common missense mutation identified in classic Rett syndrome. Characteristics include slowed head growth, loss of language skills, hand stereotypes and loss of purposeful hand skills, irregular breathing, motor and severe intellectual disability, and seizures (60-80% of patients), with 40% losing the ability to walk or never being able to walk. Subjects have an increased risk of premature death, with 70% surviving until age 50; and the mortality rate for classic diagnosis is higher than for atypical diagnosis. No neuronal loss or neurodegeneration is observed post-mortem in Rett patients, indicating no significant changes in brain structure; therefore, treatment after the onset of developmental regression may potentially reduce the severity of symptoms. Rett syndrome typically presents with developmental regression between 6 and 18 months of age, with a stable phase usually following the regression. The median age of seizures is 50 months. Diagnosis is based on clinical findings (using the RTT diagnostic criteria worksheet) and can also be determined through genetic testing. Although genetic testing is common in current diagnostic approaches, many older women are not yet tested.

[0004] To date, there are no approved therapies specifically for Rett syndrome. Management includes occupational, speech, and physical therapies, as well as medications for seizures, muscle stiffness, anxiety, or sleep. Seizures affect 60–80% of patients with Rett syndrome, and drug resistance is present in approximately one-third of cases. There is a need for therapeutic agents that can be used to treat this condition or disease by increasing the activity of the MeCP2 protein. Summary of the Invention

[0005] This article provides therapeutic agents that can be used to increase the activity of MeCP2 protein and treat symptoms or diseases.

[0006] In some respects, this paper provides a compound of formula (I): X A X N1 X N2 X N3 X5 X6 X7 X8 X9 X 10 X11 X 12 X 13 X 14 X15X 16 X 17 X 18 X 19 X 20 X C1 X C2 X C3 X B , where X A for And B A The following conditions must be met: (i) When X N1、 X N2 and X N3 And when X5 does not exist, it is (ii) When X N1、 X N2 and X N3 When it does not exist and X5 exists, it is: (iii) When X N1 and X N2 It does not exist and X N3 When X5 exists, it is (iv) When X N1 It does not exist and X N2 and X N3 And when X5 exists, it is ; or (v) when X N1 X N2 and X N3 And when X5 exists, it is ;X N1 for Or it does not exist, where if X N1 If it exists, then X N2 and X N3 And X5 exists; X N2 for Or it does not exist, where if X N2 If it exists, then X N3 And X5 exists; X N3 for Or it does not exist, where if X N3 If it exists, then X5 exists; X5 is Or it may not exist; X6 is X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for , or ;X 14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X 18 for ;X 19 for ;X 20 for Or it may not exist; X C1 for Or it does not exist, where if X C1 If it exists, then X 20 Existence; X C2 for Or it does not exist, where if X C2 If it exists, then X C1 and X 20 Existence; X C3 for Or it does not exist, where if X C3 If it exists, then X C1 and X C2 and X 20 Exists; and X B for And B B The following conditions must be met: (i) When X 20 and X C1 X C2 and X C3 When it exists, it is (ii) When X 20 and X C1 and X C2 Existence and X C3 When it does not exist, it is (iii) When X 20 and X C1 Existence and X C2 and X C3 When it does not exist, it is (iv) When X 20 Existence and X C1 X C2 and X C3 When it does not exist, it is ; or (v) when X 20 and X C1 X C2 and X C3 When it does not exist, it is .

[0007] In some respects, B A For (i) ;X N1 Does not exist; X N2 Does not exist; X N3 X5 does not exist; X6 is not present. X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for or ;X 14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X 18 for ;X 19 for ;X 20 Does not exist; X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (v) .

[0008] In some respects, B A For (iii) ;X N1 Does not exist; X N2 Does not exist; X N3 for X5 is X6 is X7 is or X8 is X9 is ;X10 for or ;X 11 for ;X 12 for ;X 13 for or ;X 14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X 18 for ;X 19 for ;X 20 Does not exist; X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (v) .

[0009] In some respects, B A For (iii) ;X N1 Does not exist; X N2 Does not exist; X N3 for X5 is X6 is X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for or ;X 14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X18 for ;X 19 for ;X 20 for ;X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (iv) .

[0010] In some respects, B A For (iii) ;X N1 Does not exist; X N2 Does not exist; X N3 for X5 is X6 is X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for or ;X 14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X 18 for ;X 19 for ;X 20 for ;X C1 for ;X C2 Does not exist; X C3 It does not exist; and B B For (iii) .

[0011] In some respects, B A For (iv) ;X N1 Does not exist; X N2 for ;X N3 for X5 is X6 is X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for or ;X 14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X 18 for ;X 19 for ;X 20 Does not exist; X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (v) .

[0012] In some respects, B A For (iv) ;X N1 Does not exist; X N2 for ;X N3 for X5 is X6 is X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for or ;X14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X 18 for ;X 19 for ;X 20 for ;X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (iv) .

[0013] In some respects, B A For (v) ;X N1 for ;X N2 for ;X N3 for X5 is X6 is X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for or ;X 14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X 18 for ;X 19 for ;X 20 Does not exist; X C1 Does not exist; X C2 Does not exist; XC3 It does not exist; and B B For (v) .

[0014] In some respects, B A For (ii) ;X N1 Does not exist; X N2 Does not exist; X N3 Does not exist; X5 is X6 is X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for or ;X 14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X 18 for ;X 19 for ;X 20 for ;X C1 for ;X C2 for ;X C3 It does not exist; and B B For (ii) .

[0015] In some respects, B A For (ii) ;X N1 Does not exist; X N2 Does not exist; X N3 Does not exist; X5 is X6 is X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for or ;X 14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X 18 for ;X 19 for ;X 20 for ;X C1 for ;X C2 Does not exist; X C3 It does not exist; and B B For (iii) .

[0016] In some respects, B A For (ii) ;X N1 Does not exist; X N2 Does not exist; X N3 Does not exist; X5 is X6 is X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for or ;X 14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X18 for ;X 19 for ;X 20 for ;X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (iv) .

[0017] In some respects, B A For (ii) ;X N1 Does not exist; X N2 Does not exist; X N3 Does not exist; X5 is X6 is X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for or ;X 14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X 18 for ;X 19 for ;X 20 Does not exist; X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (v) .

[0018] In some respects, B A For (i) ;X N1 Does not exist; X N2 Does not exist; X N3 X5 does not exist; X6 is not present. X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for or ;X 14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X 18 for ;X 19 for ;X 20 for ;X C1 for ;X C2 for ;X C3 for And B B For (i) .

[0019] In some respects, B A For (i) ;X N1 Does not exist; X N2 Does not exist; X N3 X5 does not exist; X6 is not present. X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for or ;X 14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X 18 for ;X 19 for ;X 20 for ;X C1 for ;X C2 for ;X C3 It does not exist; and B B For (ii) .

[0020] In some respects, B A For (i) ;X N1 Does not exist; X N2 Does not exist; X N3 X5 does not exist; X6 is not present. X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for or ;X 14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X 18 for ;X 19 for ;X 20 for ;X C1 for ;X C2 Does not exist; X C3 It does not exist; and B B For (iii) .

[0021] In some respects, B A For (i) ;X N1 Does not exist; X N2 Does not exist; X N3 X5 does not exist; X6 is not present. X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for or ;X 14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X 18 for ;X 19 for ;X 20 for ;X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (iv) .

[0022] In some respects, B A For (i) ;X N1 Does not exist; X N2 Does not exist; X N3 X5 does not exist; X6 is not present. X7 is X8 is X9 is ;X 10 for ;X 11 for ;X 12 for ;X 13 for ;X 14 for ;X 15 for ;X 16 for ;X 17 for ;X 18 for ;X 19 for ;X 20 Does not exist; X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (v) .

[0023] In some respects, B A For (i) ;X N1 Does not exist; X N2 Does not exist; X N3 X5 does not exist; X6 is not present. X7 is X8 is X9 is ;X 10 for ;X 11 for ;X 12 for ;X 13 for ;X 14 for or ;X 15 for ;X 16 for ;X 17 for ;X 18 for ;X 19 for ;X 20 Does not exist; X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (v) .

[0024] In some respects, B A For (i) ;X N1 Does not exist; X N2 Does not exist; X N3 X5 does not exist; X6 is not present. X7 is X8 is X9 is ;X 10 for ;X 11 for ;X 12 for ;X 13 for ;X 14 for or ;X 15 for ;X 16 for ;X 17 for ;X 18 for ;X 19 for ;X 20 Does not exist; X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (v) .

[0025] In some respects, B A For (i) ;X N1 Does not exist; X N2 Does not exist; X N3 X5 does not exist; X6 is not present. X7 is X8 is X9 is ;X 10 for ;X 11 for ;X 12 for ;X 13 for ;X 14 for ;X 15 for ;X 16 for ;X 17 for ;X 18 for ;X 19 for ;X 20 Does not exist; X C1 Does not exist; XC2 Does not exist; X C3 It does not exist; and B B For (v) .

[0026] In some respects, B A For (i) ;X N1 Does not exist; X N2 Does not exist; X N3 X5 does not exist; X6 is not present. X7 is X8 is X9 is ;X 10 for ;X 11 for ;X 12 for ;X 13 for ;X 14 for ;X 15 for ;X 16 for ;X 17 for ;X 18 for ;X 19 for ;X 20 Does not exist; X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (v) .

[0027] In some respects, B A For (iii) ;X N1 Does not exist; X N2 Does not exist; X N3 for X5 is X6 is X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for or ;X 14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X 18 for ;X 19 for ;X 20 for ;X C1 for ;X C2 Does not exist; X C3 It does not exist; and B B For (iii) .

[0028] In some respects, B A For (ii) ;X N1 Does not exist; X N2 Does not exist; X N3 Does not exist; X5 is X6 is X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for or ;X 14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X 18 for ;X 19 for ;X 20 for ;X C1 for ;X C2 for ;X C3 It does not exist; and B B For (ii) .

[0029] In some respects, B A For (ii) ;X N1 Does not exist; X N2 Does not exist; X N3 Does not exist; X5 is X6 is X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for or ;X 14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X 18 for ;X 19 for ;X 20 for ;X C1 for ;X C2 Does not exist; X C3 It does not exist; and B B For (iii) .

[0030] In some respects, B A For (i) ;X N1 Does not exist; X N2 Does not exist; X N3 X5 does not exist; X6 is not present. X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for or ;X 14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X 18 for ;X 19 for ;X 20 for ;X C1 for ;X C2 for ;X C3 for And B B For (i) .

[0031] In some respects, B A For (i) ;X N1 Does not exist; X N2 Does not exist; X N3 X5 does not exist; X6 is not present. X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for or ;X 14 for , or ;X 15 for ;X 16 for ;X 17for or ;X 18 for ;X 19 for ;X 20 for ;X C1 for ;X C2 for ;X C3 It does not exist; and B B For (ii) .

[0032] In some respects, B A For (i) ;X N1 Does not exist; X N2 Does not exist; X N3 X5 does not exist; X6 is not present. X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for or ;X 14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X 18 for ;X 19 for ;X 20 for ;X C1 for ;X C2 Does not exist; X C3 It does not exist; and B B For (iii) .

[0033] In some respects, this paper provides a compound of formula (I): X A X N1 XN2 X N3 X5 X6 X7 X8 X9 X 10 X 11 X 12 X 13 X 14 X15X 16 X 17 X 18 X 19 X 20 X C1 X C2 X C3 X B , where X A for And B A The following conditions must be met: (i) When X N1、 X N2 and X N3 And when X5 does not exist, it is (ii) When X N1、 X N2 and X N3 When it does not exist and X5 exists, it is: (iii) When X N1 and X N2 It does not exist and X N3 When X5 exists, it is (iv) When X N1 It does not exist and X N2 and X N3 And when X5 exists, it is ; or (v) when X N1 X N2 and X N3 And when X5 exists, it is ;X N1 for Or it does not exist, where if X N1 If it exists, then X N2 and X N3 And X5 exists; X N2 for Or it does not exist, where if X N2 If it exists, then X N3 And X5 exists; X N3 for Or it does not exist, where if X N3 If it exists, then X5 exists; X5 is Or it may not exist; X6 is X7 is X8 is X9 is ;X 10 for ;X 11 for ;X 12 for ;X 13 for ;X 14 for ;X 15 for ;X 16 for ;X 17 for ;X 18 for ;X 19 for ;X 20 for Or it may not exist; X C1 for Or it does not exist, where if X C1 If it exists, then X 20 Existence; X C2 for Or it does not exist, where if X C2 If it exists, then X C1 and X 20 Existence; X C3 for Or it does not exist, where if X C3 If it exists, then X C1 and X C2 and X 20 Exists; and X B for And B B The following conditions must be met: (i) When X C1 X C2 and X C3 and X 20 When it exists, it is (ii) When X C1 and X C2 and X 20 Existence and X C3 When it does not exist, it is (iii) When X C1 and X 20 Existence and X C2 and X C3 When it does not exist, it is (iv) When X 20 Existence and X C1 X C2 and X C3 When it does not exist, it is ; or (v) when X 20 and XC1 X C2 and X C3 When it does not exist, it is .

[0034] In some respects, B A For (i) ;X N1 Does not exist; X N2 Does not exist; X N3 X5 does not exist; X6 is not present. X7 is X8 is X9 is ;X 10 for ;X 11 for ;X 12 for ;X 13 for ;X 14 for ;X 15 for ;X 16 for ;X 17 for ;X 18 for ;X 19 for ;X 20 Does not exist; X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (v) .

[0035] In some respects, B A For (i) ;X N1 Does not exist; X N2 Does not exist; X N3 X5 does not exist; X6 is not present. X7 is X8 is X9 is ;X 10 for ;X 11 for ;X 12 for ;X 13 for ;X 14 for ;X 15for ;X 16 for ;X 17 for ;X 18 for ;X 19 for ;X 20 for ;X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (iv) .

[0036] In some respects, B A For (i) ;X N1 Does not exist; X N2 Does not exist; X N3 X5 does not exist; X6 is not present. X7 is X8 is X9 is ;X 10 for ;X 11 for ;X 12 for ;X 13 for ;X 14 for ;X 15 for ;X 16 for ;X 17 for ;X 18 for ;X 19 for ;X 20 for ;X C1 for ;X C2 Does not exist; X C3 It does not exist; and B B For (iii) .

[0037] In some respects, B A For (i) ;X N1 Does not exist; X N2 Does not exist; X N3 X5 does not exist; X6 is not present. X7 is X8 is X9 is ;X 10 for ;X 11 for ;X 12 for ;X 13 for ;X 14 for ;X 15 for ;X 16 for ;X 17 for ;X 18 for ;X 19 for ;X 20 for ;X C1 for ;X C2 for ;X C3 It does not exist; and B B For (ii) .

[0038] In some respects, B A For (i) ;X N1 Does not exist; X N2 Does not exist; X N3 X5 does not exist; X6 is not present. X7 is X8 is X9 is ;X 10 for ;X 11 for ;X 12 for ;X 13 for ;X 14 for ;X 15 for ;X 16 for ;X 17 for ;X 18 for ;X 19 for ;X 20 for ;XC1 for ;X C2 for ;X C3 for And B B For (i) .

[0039] In some respects, B A For (ii) ;X N1 Does not exist; X N2 Does not exist; X N3 Does not exist; X5 is X6 is X7 is X8 is X9 is ;X 10 for ;X 11 for ;X 12 for ;X 13 for ;X 14 for ;X 15 for ;X 16 for ;X 17 for ;X 18 for ;X 19 for ;X 20 Does not exist; X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (v) .

[0040] In some respects, B A For (ii) ;X N1 Does not exist; X N2 Does not exist; X N3 Does not exist; X5 is X6 is X7 is X8 is X9 is ;X 10 for ;X 11 for ;X 12 for ;X 13 for ;X 14 for ;X 15 for ;X 16 for ;X 17 for ;X 18 for ;X 19 for ;X 20 for ;X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (iv) .

[0041] In some respects, B A For (ii) ;X N1 Does not exist; X N2 Does not exist; X N3 Does not exist; X5 is X6 is X7 is X8 is X9 is ;X 10 for ;X 11 for ;X 12 for ;X 13 for ;X 14 for ;X 15 for ;X 16 for ;X 17 for ;X 18 for ;X 19 for ;X 20 for ;X C1 for ;X C2 Does not exist; X C3 It does not exist; and B B For (iii) .

[0042] In some respects, B AFor (iii) ;X N1 Does not exist; X N2 Does not exist; X N3 for X5 is X6 is X7 is X8 is X9 is ;X 10 for ;X 11 for ;X 12 for ;X 13 for ;X 14 for ;X 15 for ;X 16 for ;X 17 for ;X 18 for ;X 19 for ;X 20 Does not exist; X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (v) .

[0043] In some respects, B A For (iii) ;X N1 Does not exist; X N2 Does not exist; X N3 for X5 is X6 is X7 is X8 is X9 is ;X 10 for ;X 11 for ;X 12 for ;X 13 for ;X 14 for ;X 15 for ;X 16 for ;X 17 for ;X 18 for ;X 19 for ;X 20 for ;X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (iv) .

[0044] In some respects, B A For (iii) ;X N1 Does not exist; X N2 Does not exist; X N3 for X5 is X6 is X7 is X8 is X9 is ;X 10 for ;X 11 for ;X 12 for ;X 13 for ;X 14 for ;X 15 for ;X 16 for ;X 17 for ;X 18 for ;X 19 for ;X 20 for ;X C1 for ;X C2 Does not exist; X C3 It does not exist; and B B For (iii) .

[0045] In some respects, B A For (iv) ;X N1 Does not exist; X N2 for ;X N3 for X5 is X6 is X7 is X8 is X9 is ;X 10 for ;X 11 for ;X 12 for ;X 13 for ;X 14 for ;X 15 for ;X 16 for ;X 17 for ;X 18 for ;X 19 for ;X 20 Does not exist; X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (v) .

[0046] In some respects, B A For (iv) ;X N1 Does not exist; X N2 for ;X N3 for X5 is X6 is X7 is X8 is X9 is ;X 10 for ;X 11 for ;X 12 for ;X 13 for ;X 14 for ;X 15 for ;X 16 for ;X 17 for ;X 18 for ;X 19 for ;X 20 for ;XC1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (iv) .

[0047] In some respects, B A For (v) ;X N1 for ;X N2 for ;X N3 for X5 is X6 is X7 is X8 is X9 is ;X 10 for ;X 11 for ;X 12 for ;X 13 for ;X 14 for ;X 15 for ;X 16 for ;X 17 for ;X 18 for ;X 19 for ;X 20 Does not exist; X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (v) .

[0048] In some respects, this article provides a compound selected from the group consisting of:

[0049] , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and .

[0050] In some respects, this document provides a compound that is an antisense oligonucleotide (ASO) having a sequence according to SEQ ID NO: 103 or 211, wherein the ASO comprises a main chain modification comprising a phosphorylguanidine diester or a derivative thereof.

[0051] In some aspects, the ASO includes CnTACAGAAGCAAGGTG, CTnACAGAAGCAAGGTG, CTnACAGAAGCAAGGTG, CTACnAGAAGCAAGGTG, CTACAnGAAGCAAGGTG, CTACAGnAAGCAAGGTG, CTACAGAnAGCAAGGTG, CTACAGAAnGCAAGGTG, CTACAGAAGnCAAGGTG, CTACAGAA CTA CAnGnAAGCAAGGTnG, CTACAGnAnAGCAAGGTnG, CTACAGAnAnGCAAGGTnG, CTACAGAAnGnCAAGGTnG, CTACAGAAGnCnAAGGTnG, CTACAGAAGCnAnAGGTnG, CTACAGAAGCAAnGnGTnG, CTACAGAAGCAnAnGGnTG, CTACAGAAGCnAnAGnGTG, CnTnAC nAGAAGCAAGGTG, CTnACnAnGAAGCAAGGTG, CTAnCAnGnAAGCAAGGTG, CTACnAGnAnAGCAAGGTG, CTACAnGAnAnGCAAGGTG, CnTACAGAAGCAAGGTnG, CTnACAGAAGCAAGGnTG, CTACAGAAGCAAGGnTG or CTACAGAAGCAAGGTnG, where n is a phosphorylguanidine diester or a derivative thereof.

[0052] In some respects, the ASO further comprises a main chain modification comprising a thiophosphate (PS) bond or an aminophosphate bond.

[0053] In some aspects, the phosphorylguanidine diester or its derivatives comprise (1,3-dimethylimidazolidine-2-ylidene)aminophosphate; ((4-acetamidophenyl)sulfonyl)aminophosphate; (1,3-dimethyltetrahydropyrimidine-2(1H)-ylidene)aminophosphate; (1,3-dimethyl-1,3-diazacycloheptane-2-ylidene)aminophosphate; or (di(pyrrolidine-1-yl)methylene)aminophosphate.

[0054] In some respects, the ASO comprises a 2'-O-methyl, 2'-fluoro, and / or 2'-O-methoxyethyl moiety.

[0055] In some respects, the ASO comprises at least one modified sugar moiety.

[0056] In some respects, the compound is conjugated with lipids.

[0057] In some respects, this article provides a compound that is an antisense oligonucleotide (ASO) having a sequence according to SEQ ID NO: 103 or 211, wherein the ASO is conjugated to a lipid.

[0058] In some respects, the lipid is conjugated to the 5' or 3' end of the compound.

[0059] In some respects, the lipid is conjugated to the compound via a phosphate ester, aminophosphate ester, or thiophosphate ester.

[0060] In some respects, the lipids are conjugated to the compounds via connectors.

[0061] In some respects, the adapter is selected from the group consisting of: proline-based adapters, aminohexyl adapters, and glycerol-based adapters.

[0062] In some respects, the connector is selected from the group consisting of: , , and .

[0063] In some respects, the lipids are selected from the group consisting of: stearic acid, oleic acid, trans oleic acid, linoleic acid, trans linoleic acid, linolenic acid, arachidic acid, myristic acid, capric acid, lauric acid, palmitic acid, arachidonic acid, and eicosenoic acid.

[0064] In some respects, the lipid is stearic acid.

[0065] In some respects, the compound has a structure according to formula (I): lipid-connector-ASO.

[0066] In some respects, the compound has a structure selected from the group consisting of:

[0067]

[0068] .

[0069] In some respects, the compound has a structure according to formula (II): ASO-connector-lipid.

[0070] In some respects, the compound has a structure selected from the group consisting of:

[0071]

[0072] By incorporating references

[0073] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the same extent, as if specifically and individually indicated that each individual publication, patent or patent application is incorporated by reference. Attached Figure Description

[0074] The novel features of this disclosure are set forth in the appended claims. A better understanding of the features and advantages of this disclosure will be obtained by referring to the following detailed description of illustrative embodiments, which utilize the principles of this disclosure, and the accompanying drawings:

[0075] Figures 1A-1B The splicing isotype of MECP2 precursor mRNA was described. Figure 1A ) and protein domains ( Figure 1B A schematic diagram of ( ).

[0076] Figure 2 This is a schematic diagram depicting a targeting strategy for regulating alternative splicing of MECP2 precursor mRNA.

[0077] Figure 3 It is an ASO step for the depicted MECP2 region.

[0078] Figure 4 ASO walking for the MECP2 exon 2 region was described.

[0079] Figure 5 The results of RT-PCR for MECP2 exon 2 inclusion using RNA from HEK293 cells were depicted. Cells were treated with 20 µM of ASO as indicated and allowed free uptake of ASO for 72 h.

[0080] Figure 6 The results of RT-PCR for MECP2 exon 2 inclusion using RNA from HEK293 cells were depicted. Cells were treated with 20 µM of ASO as indicated and allowed free uptake of ASO for 72 h.

[0081] Figure 7 The results of RT-PCR for MECP2 exon 2 inclusion using RNA from HEK293 cells were depicted. Cells were treated with 20 µM of ASO as indicated and allowed free uptake of ASO for 72 h.

[0082] Figure 8 The results of RT-PCR for MECP2 exon 2 inclusion using RNA from HEK293 cells were depicted. Cells were treated with 20 µM of ASO as indicated and allowed free uptake of ASO for 72 h.

[0083] Figure 9 The results of in vitro conversion of MECP2 isotypes and increase of MECP2 protein expression by the indicated compounds (numbers 8 and 50) are shown.

[0084] Figure 10A The results of RT-PCR for MECP2 exon 2 inclusion using RNA from HEK293 cells treated with increasing concentrations of the indicated compounds (numbered 8, 8-1, 8-2, and 8-3) are depicted.

[0085] Figure 10B Describing the comparison from Figure 10A The result is shown in the graph.

[0086] Figure 11 Results are shown for adult WT and T158M / + mice treated with compound number 8. T158M / + mice exhibited similar pharmacological effects to WT mice. Treatment with compound number 8 upregulated protein expression in adult WT and MECP2 T158M / + mice.

[0087] Figure 12 The results of RT-PCR for MECP2 exon 2 inclusion using RNA from HEK293 cells were depicted. Cells were nuclearly transfected with 3 µM lipid-modified ASO for 24 h, or treated with 30 μM lipid-modified ASO and allowed free ASO uptake for 72 h.

[0088] Figure 13 The results of RT-PCR for MECP2 exon 2 inclusion using RNA from HEK293 cells were depicted. Cells were nuclearly transfected with 1 µM stearate-conjugated ASO for 24 h, or treated with 30 μM stearate-conjugated ASO and allowed free ASO uptake for 72 h.

[0089] Figure 14A An exemplary structure of ASO having a P3'→N5' aminophosphate (PN) bond is depicted. Figure 14B An exemplary structure of a PN bond is depicted.

[0090] Figure 15The results of RT-PCR for MECP2 exon 2 inclusion using RNA from HEK293 cells were depicted. Cells were nuclear transfected with 1 µM of PN-modified ASO for 24 h, or treated with 20 μM of PN-modified ASO and allowed free ASO uptake for 72 h.

[0091] Figure 16 The results of RT-PCR for MECP2 exon 2 inclusion using RNA from HEK293 cells were depicted. Cells were nuclear transfected with 1 µM of PN-modified ASO for 24 h, or treated with 20 μM of PN-modified ASO and allowed free ASO uptake for 72 h.

[0092] Figures 17A-17D The results of RT-PCR for MECP2 exon 2 inclusion using RNA from neonatal WT mice on day 8 ( Figure 17A Day 15 Figure 17B Day 29 Figure 17C ) and day 114 ( Figure 17D Mice were treated with compound number 8 or compound number 8-1. PBS was used as a control group. ICV injection was performed on day 2 postnatal (PND2). Mice were euthanized at PND9, PND16, PND30, and PND114 to collect cortical tissue. Data are presented as mean with SD. Statistical analysis was performed using one-way ANOVA with Sidak correction.

[0093] Figures 17E-17H The protein expression results of MeCP2 from neonatal MECP2 mutant mice were depicted on day 8 ( Figure 17E Day 15 Figure 17F Day 29 Figure 17G ) and day 114 ( Figure 17H Mice were treated with compound number 8 or compound number 8-1. PBS was used as a control group. ICV injection was performed at PND2. Mice were euthanized at PND9, PND16, PND30, and PND114 to collect cortical tissue. Data are presented as mean with SD. Statistical analysis was performed using one-way ANOVA with Sidak correction.

[0094] Figures 18A-18B An exemplary process for synthesizing the 5' conjugate of ASO is described. The aminohexyl-linked oligonucleotide is an intermediate in the first part ( Figure 18A ), and lipoylated ASO is a product from the second part ( Figure 18B ).

[0095] Figures 19A-19B An exemplary procedure for synthesizing the 3' conjugate of ASO is described. The aminohexyl-linked oligonucleotide is an intermediate in the first part ( Figure 19A ), and lipoylated ASO is a product from the second part ( Figure 19B ).

[0096] Figure 20 An exemplary process for synthesizing PN bonds is described.

[0097] Figures 21A-21D This is a histogram showing neurons expressing wild-type MeCP2 protein treated with a mimicry (Wt x mimicry), human MeCP2 T158M neurons treated with a mimicry (mutant x mimicry), and the tested ASO compound: compound number 8 ( Figure 21A Compound number 8-1 Figure 21B Compound number 8-4 Figure 21C ) and compound number 8-5 ( Figure 21D Relative MeCP2 exon 2 expression in human MeCP2 T158M neurons treated at various concentrations (0.001 µM, 1.0 µM, 10.0 µM, and 20.0 µM). All values ​​are normalized relative to the mutant x mimic control. The number at the top of each bar represents the mean fold change relative to the mutant x mimic control. Each data point is the mean of 25 fields of view per well, normalized relative to neuron count / well. The number at the bottom of each bar is the number of wells imaged.

[0098] Figures 22A-22D This is a histogram showing neurons expressing wild-type MeCP2 protein treated with a mimicry (Wt x mimicry), human MeCP2 T158M neurons treated with a mimicry (mutant x mimicry), and the tested ASO compound: compound number 8 ( Figure 22A Compound number 8-1 Figure 22B Compound number 8-4 Figure 22C ) and compound number 8-5 ( Figure 22D Relative protein expression in human MeCP2 T158M neurons treated at different concentrations (0.001 µM, 1.0 µM, 10.0 µM, and 20.0 µM). All values ​​are normalized relative to the mutant x mimic control. The number at the top of each bar represents the mean fold change relative to the mutant x mimic control. Each data point is the average of 25 fields of view per well, normalized relative to neuron count / well. The number at the bottom of each bar is the number of wells imaged.

[0099] Figures 23A-23DThis is a histogram showing neurons expressing wild-type MeCP2 protein treated with a mimicry (Wt x mimicry), human MeCP2 T158M neurons treated with a mimicry (mutant x mimicry), and the tested ASO compound: compound number 8 ( Figure 23A Compound number 8-1 Figure 23B Compound number 8-4 Figure 23C ) and compound number 8-5 ( Figure 23D Relative dendritic branching measurements in human MeCP2 T158M neurons treated at different concentrations (0.001 µM, 1.0 µM, 10.0 µM, and 20.0 µM). All values ​​are normalized relative to the mutant x mimic control. The number at the top of each bar represents the mean fold change relative to the mutant x mimic control. Each data point is the average of 25 fields of view per well, normalized relative to neuron count / well. The number at the bottom of each bar is the number of wells imaged.

[0100] Figures 24A-24D This is a histogram showing neurons expressing wild-type MeCP2 protein treated with a mimicry (Wt x mimicry), human MeCP2 T158M neurons treated with a mimicry (mutant x mimicry), and the tested ASO compound: compound number 8 ( Figure 24A Compound number 8-1 Figure 24B Compound number 8-4 Figure 24C ) and compound number 8-5 ( Figure 24D Relative synaptic point counts in human MeCP2 T158M neurons treated at different concentrations (0.001 µM, 1.0 µM, 10.0 µM, and 20.0 µM). All values ​​are normalized relative to the mutant x mimic control. The number at the top of each bar represents the mean fold change relative to the mutant x mimic control. Each data point is the average of 25 fields of view per well, normalized relative to neuron count / well. The number at the bottom of each bar is the number of wells imaged. Detailed Implementation

[0101] Certain specific details have been set forth in this specification to provide a thorough understanding of the various embodiments. However, those skilled in the art will understand that this disclosure can be practiced without these details. In other instances, well-known structures have not been shown or described in detail to avoid unnecessarily obscuring the description of the embodiments. Unless the context requires otherwise, throughout the following specification and the appended claims, the words “comprise” and its variations, such as “comprises” and “comprising”, will be interpreted in an open-ended, inclusive sense, meaning “including but not limited to”. Furthermore, the headings provided herein are for convenience only and do not constitute an explanation of the scope or meaning of the claimed disclosure.

[0102] As used in this specification and the appended claims, unless the context clearly indicates otherwise, the singular forms “an,” “an,” and “the” include plural references. It should also be noted that the term “or” is generally used to include the meaning of “and / or” unless the context clearly indicates otherwise.

[0103] The coordinates used in this article refer to the coordinates of the genome reference assembly GRCh38 (Genome Research Consortium human build 38), also known as Hg38 (Human Genome 38).

[0104] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Although similar or equivalent methods and materials described herein may be used to practice or test this disclosure, suitable methods and materials are described below.

[0105] As used herein, the variable 3' splice site of an intron is equivalent to the variable 5' splice site of an exon immediately downstream of the intron.

[0106] As used herein, the variable 5' splice site of an intron is equivalent to the variable 3' splice site of an exon immediately upstream of the intron.

[0107] Unless specifically defined, the nomenclature, procedures, and techniques used in conjunction with the analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry described herein are those well-known and commonly used in the art. Standard techniques are applicable to chemical synthesis and chemical analysis.

[0108] Unless otherwise stated, the following terms have the following meanings:

[0109] "Administration" can mean providing a drug to an animal, and includes, but is not limited to, administration by a medical professional and self-administration. "Improvement" means the reduction, slowing, cessation, or reversal of at least one indicator of the severity of a syndrome or symptom. The severity of the indicator can be determined by subjective or objective measurements known to those skilled in the art.

[0110] "Animal" can refer to human or non-human animals, including but not limited to mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including but not limited to monkeys and chimpanzees.

[0111] "Antisense oligomers" can refer to oligomeric compounds that can undergo hybridization with target nucleic acids via hydrogen bonding. Examples of antisense oligomers include single-stranded and double-stranded compounds such as antisense oligonucleotides, siRNA, shRNA, and ssRNA.

[0112] "Antisense inhibition" or "inhibition" can refer to a reduction in the level of the target nucleic acid compared to the target nucleic acid level, either in the presence of an antisense oligomer that complements the target nucleic acid or in the absence of an antisense oligomer.

[0113] "Antisense mechanism" can refer to all those mechanisms by which a compound hybridizes with a target nucleic acid, where the result or effect of hybridization is target degradation or target occupation, accompanied by the stagnation of cellular mechanisms such as transcription or splicing. The antisense oligomers presented in this article can "antisense" with target nucleic acids, meaning that the antisense oligomers can hybridize with the target nucleic acid via hydrogen bonding.

[0114] "Antisense oligonucleotide" can refer to a single-stranded oligonucleotide with a nucleobase sequence that allows hybridization with the corresponding segment of a target nucleic acid.

[0115] "Base complementarity" can refer to the ability of an antisense oligonucleotide to precisely pair (i.e., hybridize) with the corresponding nucleobases in a target nucleic acid, and is mediated by Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen binding between the corresponding nucleobases.

[0116] "Bicyclic sugar" can refer to a furanose ring modified by bridging two atoms. Bicyclic sugars are modified sugars.

[0117] "Bicyclic nucleoside" (also known as "BNA") can refer to a nucleoside having a sugar moiety containing a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic system. In some embodiments, the bridge connects the 4' and 2' carbons of the sugar ring.

[0118] "Cap structure" or "terminal cap portion" may refer to a chemical modification at any end of an antisense oligomer. "cEt" or "restricted ethyl" may refer to a bicyclic nucleoside having a sugar moiety comprising a bridge connecting a 4'-carbon and a 2'-carbon, wherein the bridge has the following formula: 4'-CH(CH3)-O-2.

[0119] "Restricted ethyl nucleoside" (also known as cEt nucleoside) can refer to a nucleoside containing a bicyclic sugar moiety containing a 4'-CH(CH3)-O-2' bridge.

[0120] "Chimeric antisense oligomers" can refer to antisense oligomers having at least two chemically distinct regions, each with multiple subunits.

[0121] "Complementarity" can refer to the ability of the nucleobases of the first nucleic acid to pair with those of the second nucleic acid.

[0122] "Continuous nucleobases" can refer to nucleobases that are adjacent to each other.

[0123] "Diluent" can refer to a component in a composition that lacks pharmacological activity but is pharmaceutically necessary or desirable. For example, in injectable drugs, the diluent can be a liquid, such as a saline solution.

[0124] In the context of modulating activity or treating or preventing symptoms, "effective amount" can mean the amount of a drug administered, either as a single dose or as part of a series, to an individual requiring such modulation, treatment, or prevention, which is effective in modulating the effect or in treating, preventing, or improving the symptoms. Effective amounts can vary between individuals depending on the health and physical condition of the individual being treated, the individual's classification group, the formulation of the composition, the assessment of the individual's medical condition, and other relevant factors.

[0125] In this article, the terms "efficacy" or "potency" can be used interchangeably to refer to the ability to produce the desired effect.

[0126] "Expression" can include all the processes by which the coding information of a gene is transformed into structures that exist and operate within the cell. Such structures include, but are not limited to, the products of transcription and translation.

[0127] A “gapmer” can refer to a chimeric antisense oligomer in which an internal region containing multiple nucleotides that support RNase H cleavage is located between an external region containing one or more nucleotides, wherein the nucleotides containing the internal region may be chemically different from the one or more nucleotides containing the external region. The internal region may be referred to as a “gap” and the external region may be referred to as a “wing”.

[0128] "Hybridization" can refer to the annealing of complementary nucleic acid molecules. In some embodiments, complementary nucleic acid molecules include, but are not limited to, antisense oligomers and target nucleic acids. In some embodiments, complementary nucleic acid molecules include, but are not limited to, antisense oligonucleotides and nucleic acid targets.

[0129] "Individual" can refer to a person or non-human animal chosen for treatment or therapy.

[0130] "Inhibit SYNGAP" or "inhibit SYNGAP" can mean reducing the level or expression of SYNGAP mRNA and / or SYNGAP protein. In some embodiments, the level of SYNGAP mRNA and / or SYNGAP protein is inhibited in the presence of an antisense oligomer that targets SYNGAP, including an antisense oligonucleotide, compared to the expression level of SYNGAP mRNA and / or SYNGAP protein in the absence of an antisense oligomer such as an antisense oligonucleotide.

[0131] "Inhibition of expression or activity" can refer to a reduction or blockage of expression or activity, and does not necessarily indicate the complete elimination of expression or activity.

[0132] "Nucleoside bond" can refer to the chemical bond between nucleosides.

[0133] "Intracerebellomedullary cistern" or "ICM" injection or delivery can refer to the injection of the agents or pharmaceutical compositions described herein into the subarachnoid space filled with cerebrospinal fluid (CSF) between the dorsal side of the cerebellum and medulla oblongata.

[0134] "Linked nucleosides" can refer to adjacent nucleosides linked together by internucleotide bonds.

[0135] "SYNGAP antisense oligomers" can refer to antisense oligomers that target SYNGAP mRNA.

[0136] "Mismatch" or "non-complementary nucleobases" can refer to a situation where the nucleobases of the first nucleic acid cannot pair with the corresponding nucleobases of the second or target nucleic acid.

[0137] "Modified nucleoside interbonds" can refer to the substitution or any change of naturally occurring nucleoside interbonds (i.e., phosphodiester nucleoside interbonds).

[0138] "Modified nucleobases" can refer to any nucleobase other than adenine, cytosine, guanine, thymine, or uracil. "Unmodified nucleobases" refer to the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).

[0139] "Modified nucleosides" can refer to nucleosides that independently have modified sugar moieties and / or modified nucleobases.

[0140] "Modified nucleotides" can refer to nucleotides that independently have modified sugar moieties, modified nucleoside bonds, and / or modified nucleobases.

[0141] "Modified antisense oligonucleotides" can refer to oligonucleotides containing at least one modified nucleoside bond, a modified sugar, and / or a modified nucleobase.

[0142] "Modified sugar" can refer to the substitution and / or any change of the natural sugar portion.

[0143] "Monomer" can refer to a single unit of an oligomer. Monomers include, but are not limited to, naturally occurring or modified nucleosides and nucleotides. "Modulus" refers to the pattern of unmodified and modified nucleosides in an antisense oligomer.

[0144] "Natural sugar moieties" can refer to sugar moieties that exist in DNA (2'-H) or RNA (2'-OH).

[0145] "Naturally occurring nucleoside inter-bonds" can refer to 3' to 5' phosphodiester bonds.

[0146] "Non-complementary nucleobases" can refer to a pair of nucleobases that do not form hydrogen bonds with each other or support hybridization in other ways.

[0147] "Nucleic acid" can refer to molecules that contain monomeric nucleotides. Nucleic acids include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), single-stranded nucleic acids, double-stranded nucleic acids, small interfering RNA (siRNA), and microRNA (miRNA).

[0148] "Nucleobase" can refer to a heterocyclic portion capable of pairing with a base of another nucleic acid. "Nucleobase complementarity" can refer to nucleobases capable of pairing with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In some embodiments, a complementary nucleobase refers to a nucleobase of an antisense oligomer capable of pairing with a nucleobase of its target nucleic acid. For example, if a nucleobase at a position in an antisense oligomer can hydrogen-bond with a nucleobase at a position in the target nucleic acid, the position of the hydrogen bond between the oligonucleotide and the target nucleic acid is considered complementary at said nucleobase pair.

[0149] "Nucleobase sequence" can refer to a sequence of consecutive nucleobases that is independent of any sugar, bond, and / or nucleobase modification.

[0150] "Nucleoside" can refer to a nucleobase linked to a sugar.

[0151] "Nucleoside mimics" can include those structures used to replace sugars or sugar and bases, and are not necessarily linked at one or more positions in an oligomer, such as nucleoside mimics having morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranoyl, bicyclic, or tricyclic sugar mimics, for example, non-furanose sugar units. Nucleotide mimics include those structures used to replace bonds at one or more positions in a nucleoside and oligomer, such as peptide nucleic acids or morpholino (morpholino linked by -N(H)-C(=O)-O- or other non-phosphodiester bonds). Sugar substitutions overlap with the slightly broader term nucleoside mimics, but are intended only to indicate the substitution of sugar units (furanose rings). The tetrahydropyranoyl rings provided herein illustrate examples of sugar substitutions in which the furanose sugar group has been replaced by a tetrahydropyranoyl ring system. "Mimetic" can refer to groups substituted by sugar, nucleobase, and / or nucleoside inter-bonds. Typically, analogues can be used to replace sugars or sugar-nucleoside bonds, and nucleobases can be maintained to hybridize with the selected target.

[0152] "Nucleotide" can refer to a nucleoside that has a phosphate ester group covalently linked to the sugar moiety of the nucleoside.

[0153] The terms “oligomeric compound” or “oligomer” as used interchangeably in this document can refer to a polymer of linked monomeric subunits capable of hybridizing with at least one region of a nucleic acid molecule.

[0154] "Oligonucleotide" can refer to a polymer of linked nucleosides, each of which may be modified or unmodified independently of the others.

[0155] "Parenteral administration" can refer to administration by injection (e.g., bolus injection) or infusion. Parenteral administration can include subcutaneous administration, intravenous administration, intramuscular administration, intra-arterial administration, intraperitoneal administration, or intracranial administration, such as intrathecal, intraventricular, or cerebellomedullary cistern administration.

[0156] "Peptide" can refer to a molecule formed by linking at least two amino acids together via an amide bond. It is not limited to this definition; as used herein, peptide refers to both polypeptides and proteins.

[0157] "Pharmaceutical" can refer to a substance that provides therapeutic benefit when administered to an individual. For example, in some embodiments, an antisense oligonucleotide targeting SYNGAP is a pharmaceutical.

[0158] "Pharmaceutical composition" can refer to a mixture of substances suitable for individual administration. For example, a pharmaceutical composition may contain antisense oligonucleotides and a sterile aqueous solution.

[0159] "Pharmaceutical acceptable salt" can refer to a physiologically and pharmaceutically acceptable salt of a pharmaceutically active ingredient (e.g., the antisense oligomers provided herein), such as a salt that retains the desired biological activity of the active ingredient and does not confer it undesirable toxicological effects.

[0160] A "thiophosphate bond" can refer to a bond between nucleosides, where the phosphodiester bond is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom. A thiophosphate bond is a modified nucleoside bond.

[0161] "Partial" can refer to a defined number of consecutive (i.e., linked) nucleobases of a nucleic acid. In some embodiments, a partial portion is a defined number of consecutive nucleobases of the target nucleic acid. In some embodiments, a partial portion is a defined number of consecutive nucleobases of the antisense oligomer.

[0162] "Prevention" or "preventing" can refer to delaying or stopping the onset or development of a condition or syndrome for a period of time ranging from minutes to days, weeks to months, or indefinitely.

[0163] "Effective dose" can refer to the amount of medicine that provides preventive or therapeutic benefits to animals.

[0164] "Ribosonucleotide" can refer to a nucleotide that has a hydroxyl group at the 2' position of the sugar moiety. Ribonucleotides can be modified with any substituent from a variety of substituents.

[0165] A “segment” is defined as a smaller or sub-region within the target nucleic acid.

[0166] "Targeting" or "targeted" can refer to the process of designing and selecting antisense oligomers that will specifically hybridize with a target nucleic acid and induce the desired effect.

[0167] "Target nucleic acid," "target RNA," "target RNA transcript," and "nucleic acid target" can all refer to nucleic acids that can be targeted by antisense oligomers. In some embodiments, the target nucleic acid is UBE2A nucleic acid.

[0168] "Target region" can refer to a portion of a target nucleic acid that targets one or more antisense oligomers.

[0169] "Target segment" can refer to the nucleotide sequence of the target nucleic acid that targets the antisense oligomer. "5' target site" refers to the 5th nucleotide of the target segment. "3' target site" refers to the 3rd nucleotide of the target segment.

[0170] "Therapeutic effective dose" can refer to the amount of medicine that provides therapeutic benefit to an individual. "Treatment" or "treating" can refer to the administration of a composition to achieve an alteration or improvement in a symptom or syndrome.

[0171] "Unmodified nucleobases" can refer to purine bases adenine (A) and guanine (G), and pyrimidine bases thymine (T), cytosine (C), and uracil (U).

[0172] "Unmodified nucleotide" can refer to a nucleotide that contains a naturally occurring nucleobase, a sugar moiety, and a nucleoside bond. In some embodiments, the unmodified nucleotide is an RNA nucleotide (i.e., β-D-ribonucleoside) or a DNA nucleotide (i.e., β-D-deoxyribonucleoside).

[0173] "Winged segments" can refer to multiple nucleosides that have been modified to impart oligonucleotide properties, such as enhanced inhibitory activity, increased binding affinity to target nucleic acids, or resistance to degradation by nucleases in vivo.

[0174] In some embodiments, the antisense oligomer of the target nucleic acid is 12 to 30 subunits in length. In some embodiments, the antisense oligomer of the target nucleic acid is 12 to 25 subunits in length. In some embodiments, the antisense oligomer of the target nucleic acid is 12 to 22 subunits in length. In some embodiments, the antisense oligomer of the target nucleic acid is 14 to 20 subunits in length. In some embodiments, the antisense oligomer of the target nucleic acid is 15 to 25 subunits in length. In some embodiments, the antisense oligomer of the target nucleic acid is 18 to 22 subunits in length. In some embodiments, the antisense oligomer of the target nucleic acid is 19 to 21 subunits in length. In some embodiments, the antisense oligomer has a length of 8 to 80, 12 to 50, 13 to 30, 13 to 50, 14 to 30, 14 to 50, 15 to 30, 15 to 50, 16 to 30, 16 to 50, 17 to 30, 17 to 50, 18 to 30, 18 to 50, 19 to 30, 19 to 50, or 20 to 30 connected subunits.

[0175] In some embodiments, the antisense oligomer of the target nucleic acid is 12 subunits long. In some embodiments, the antisense oligomer of the target nucleic acid is 13 subunits long. In some embodiments, the antisense oligomer of the target nucleic acid is 14 subunits long. In some embodiments, the antisense oligomer of the target nucleic acid is 15 subunits long. In some embodiments, the antisense oligomer of the target nucleic acid is 16 subunits long. In some embodiments, the antisense oligomer of the target nucleic acid is 17 subunits long. In some embodiments, the antisense oligomer of the target nucleic acid is 18 subunits long. In some embodiments, the antisense oligomer of the target nucleic acid is 19 subunits long. In some embodiments, the antisense oligomer of the target nucleic acid is 20 subunits long. In some embodiments, the antisense oligomer of the target nucleic acid is 21 subunits long. In some embodiments, the antisense oligomer of the target nucleic acid is 22 subunits long. In some embodiments, the antisense oligomer of the target nucleic acid is 23 subunits long. In some embodiments, the antisense oligomer of the target nucleic acid is 24 subunits long. In some embodiments, the antisense oligomer of the target nucleic acid is 25 subunits long. In some embodiments, the antisense oligomer of the target nucleic acid is 26 subunits long. In some embodiments, the antisense oligomer of the target nucleic acid is 27 subunits long. In some embodiments, the antisense oligomer of the target nucleic acid is 28 subunits long. In some embodiments, the antisense oligomer of the target nucleic acid is 29 subunits long. In some embodiments, the antisense oligomer of the target nucleic acid is 30 subunits long. In some embodiments, the antisense oligomer targeting the target nucleic acid has a length of 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 linked subunits, or within a range defined by any two of the above values. In some embodiments, the antisense oligomer is an antisense oligonucleotide, and the linked subunits are nucleosides.

[0176] The antisense oligomers provided herein can have nucleotides that mismatch the target sequence. For example, an antisense oligonucleotide of 25 nucleotides in length can have 8 or 11 mismatched bases near its end while still being able to guide specific cleavage of the target mRNA, albeit to a lesser extent than an antisense oligonucleotide without mismatches. In some cases, the antisense oligonucleotides provided herein are 12 to 30 subunits (e.g., nucleotides) in length, including those with 1 or 3 mismatches.

[0177] Chemically modified antisense oligomers

[0178] In some embodiments, the antisense oligomers provided herein have chemically modified subunits arranged in a pattern or motif to impart properties to the antisense oligomers, such as enhanced inhibitory activity, increased binding affinity to target nucleic acids, or resistance to degradation by nucleases in vivo.

[0179] In some cases, this article provides chimeric antisense oligomers. For example, chimeric antisense oligomers may contain at least one modified region to confer increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity to target nucleic acids, and / or increased inhibitory activity. A second region of the chimeric antisense oligomer may optionally serve as a substrate for RNase H, a cellular endonuclease that cleaves the RNA strand of the RNA:DNA duplex.

[0180] In some cases, the antisense oligomers provided herein possess a spacer motif. Antisense oligomers possessing a spacer motif can be considered chimeric antisense oligomers. Within the spacer, an internal region containing multiple nucleotides supporting RNase H cleavage can be positioned between external regions containing multiple nucleotides, said multiple nucleotides being chemically distinct from the nucleosides of the internal regions. In the case of antisense oligonucleotides possessing a spacer motif, the spacer segment can act as a substrate for endonuclease cleavage, while the wing segments contain modified nucleosides. In some embodiments, the regions of the spacer are distinguished by the type of sugar moiety comprising each distinct region. The types of sugar moieties used to distinguish the regions of the interstitial body can include β-D-ribonucleosides, β-D-deoxyribonucleosides, 2'-modified nucleosides (such 2'-modified nucleosides can include 2'-MOE and 2'-O-CH3, etc.), and bicyclic sugar-modified nucleosides (such bicyclic sugar-modified nucleosides can include nucleosides having the following: 4'-(CH2)n-O-2' bridge, where n = 1 or n = 2 and 4'-CH2-O-CH2-2'). In some embodiments, the wing includes several modified sugar moieties, including, for example, 2'-MOE. In some embodiments, the wing includes several modified and unmodified sugar moieties. In some embodiments, the wing includes various combinations of 2'-MOE nucleosides and 2'-deoxyribonucleosides.

[0181] Each distinct region may contain a homogeneous sugar motif, a variant sugar motif, or an alternating sugar motif. Wing-gap-wing motifs are often described as “XYZ”, where “X” represents the length of the 5' wing, “Y” represents the length of the gap, and “Z” represents the length of the 3' wing. “X” and “Z” may contain homogeneous, variant, or alternating sugar motifs. In some embodiments, “X” and “Y” comprise one or more 2'-deoxynucleotides. “Y” may contain a 2'-deoxynucleotide. As used herein, the gap body described as “XYZ” may have a configuration such that the gap is immediately adjacent to each of the 5' and 3' wings. Therefore, there may be no intermediate nucleotide between the 5' wing and the gap, or between the gap and the 3' wing. Any antisense compound in the antisense oligomers described herein may have a gap body motif. In some embodiments, “X” and “Z” are the same; in others, they are different.

[0182] In some embodiments, the spacer provided herein comprises, for example, a 20-mer having a 5-10-5 motif in the form of "XYZ", as described herein. In some embodiments, the spacer provided herein comprises, for example, a 19-mer having a 5-9-5 motif in the form of "XYZ", as described herein. In some embodiments, the spacer provided herein comprises, for example, an 18-mer having a 5-8-5 motif in the form of "XYZ", as described herein. In some embodiments, the spacer provided herein comprises, for example, an 18-mer having a 4-8-6 motif in the form of "XYZ", as described herein. In some embodiments, the spacer provided herein comprises, for example, an 18-mer having a 6-8-4 motif in the form of "XYZ", as described herein. In some embodiments, the spacer provided herein comprises, for example, an 18-mer having a 5-7-6 motif in the form of "XYZ", as described herein.

[0183] In some cases, the antisense oligomer comprises: a 5' region consisting of three, four, five, or six linked nucleosides (e.g., the "X" portion discussed above); a central region consisting of eight, nine, ten, eleven, or twelve linked nucleosides (e.g., the "Y" portion discussed above); and a 3' region consisting of three, four, five, or six linked nucleosides (e.g., the "Z" portion discussed above). In some cases, each of the three, four, five, or six linked nucleosides in the 5' region and each of the three, four, five, or six linked nucleosides in the 3' region comprises a modified sugar moiety, and each of the eight, nine, ten, eleven, or twelve linked nucleosides in the central region is a deoxyribonucleoside. In some cases, the modified sugar moiety comprises a 2'-O-methyl moiety, a 2'-fluoro moiety, a 2'-O-methoxyethyl moiety, a 2'-NMA moiety, or any combination thereof. In some cases, one or more nucleotides in the 5' and 3' regions further include other modifications as disclosed herein. In some cases, all nucleotides in the 5' and 3' regions further include other modifications as disclosed herein.

[0184] Complementarity

[0185] When a sufficient number of nucleobases in a polynucleotide sequence (e.g., antisense oligomers) can hydrogen bond with the corresponding nucleobases of the target nucleic acid, the agents provided herein can have polynucleotide sequences complementary to the target nucleic acid, enabling the desired effect (e.g., antisense inhibition of the target nucleic acid, such as SYNGAP nucleic acid).

[0186] The non-complementary nucleobases between the agent (e.g., antisense oligomer) and the target nucleic acid can be tolerated, provided that the agent (e.g., antisense oligomer) is still able to specifically hybridize with the target nucleic acid. Furthermore, the agent (e.g., antisense oligomer) can hybridize in one or more segments of the target nucleic acid such that intermediate or adjacent segments are not involved in hybridization events (e.g., loop structures, mismatches, or hairpin structures).

[0187] In some embodiments, the agents provided herein (e.g., antisense oligomers) or designated portions thereof are complementary to the SYNGAP nucleic acid, target region, target segment, or designated portions thereof, or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary. The percentage of complementarity between the antisense oligomer and the target nucleic acid can be determined using conventional methods, such as using the BLAST (Basic Local Alignment Search) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403410; Zhang and Madden, Genome Res., 1997, 7, 649656). The percentage of homology, sequence identity, or complementarity can be obtained, for example, through the Gap program (University Research Park, Madison Wisconsin, Genetics Computer Group, Unix version 8, Wisconsin Sequence Analysis Package), which uses default settings and employs the Smith and Waterman algorithm (Advances in Appl. Math., 1981, 2, 482 489).

[0188] In some embodiments, the agents provided herein (e.g., antisense oligomers) or designated portions thereof are completely complementary (i.e., 100% complementary) to the target nucleic acid or designated portions thereof. For example, the agents provided herein (e.g., antisense oligomers) may be completely complementary to the SYNGAP nucleic acid or its target region or target segment or target sequence. As used herein, “completely complementary” may mean that each nucleobase of the antisense oligomer is precisely base-paired with the corresponding nucleobase of the target nucleic acid.

[0189] The non-complementary nucleobases can be located at the 5' or 3' end of the antisense oligomer. Alternatively, the non-complementary nucleobases and nucleobases can be located within the antisense oligomer. When two or more non-complementary nucleobases are present, the nucleobases can be continuous (i.e., linked) or discontinuous. In one embodiment, the non-complementary nucleobases are located in the wing region of the interstitial antisense oligonucleotide.

[0190] In some embodiments, the antisense oligomers provided herein, having a length of 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases relative to the target nucleic acid or a designated portion thereof, contain no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobases.

[0191] In some embodiments, the antisense oligomers provided herein with a length of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides relative to the target nucleic acid or a designated portion thereof contain no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleotides.

[0192] The agents provided herein (e.g., antisense oligomers) may also include agents complementary to a portion of a target nucleic acid. As used herein, "portion" may refer to a defined number of consecutive (i.e., linked) nucleobases within a region or segment of the target nucleic acid. "Portion" may also refer to a defined number of consecutive nucleobases of an antisense oligomer. In some embodiments, the agent (e.g., antisense oligomer) is complementary to at least 8 nucleobase portions of a target segment. In some embodiments, the agent (e.g., antisense oligomer) is complementary to at least 9 nucleobase portions of a target segment. In some embodiments, the agent (e.g., antisense oligomer) is complementary to at least 10 nucleobase portions of a target segment. In some embodiments, the agent (e.g., antisense oligomer) is complementary to at least 11 nucleobase portions of a target segment. In some embodiments, the agent (e.g., antisense oligomer) is complementary to at least 12 nucleobase portions of a target segment. In some embodiments, the agent (e.g., an antisense oligomer) is complementary to at least 13 nucleobase moieties of the target segment. In some embodiments, the agent (e.g., an antisense oligomer) is complementary to at least 14 nucleobase moieties of the target segment. In some embodiments, the agent (e.g., an antisense oligomer) is complementary to at least 15 nucleobase moieties of the target segment. Antisense oligomers complementary to at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleobase moieties of the target segment, or ranges defined by any two of these values, are also contemplated.

[0193] The agents (e.g., antisense oligomers) provided herein may also have a defined percentage of identity with a specific nucleotide sequence, SEQ ID NO, or a portion thereof. As used herein, an antisense oligomer is considered identical to the sequence disclosed herein if it has the same nucleobase pairing ability. For example, RNA containing uracil instead of thymidine in a disclosed DNA sequence would be considered identical to the DNA sequence because both uracil and thymidine pair with adenine. Shortened and extended forms of the antisense oligomers described herein, as well as oligomers having different bases relative to the antisense oligomers provided herein, are also contemplated. Different bases may be adjacent to each other or dispersed throughout the antisense oligomer. The percentage of identity of an antisense oligomer is calculated based on the number of bases having the same base pairing ability relative to the sequence with which it is being compared.

[0194] In some embodiments, the agent (e.g., an antisense oligomer) or a portion thereof is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one or more of the agents (e.g., antisense oligomers) or SEQ ID NO or a portion thereof disclosed herein.

[0195] In some embodiments, a portion of the agent (e.g., an antisense oligomer) is compared to an equal-length portion of the target nucleic acid. In some embodiments, a portion of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobases is compared to an equal-length portion of the target nucleic acid.

[0196] In some embodiments, a portion of the antisense oligonucleotide is compared to an equal-length portion of the target nucleic acid. In some embodiments, a portion of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobases is compared to an equal-length portion of the target nucleic acid.

[0197] Modification

[0198] In some embodiments, the antisense oligomers provided herein may have one or more chemical modifications compared to naturally occurring nucleotides (or the natural form of antisense oligomers) having the same or equivalent polynucleotide sequences. Modifications to antisense oligomers encompass substitution or alteration of nucleotide internucleotide bonds, sugar moieties, or nucleobases. Modified antisense oligomers may be preferred over the natural form due to desired properties such as enhanced cellular uptake, enhanced affinity for nucleic acid targets, increased stability in the presence of nucleases, or increased inhibitory activity.

[0199] Chemically modified nucleosides can be used to increase the binding affinity of shortened or truncated antisense oligonucleotides to their target nucleic acids. Therefore, comparable results can often be obtained with shorter antisense oligomers containing such chemically modified nucleosides.

[0200] Nucleosides can be base-sugar combinations. The nucleobase (also called the base) portion of a nucleoside can be a heterocyclic base portion in its native form. A nucleotide is a nucleoside that further includes a phosphate ester group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include pentofuranosyl sugars, the phosphate ester group can be linked to the 2', 3', or 5' hydroxyl portion of the sugar. Oligonucleotides are formed by covalent bonds between adjacent nucleosides to form linear polymeric oligonucleotides. Within the oligonucleotide structure, the phosphate ester group is generally referred to as the nucleoside internucleotide bond that forms the oligonucleotide.

[0201] Modified nucleoside interbonds

[0202] The naturally occurring nucleotide internucleotide bonds in RNA and DNA are 3' to 5' phosphodiester bonds. The antisense oligomers presented herein may have one or more modified, i.e., non-naturally occurring nucleotide internucleotide bonds. Antisense oligomers with one or more modified nucleotide internucleotide bonds may possess desired properties, such as enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases.

[0203] Oligonucleotides with modified nucleoside bonds can include both phosphorus-retaining and phosphorus-free nucleoside bonds. Representative phosphorus-containing nucleoside bonds include, but are not limited to, phosphate diesters, phosphate triesters, methylphosphonates, aminophosphates, and thiophosphates.

[0204] In some embodiments, the antisense oligomer targeting SYNGAP nucleic acid comprises one or more modified nucleoside internucleotide bonds. In some embodiments, the modified nucleoside internucleotide bonds are dispersed throughout the antisense oligomer. In some embodiments, the modified nucleoside internucleotide bonds are phosphate thioester bonds. In some embodiments, each nucleoside internucleotide bond in the antisense oligomer is a phosphate thioester nucleoside internucleotide bond.

[0205] Modified sugar portion

[0206] The antisense oligomers provided herein may contain one or more nucleosides, wherein the glycosyl group has been modified. Such glycomodified nucleosides can confer enhanced nuclease stability, increased binding affinity, or other beneficial biological properties to the antisense oligomers. In some embodiments, the nucleosides comprise chemically modified furanose ring moieties.

[0207] Examples of chemically modified furanose rings include, but are not limited to, the addition of substituent groups (including 5' and / or 2' substituent groups; bridging non-uniform ring atoms to form bicyclic nucleic acids (BNAs); and the use of S, N(R) or C(R1)(R2) (R, R1, and R2 are each independently H, C1-C). 12 Alkyl or protecting groups) replace the ribosyl epoxy atom; and combinations thereof. Examples of chemically modified sugars include 2'-F-5'-methyl substituted nucleosides (for other disclosed 5',2'-disubstituted nucleosides, see PCT International Application WO 2008 / 101157) or ribosyl epoxy atoms replaced with S and further substituted at the 2' position (see U.S. Patent Application US2005-0130923, published June 16, 2005), or alternatively, 5'-substituted BNA (see PCT International Application WO 2007 / 134181, wherein LNA is substituted, for example, with 5'-methyl or 5'-vinyl).

[0208] Examples of nucleosides having modified sugar moieties include, but are not limited to, nucleosides containing 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH3, 2'-OCH2CH3, 2'-OCH2CH2F, 2'-NMA, and 2'-O(CH2)2OCH3 substituents. The substituent at the 2' position may also be selected from: allyl, amino, azide, thio, O-allyl, O-Cl-C. 10 Alkyl, OCF3, OCH2F, O(CH2)2SCH3, O(CH2)2-ON(R) m (R) n O-CH2-C(=O)-N(R) m (R) n ) and O-CH2-C(=O)-N(R l )-(CH2)2-N(R m (R) n ), where each R l、 R m and R n Independently H or substituted or unsubstituted C1-C 10 alkyl.

[0209] As used herein, "bicyclic nucleoside" can refer to a modified nucleoside comprising a bicyclic sugar moiety. Examples of bicyclic nucleosides include, but are not limited to, nucleosides comprising a bridge between 4' and 2' ribosyl ring atoms. In some embodiments, the antisense oligomers provided herein comprise one or more bicyclic nucleosides comprising a 4' to 2' bridge. Examples of such 4' to 2' bridged bicyclic nucleotides include, but are not limited to, those described below: U.S. Patent Nos. 7,399,845 and 8,278,283; U.S. Patent Applications Nos. 7,696,345, 7,427,672, 8,278,426, 6,268,490, 6,525,191, 6,670,461, 6,770,748, 6,794,499, 7,034,133, 7,053,207, 7,399,845, 7,547,684, 7,741,457, and 7,696,345; U.S. Patent No.; U.S. Patent Publication No. US2008-0039618; and Chattopadhyaya et al., *Journal of Organic Chemistry*, 2009, 74. 1 18-134; Singh et al., *Chem. Commun.*, 1998, 4, 455-456; Koshkin et al., *Tetrahedron*, 1998, 54, 3607-3630; Wahlestedt et al., *Proceedings of the National Academy of Sciences of the United States of America*, 2000, 97, 5633-5638; Kumar et al., *Bioorg. Med. Chem. Lett.*, 1998, 8, 2219-2222; Singh et al., *Journal of Organic Chemistry*, 1998, 63, 10035-10039; Srivastava et al., *Journal of the American Chemical Society*, 2007, 129(26) 8362-8379; Elayadi et al., "Recent Opinions on Drugs", 2001, 2, 558-561; Braasch et al., "Chem. Biol", 2001, 8, 1-7; and Orum et al., "New Insights into Molecular Therapy", 2001, 3, 239-243.Each of the aforementioned bicyclic nucleotides can be prepared to have one or more stereochemical sugar configurations, including, for example, α-L-ribofranose and β-D-ribofranose (see PCT International Application PCT / DK98 / 00393, published March 25, 1999 as WO 99 / 14226). In some embodiments, the bicyclic sugar portion of the BNA nucleotide includes, but is not limited to, that described in U.S. Patent No. 11,129,844.

[0210] The synthesis and preparation of methyleneoxy (4'-CH2-O-2')BNA monomers adenine, cytosine, guanine, 5-methylcytosine, thymine, and uracil, as well as their oligomerization and nucleic acid recognition properties, have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630).

[0211] As used herein, “4'-2' bicyclic nucleoside” or “4' to 2' bicyclic nucleoside” can refer to a bicyclic nucleoside containing a furanose ring comprising a bridge connecting the two carbon atoms of the furanose ring, connecting the 2' and 4' carbon atoms of the ring.

[0212] As used herein, "monocyclic nucleoside" can refer to a nucleoside that includes a modified sugar moiety that is not a bicyclic sugar moiety. In some embodiments, the sugar moiety of the nucleoside or a sugar moiety analogue is modified or substituted at any position.

[0213] As used herein, “2'-modified sugar” can mean a furanyl sugar modified at the 2' position. In some embodiments, such modification includes substituents selected from: halides, including but not limited to substituted and unsubstituted alkoxy groups, substituted and unsubstituted thioalkyl groups, substituted and unsubstituted aminoalkyl groups, substituted and unsubstituted alkyl groups, substituted and unsubstituted allyl groups, and substituted and unsubstituted alkynyl groups. In some embodiments, the 2' modification is selected from substituents including but not limited to: O[(CH2)] n O] m CH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n F, O(CH2) n ONH2, OCH2C(=O)N(H)CH3 and O(CH2) n ON[(CH2) n [CH3]2, where n and m are 1 to approximately 10. Other 2'-substituent groups may also be selected from: C1-C 12Alkyl, substituted alkyl, alkenyl, alkynyl, aryl, aralkyl, O-aryl or O-aralkyl, SH, SCH3, OCN, CI, Br, CN, F, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocyclic alkyl, heterocyclic aryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group, reporter group, intercalator, group for improving pharmacokinetic properties, or group for improving the pharmacokinetic properties of antisense oligomers, and other substituents with similar properties. In some embodiments, the modified nucleoside contains a 2'-MOE side chain (Baker et al., *Journal of Biochemistry*, 1997, 272, 11944-12000). Such 2'-MOE substitutions have been described as exhibiting improved binding affinity compared to unmodified nucleosides and other modified nucleosides such as 2'-O-methyl, O-propyl, and O-aminopropyl. Oligonucleotides with 2'-MOE substituents have also been shown to be promising antisense inhibitors of gene expression with promising features for in vivo use (Martin, Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides, 1997, 16, 917-926).

[0214] As used herein, “2'-NMA” may refer to the -O-CH2-C(=O)-NH-CH3 group replacing the 2'-OH group of the ribosyl sugar moiety. “2'-NMA sugar moiety” or “2'-NMA moiety” is a sugar moiety in which the 2'-OH group of the ribosyl sugar moiety is replaced by the 2'-O-CH2-C(=O)-NH-CH3 group. Unless otherwise stated, the 2'-NMA sugar moiety is β-D configured. “NMA” may refer to ON-methylacetamide.

[0215] As used in this article, "2'-NMA nucleoside" can refer to a nucleoside containing the 2'-NMA sugar moiety.

[0216] As used herein, “2'-F” can refer to a nucleoside containing a sugar that contains a fluorine group at the 2' position.

[0217] As used herein, “2'-OMe”, “2'-OCH3”, or “2'-O-methyl” can each refer to a nucleoside containing a sugar having a -OCH3 group at the 2' position of the sugar ring.

[0218] As used herein, “MOE” or “2'-MOE” or “2'-OCH2CH2OCH3” or “2'-O-methoxyethyl” each refer to a nucleoside containing a sugar having a -OCH2CH2OCH3 group at the 2' position of the sugar ring.

[0219] In some embodiments, one or more of the multiple nucleosides are modified. In some embodiments, the oligonucleotide comprises one or more ribonucleosides (RNA) and / or deoxyribonucleosides (DNA). In some embodiments, the oligonucleotide comprises a mixture of one or more ribonucleosides (RNA) and deoxyribonucleosides (DNA).

[0220] Many other bicyclic and tricyclic sugar substitute ring systems are also known in the art, which can be used to modify nucleosides for incorporation into antisense oligomers (see, for example, review article: Leumann, Bioorganic and Medicinal Chemistry, 2002, 10, 841-854). Such ring systems can undergo various further substitutions to enhance activity.

[0221] The methods used to prepare modified sugars are well known to those skilled in the art.

[0222] In nucleotides with modified sugar moieties, the nucleobase moieties (natural, modified, or a combination thereof) are maintained to hybridize with appropriate nucleic acid targets.

[0223] In some embodiments, the antisense oligomer comprises one or more nucleosides having a modified sugar moiety. In some embodiments, the modified sugar moiety is 2'-MOE. In some embodiments, the 2'-MOE-modified nucleoside is arranged within a spacer motif. In some embodiments, the modified sugar moiety is a bicyclic nucleoside having a (4'-CH(CH3)-O-2') bridging group. In some embodiments, the (4'-CH(CH3)-O-2')-modified nucleoside is arranged throughout the entire wing of the spacer motif.

[0224] "5'-methylcytosine" can refer to cytosine modified with a methyl group attached to the 5' position. 5'-methylcytosine is a modified nucleobase.

[0225] "5'-methyluracil" can refer to uracil modified with a methyl group attached to the 5' position. 5'-methyluracil is a modified nucleobase.

[0226] "5'-methylthymine" can refer to thymine modified with a methyl group attached to the 5' position. 5'-methylthymine is a modified nucleobase.

[0227] In some cases, the antisense oligomers provided herein comprise 5'-methylcytosine, 5'-methyluracil, 5'-methylthymine, or combinations thereof. In some cases, each cytosine in the antisense oligomer is methylated, i.e., has a methyl group attached to the 5' position. In some cases, each uracil in the antisense oligomer is methylated, i.e., has a methyl group attached to the 5' position. In some cases, the antisense oligomer has 1, 2, 3, 4, 5, 6, 7, 8, or more 5'-methylcytosines. In some cases, the antisense oligomer has 1, 2, 3, 4, 5, 6, 7, 8, or more 5'-methyluracils. In some cases, the antisense oligomer has both methylcytosine and methyluracil.

[0228] Pharmaceutical compositions and treatment methods

[0229] In some respects, this document provides pharmaceutical compositions comprising pharmaceutical agents of this disclosure, such as antisense oligomers or carriers encoding pharmaceutical agents.

[0230] Pharmaceutical agents (e.g., antisense oligomers) comprising the described compositions or carriers encoding pharmaceutical agents, as well as pharmaceutical compositions or formulations used in any of the methods described herein, may be prepared according to conventional techniques well known in the pharmaceutical industry and described in published literature. In some embodiments, pharmaceutical compositions or formulations for treating a subject comprise an effective amount of any antisense oligomer as described herein or a pharmaceutically acceptable salt, solvate, hydrate, or ester thereof. Pharmaceutical formulations comprising antisense oligomers may further comprise pharmaceutically acceptable excipients, diluents, or carriers.

[0231] The agents (e.g., antisense oligomers) or carriers provided herein can be mixed with pharmaceutically acceptable active or inert substances to prepare pharmaceutical compositions or formulations. Agents targeting SYNGAP nucleic acids (e.g., antisense oligomers) can be used in pharmaceutical compositions by combining the agent with a suitable pharmaceutically acceptable diluent or carrier. Pharmaceutically acceptable diluents may include phosphate-buffered saline (PBS), artificial cerebrospinal fluid (aCSF), physiological saline, or any other suitable solution.

[0232] Pharmaceutically acceptable salts are suitable for contact with tissues in humans and lower animals without excessive toxicity, irritation, allergic reactions, etc., and are proportionate to a reasonable benefit / risk ratio. (See, for example, SM Berge et al., *Journal of Pharmaceutical Sciences*, 66: 1-19 (1977), which is incorporated herein by reference for this purpose). Salts can be prepared in situ during the final isolation and purification of the compound, or prepared separately by reacting the free base form with a suitable organic acid. Examples of pharmaceutically acceptable, non-toxic acid addition salts are salts formed by amino groups with acids or by other methods described in the literature, such as ion exchange: inorganic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid; or organic acids, such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucono-heptate, glyceryl phosphate, gluconate, hemisulfate, heptaate, hydroiodate, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, dihydroxynaphthalate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, neopentanoate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium. Where appropriate, other pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations that resist the formation of counterions, such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates.

[0233] In some embodiments, the composition is formulated into any of a number of possible dosage forms, such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. In embodiments, the composition is formulated into a suspension in an aqueous, non-aqueous, or mixed culture medium. Aqueous suspensions may further contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethyl cellulose, sorbitol, and / or dextran. The suspension may also contain stabilizers. In embodiments, the pharmaceutical formulations or compositions of this disclosure include, but are not limited to, solutions, emulsions, microemulsions, foams, or formulations containing liposomes (e.g., cationic or non-cationic liposomes).

[0234] The pharmaceutical compositions or formulations described herein may contain one or more suitable and well-known to those skilled in the art or described in published literature, such as permeation enhancers, carriers, excipients, or other active or inactive ingredients. In embodiments, liposomes may also include spatially stable liposomes, such as liposomes comprising one or more specialized lipids. These specialized lipids produce liposomes with enhanced cycle life. In embodiments, spatially stable liposomes comprise one or more glycolipids or are derived from one or more hydrophilic polymers, such as polyethylene glycol (PEG) moiety. In some embodiments, surfactants are included in the pharmaceutical formulation or composition. The use of surfactants in pharmaceuticals, formulations, and emulsions is well-known in the art. In embodiments, this disclosure employs permeation enhancers to achieve efficient delivery of antisense oligonucleotides, for example, to aid diffusion across cell membranes and / or enhance the permeability of lipophilic drugs. In some embodiments, the permeation enhancer is a surfactant, fatty acid, bile salt, chelating agent, or non-chelating non-surfactant.

[0235] In some embodiments, the pharmaceutical formulation comprises multiple agents (e.g., antisense oligomers). In embodiments, the agent (e.g., antisense oligomer) or a carrier encoding the agent is administered in combination with another agent or therapeutic agent.

[0236] Pharmaceutical compositions comprising antisense oligomers may encompass any pharmaceutically acceptable salt, ester, or salt of such esters, or any other oligonucleotide capable of providing (directly or indirectly) a biologically active metabolite or its residues when administered to animals, including humans. Thus, for example, this disclosure is also formulated as pharmaceutically acceptable salts of antisense oligomers, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

[0237] The prodrug may include an additional nucleoside incorporated at one or both ends of the antisense oligomer, which is cleaved by endogenous nucleases in the body to form the active antisense oligomer.

[0238] The antisense oligomers disclosed herein can be covalently linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the resulting antisense oligomers. Conjugate groups may include cholesterol and lipid moieties. Other conjugate groups may include carbohydrates, phospholipids, biotin, phenazine, folic acid, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes.

[0239] The antisense oligomers disclosed herein can also be modified to have one or more stabilizing groups, which are typically attached to one or both ends of the antisense oligomer to enhance properties such as nuclease stability. The stabilizing groups include cap structures. These end modifications can protect antisense oligomers with terminal nucleic acids from exonuclease degradation and can facilitate intracellular delivery and / or localization. The cap can be present at the 5' end (5' cap) or the 3' end (3' cap), or it can be present at both ends. The cap structure can include, for example, an inverted deoxy-base cap. Additional 3' and 5' stabilizing groups that can be used to cap one or both ends of the antisense oligomer to confer nuclease stability can include those disclosed in WO 03 / 004602, published January 16, 2003.

[0240] Alternative splicing in certain genes can produce nonproductive or less productive mRNA transcripts, which in turn can downregulate protein expression. Therapeutic agents that target alternative splicing events in these genes can modulate protein expression levels. Such therapeutic agents can be used to treat conditions caused by protein deficiency.

[0241] One of the alternative splicing events that can lead to unproductive or less productive mRNA transcripts is the inclusion of exons containing inefficient translation regions, which can result in inefficient translation of the mRNA transcript. In some cases, mRNA transcripts with inefficient translation regions have significantly lower translation efficiency compared to their otherwise identical counterparts without such regions.

[0242] On one hand, this disclosure provides compositions and methods for regulating alternative splicing of genes, which may have precursor mRNA having exons containing inefficiently translated regions, to increase the production of mRNA that efficiently translates and encodes a target peptide sequence and thus the translated target peptide sequence. The compositions and methods include antisense oligomers (ASOs), which may induce exon skipping and promote the generation of efficiently translatable mRNA. In various instances, the methods of this disclosure can be used to increase the target peptide sequence to treat symptoms caused by a deficiency of the target peptide sequence or related proteins.

[0243] In some embodiments, this document describes a method for regulating the expression of a target peptide sequence via cells having a precursor mRNA encoding the target peptide sequence and containing an inefficiently translated region. The method comprises contacting cells with a therapeutic agent (which binds to a target region of the precursor mRNA encoding the target peptide sequence), thereby regulating splicing from the inefficiently translated region of the precursor mRNA encoding the target peptide sequence, thereby regulating the level of a first processed mRNA lacking the inefficiently translated region and encoding the target peptide sequence, and thereby regulating the expression of the target peptide sequence in the cell, wherein the processed mRNA has higher translation efficiency for producing the target peptide sequence in the cell compared to a second processed mRNA containing the inefficiently translated region. In some cases, the second processed mRNA is otherwise identical to the first processed mRNA but contains the inefficiently translated region. In some cases, both the first and second processed mRNAs are splicing products of the same precursor mRNA. In some cases, the therapeutic agent regulates the splicing of the precursor mRNA, thereby regulating the balance between the first and second processed mRNAs in the cell.

[0244] In some embodiments, one type of alternative splicing event that can result in unproductive or low-productivity mRNA transcripts is the inclusion of an exon containing a premature stop codon (PTC) followed by a variable start codon. In these cases, the precursor mRNA has a first start codon, a second start codon (the variable start codon), and a PTC located downstream of the first start codon and upstream of the second start codon. Motifs or exons containing a PTC and a variable start codon can contribute to low mRNA transcript productivity, without being bound by any particular theory. In some embodiments, motifs or exons containing a PTC and a variable start codon cause inefficient translation of downstream exon sequences. In some embodiments, motifs or exons containing a PTC and a variable start codon result in less stable protein products expressed by downstream exon sequences, or defects in post-translational modifications, or some other process at the molecular or cellular level that may lead to defects in protein expression or function. Without being bound by a particular theory, the presence of a variable start codon may lead to nonsense-mediated decay (NMD) of mRNA transcripts, because the translating ribosomes can restart translation at the variable start codon immediately after encountering the PTC, rather than triggering the recruitment of the NMD mechanism, which may ultimately lead to the degradation of the mRNA transcript.

[0245] On one hand, this disclosure provides compositions and methods for regulating alternative splicing of genes, which may have precursor mRNA containing exons with a PTC (followed by a variable start codon) to increase the production of mature mRNA encoding a target peptide sequence and thus the translated target peptide sequence. In some cases, the composition includes an ASO, which may induce exon skipping and promote the generation of mRNA that can be efficiently translated. In various instances, the methods of this disclosure can be used to increase the target peptide sequence to treat symptoms caused by a deficiency of the target peptide sequence or related proteins.

[0246] In some embodiments, this document describes a method for regulating the expression of a target peptide sequence by means of a cell having a precursor mRNA encoding the target peptide sequence and comprising a first start codon, a second start codon, and a premature stop codon (PTC) located downstream of the first start codon and upstream of the second start codon. The method comprises contacting the cell with a therapeutic agent (which binds to a target portion of the precursor mRNA encoding the target peptide sequence), thereby regulating the splicing of the PTC and the second start codon from the precursor mRNA, thereby regulating the level of a first processed mRNA lacking the PTC and the second start codon and encoding the target peptide sequence, and thereby regulating the expression of the target peptide sequence in the cell.

[0247] RNA splicing

[0248] Inserted sequences or introns are removed by a large and highly dynamic RNA-protein complex called the spliceosome, which coordinates complex interactions between primary transcripts, small nuclear RNA (snRNA), and a large number of proteins. The spliceosome temporarily assembles in an ordered manner on each intron, beginning at a 5' splice site (5'ss) recognized by U1 snRNA or a 3' splice site (3'ss) recognized by the U2 pathway. This involves the binding of the U2 cofactor (U2AF) to the 3'ss region to facilitate the binding of U2 to the branch point sequence (BPS). U2AF is a stable heterodimer consisting of a 65 kD subunit (U2AF65) encoded by U2AF2 that binds to a polypyrimidine fragment (PPT) and a 35 kD subunit (U2AF35) encoded by U2AF1 that interacts with a highly conserved AG dinucleotide at the 3'ss and stabilizes the binding of U2AF65. In addition to the BPS / PPT unit and 3'ss / 5'ss, precise splicing requires auxiliary sequences or structures that activate or inhibit splice site recognition, called introns or exon splice enhancers or silencers. These elements allow the identification of true splice sites among a large number of cryptic or pseudo-sites in the genomes of higher eukaryotes, which have the same sequence but are an order of magnitude larger than the true sites. Although these large numbers of cryptic or pseudo-sites often have regulatory functions, the exact mechanisms of their activation or repression are poorly understood.

[0249] The decision to splice is often modeled as a stochastic rather than deterministic process, making it possible for even the most limited splicing signals to sometimes result in incorrect splicing. However, under normal conditions, precursor mRNA splicing can proceed with surprisingly high fidelity. This may be partly attributed to the activity of adjacent cis-acting auxiliary exon and intron splicing regulatory elements (ESRs or ISRs). These functional elements are typically classified as exon or intron splicing enhancers (ESEs or ISEs) or silencers (ESSs or ISSs) based on their ability to stimulate or inhibit splicing, respectively. While there is now evidence that some auxiliary cis-acting elements can function by influencing the kinetics of spliceosome assembly, such as the arrangement of the complex between U1 snRNP and 5'ss, it appears that many elements likely work synergistically with trans-acting RNA-binding proteins (RBPs). For example, the serine and arginine-rich RBP family (SR proteins) is a conserved family of proteins that plays a crucial role in defining exons. SR proteins facilitate exon recognition by recruiting components of the splicing precursor to adjacent splicing sites or by antagonizing the effects of nearby ESSs. The repression effect of ESS can be mediated by members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family and can alter the recruitment of core splicing factors to adjacent splicing sites. In addition to their role in splicing regulation, silencing elements are also thought to play a role in the repression of pseudoexons, a set of pseudointron splicing sites with typical exon intervals but no functional open reading frames. ESE and ESS, together with their homologous trans-acting RBPs, represent an important component of a splicing control group that specifies how, where, and when mRNA is assembled from its precursors.

[0250] Sequences marking exon-intron boundaries are degenerate signals of varying intensities that can occur frequently within human genes. In multi-exon genes, different splice site pairs can be linked together in many different combinations, resulting in different arrays of transcripts from a single gene. This is often referred to as alternative precursor mRNA splicing. Although most mRNA isoforms produced by alternative splicing can be exported from the nucleus and translated into functional polypeptides, the translation efficiency of different mRNA isoforms from a single gene can vary considerably. Those mRNA isoforms with a premature stop codon (PTC) located at least 50 bp upstream of the exon junction complex may be targeted for degradation via the nonsense-mediated mRNA decay (NMD) pathway. However, in some embodiments, such as illustrated in the case of the MECP2 e2 mRNA isoform (discussed below), an alternative start codon following the PTC can prevent the induction of NMD events. Mutations in conventional (BPS / PPT / 3'ss / 5'ss) and helper splicing motifs can lead to aberrant splicing, such as exon skipping or cryptic (or pseudo) exon inclusion or splicing site activation, and significantly contribute to human morbidity and mortality. Both aberrant and alternative splicing patterns can be influenced by natural DNA variants in exons and introns.

[0251] In some embodiments, the compositions and methods utilize cryptic splicing sites to modulate alternative splicing to generate desired splice isotypes for regulating the expression levels of target peptide sequences. Cryptic (or spurious) splicing sites may have the same splice recognition sequence as true splicing sites but are not used for splicing responses. These spurious splicing sites are an order of magnitude more numerous than true splicing sites in the human genome and are generally repressed by molecular mechanisms that are not well understood to date. Cryptic 5' splicing sites have a shared NNN / GUNNNN or NNN / GCNNNN, where N is any nucleotide and / or is an exon-intron boundary. Cryptic 3' splicing sites have a shared NAG / N. Activation of these splicing sites is positively influenced by surrounding nucleotides, making these splicing sites more similar to the optimal shared sequences of true splicing sites, namely MAG / GURAGU and YAG / G, where M is C or A, R is G or A, and Y is C or U.

[0252] Splice sites and their regulatory sequences can be readily identified by a person skilled in the art using publicly available and suitable algorithms, such as those described in, for example, Kralovicova, J. and Vorechovsky, I. (2007), Global control of aberrant splice site activation by auxiliary splicing sequences: evidence for a gradient in exon and intron definition. Nucleic AcidsRes., 35, 6399-6413 (available at www.ncbi.nlm.nih.gov / pmc / articles / PMC2095810 / pdf / gkm680.pdf).

[0253] Cryptant splicing sites or splicing regulatory sequences can competitively bind to RNA-binding proteins, such as U2AF. In some embodiments, the agent can bind to cryptic splicing sites or splicing regulatory sequences to prevent the binding of RNA-binding proteins, thereby facilitating the binding of RNA-binding proteins to the desired splicing site.

[0254] MECP2 mRNA splicing

[0255] In some embodiments, the methods of this disclosure utilize alternative splicing of precursor mRNA transcribed from the MECP2 gene. The MECP2 precursor mRNA may have four exons and be alternatively spliced ​​(Figure 1). Exemplary MECP2 precursor mRNA sequences include transcripts listed in Table 1. In some embodiments, the MECP2 precursor mRNA may be spliced ​​into two isoforms, which may result in the production of the MeCP2α or e1 protein isoform and the MeCP2β or e2 isoform, respectively. Skipping exon 2 may result in the production of the dominant MeCP2α or e1 isoform, while the inclusion of exon 2 may result in the production of the MeCP2β or e2 isoform. The MeCP2-e1 and e2 isoforms may have unique N-terminal sequences of 21 aa and 9 aa, respectively. The relative expression levels of these isoforms can vary in tissues, with MeCP2-e1 being more dominant in the adult brain. MeCP2-e2 is more fully expressed in the placenta, liver, and skeletal muscle. Furthermore, in some cases, deletion of MeCP2-e2 by disrupting exon 2 does not produce an RTT-related phenotype in mice. Exon 2 is an out-of-frame exon. This exon has both a PTC and a variable start codon. Translation from the downstream start codon can be very inefficient. Mutagenesis studies have shown that the presence of two start codons in the full-length (e2) transcript significantly reduces the translation efficiency of the downstream open reading frame. When the upstream ATG in exon 1 is eliminated, the translation of MeCP2-e2 can be significantly increased. In some embodiments, skipping (splicing out) exon 2 from the full-length (e2) MECP2 transcript can reduce the pool of nonproductive transcripts, thereby increasing the translation of the MeCP2-e1 protein. In the adult brain, approximately 50% of transcripts may contain exon 2, as determined by RNA-seq (Lister PMID 23828890). In some cases, isotypes containing exon 2 can be assigned in the adult brain; therefore, exon 2 skipping induced by therapeutic agents (e.g., ASO) may be a viable therapeutic strategy to increase MeCP2 expression in patients with MeCP2 protein deficiency, such as those with Rett syndrome.

[0256] In some embodiments, the therapeutic agent targets a sequence of about 4 to about 300 nucleotides upstream (or 5') of the 5' end of the undertranslation region, such as exon 2 of MECP2 precursor mRNA. In some embodiments, the therapeutic agent targets a sequence of about 1 to about 20 nucleotides, about 20 to about 50 nucleotides, about 50 to about 100 nucleotides, about 100 to about 150 nucleotides, about 150 to about 200 nucleotides, about 200 to about 250 nucleotides, or about 250 to about 300 nucleotides upstream (or 5') of the 5' end of the undertranslation region. In some embodiments, the therapeutic agent may target a sequence of more than 300 nucleotides upstream of the 5' end of the undertranslation region. In some embodiments, the therapeutic agent targets a sequence of about 4 to about 300 nucleotides downstream (or 3') of the 3' end of the undertranslation region. In some embodiments, the therapeutic agent targets a sequence of about 1 to about 20 nucleotides, about 20 to about 50 nucleotides, about 50 to about 100 nucleotides, about 100 to about 150 nucleotides, about 150 to about 200 nucleotides, about 200 to about 250 nucleotides, or about 250 to about 300 nucleotides downstream of the 3' end of the inefficient translation region. In some embodiments, the therapeutic agent targets a sequence of more than 300 nucleotides downstream of the 3' end of the inefficient translation region.

[0257] In some embodiments, the therapeutic agent targets a sequence approximately 4 to 300 nucleotides upstream (or 5') of the 5' end of an exon containing a PTC and a second start codon (variable start codon), such as exon 2 region of MECP2 precursor mRNA. In some embodiments, the therapeutic agent targets a sequence approximately 1 to 20 nucleotides, approximately 20 to 50 nucleotides, approximately 50 to 100 nucleotides, approximately 100 to 150 nucleotides, approximately 150 to 200 nucleotides, approximately 200 to 250 nucleotides, or approximately 250 to 300 nucleotides upstream (or 5') of the 5' end of an exon containing a PTC and a variable start codon. In some embodiments, the therapeutic agent may target a sequence more than 300 nucleotides upstream of the 5' end of an exon containing a PTC and a variable start codon. In some embodiments, the therapeutic agent targets a sequence approximately 4 to 300 nucleotides downstream (or 3') of the 3' end of an exon containing a PTC and a variable start codon. In some embodiments, the therapeutic agent targets a sequence approximately 1 to 20 nucleotides, approximately 20 to 50 nucleotides, approximately 50 to 100 nucleotides, approximately 100 to 150 nucleotides, approximately 150 to 200 nucleotides, approximately 200 to 250 nucleotides, or approximately 250 to 300 nucleotides downstream of the 3' end of an exon containing a PTC and a variable start codon. In some embodiments, the therapeutic agent targets a sequence more than 300 nucleotides downstream of the 3' end of an exon containing a PTC and a variable start codon.

[0258] In some embodiments, the precursor mRNA transcript that can be targeted by the methods or compositions provided herein is encoded by a gene sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a MeCP2 gene sequence such as ENSG00000169057. In some embodiments, the precursor mRNA transcript that can be targeted by the methods or compositions provided herein comprises sequences or transcripts ENST00000453960.7, ENST00000303391.11, ENST00000628176.2, ENST00000407218.5, ENST00000415944.3, and ENST000006301 listed in Table 1. 51.2, ENST00000637917.1, ENST00000369957.5, ENST00000675526.1, ENST00000674996.1 , ENST00000460227.4, ENST00000488293.4, ENST00000463644.5, ENST00000626422.2, ENS T00000627864.1, ENST00000631210.1, ENST00000611468.2, ENST00000625300.1, ENST00 000496908.5, ENST00000676382.1, ENST00000637533.1, ENST00000637791.1, ENST000006 The sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity among any one of 37467.1, ENST00000638041.1, ENST00000486506.5, ENST00000629277.1, ENST00000675841.1, ENST00000481807.3.

[0259] In some embodiments, the therapeutic agent targets intron 1, exon 2, or intron 2 of the MECP2 precursor mRNA.

[0260] In some embodiments, the therapeutic agent targets a sequence of approximately 1500 nucleotides, approximately 1000 nucleotides, approximately 800 nucleotides, approximately 700 nucleotides, approximately 600 nucleotides, approximately 500 nucleotides, approximately 400 nucleotides, approximately 300 nucleotides, approximately 200 nucleotides, approximately 100 nucleotides, approximately 80 nucleotides, approximately 70 nucleotides, approximately 60 nucleotides, or approximately 50 nucleotides upstream (or 5') of the 5' end of exon 2 of the MECP2 precursor mRNA. In some embodiments, the therapeutic agent targets a sequence of up to about 1,500 nucleotides, about 1,000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides upstream (or 5') of the 5' end of exon 2 of the MECP2 precursor mRNA.

[0261] In some embodiments, the therapeutic agent targets a sequence of approximately 1500 nucleotides, approximately 1000 nucleotides, approximately 800 nucleotides, approximately 700 nucleotides, approximately 600 nucleotides, approximately 500 nucleotides, approximately 400 nucleotides, approximately 300 nucleotides, approximately 200 nucleotides, approximately 100 nucleotides, approximately 80 nucleotides, approximately 70 nucleotides, approximately 60 nucleotides, or approximately 50 nucleotides downstream (or 3') of the 3' end of exon 2 of the MECP2 precursor mRNA. In some embodiments, the therapeutic agent targets a sequence of up to about 1,500 nucleotides, about 1,000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides downstream (or 3') of the 3' end of exon 2 of the MECP2 precursor mRNA.

[0262] In some embodiments, according to the sequences or transcripts in Table 1, ENST00000453960.7, ENST00000303391.11, ENST00000628176.2, ENST00000407218.5, ENST00000415944.3, ENST00000630151.2, ENST00000637917.1, ENST00000369957.5, ENST00000675526.1, ENST00000674996.1, ENST00000460227.4, ENST00000488293.4, ENST00000463644.5, ENST00000626422.2, ENST00000 The therapeutic agent is ASO, and the ASO has a sequence complementary to the targeting portion of the precursor mRNA. (The list of possible therapeutic agents is not translated as it is not part of the main description of the therapeutic agent.)In some embodiments, the ASO has a sequence that is related to the sequences or transcripts selected from Table 1, such as ENST00000453960.7, ENST00000303391.11, ENST00000628176.2, ENST00000407218.5, ENST00000415944.3, ENST00000630151.2, ENST0000 0637917.1, ENST00000369957.5, ENST00000675526.1, ENST00000674996.1, ENST0000046 0227.4, ENST00000488293.4, ENST00000463644.5, ENST00000626422.2, ENST00000627864 .1, ENST00000631210.1, ENST00000611468.2, ENST00000625300.1, ENST00000496908.5, ENST00000676382.1, ENST00000637533.1, ENST00000637791.1, ENST00000637467.1, ENST The sequence complement of any one of the following groups is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0263] In some embodiments, ASO targets sequences containing exon-intron boundaries (or junctions).

[0264] In some embodiments, the methods and compositions disclosed herein are used to increase the expression of MeCP2 by inducing exon 2 exon skipping of MECP2 precursor mRNA.

[0265] In some cases, the target peptide sequence is part of the MECP2 protein. In other cases, the therapeutic agent increases the level of the first processed mRNA encoding the protein and decreases the level of the second processed mRNA encoding the protein having the sequence MVAGMLGLREEKSEDQDLQGLKDKPLKFKKVKKDKKEEKEG KHEPVQPSAHHSAEPAEAGKAETSEGSGSAPAVPEASASPKQRRSIIRDRGPMYDDPTLPEGWTRKLKQRKSGRSAGKYDVYLINPQGKAFRSKVELIAYFEKLQELAEAGDAPKGAAPRDPRRPRQRVCR. In some cases, the target region of the precursor mRNA is located up to approximately 1500 nucleotides, approximately 1000 nucleotides, approximately 800 nucleotides, approximately 700 nucleotides, approximately 600 nucleotides, approximately 500 nucleotides, approximately 400 nucleotides, approximately 300 nucleotides, approximately 200 nucleotides, approximately 100 nucleotides, approximately 80 nucleotides, approximately 70 nucleotides, approximately 60 nucleotides, or approximately 50 nucleotides upstream of the sequence GCTCCATAAAAATACAGACTCACCAGTTCCTGCTTTGATGTGAC ATGTGACTCCCCAGAATACACCTTGCTTCTGTAGACCAGCTCCAACAGGATTCCATGGTAGCTGGGATGTTAGGGCTCAG. In some cases, the target region of the precursor mRNA is located at least about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides upstream of the sequence GCTCCATAAAAATACAGACTCACCAGTTCCTGCTTTGATGTGACATGTGACTCCCCAGAATACACCTTGCTTCTGTAGACCAGCTCCAACAGGATTCCATGGTAGCTGGGATGTTAGGGCTCAG. In some cases, the target region of the precursor mRNA is located upstream of the genomic site GRCh38 / hg38:chrX 154092307, which may be up to approximately 1500 nucleotides, approximately 1000 nucleotides, approximately 800 nucleotides, approximately 700 nucleotides, approximately 600 nucleotides, approximately 500 nucleotides, approximately 400 nucleotides, approximately 300 nucleotides, approximately 200 nucleotides, approximately 100 nucleotides, approximately 80 nucleotides, approximately 70 nucleotides, approximately 60 nucleotides, or approximately 50 nucleotides.In some cases, the target region of the precursor mRNA is located upstream of the genomic site GRCh38 / hg38:chrX 154092307, which is at least about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides. In some cases, the target region of the precursor mRNA is located downstream of the sequence GCTCCATAAAAATACAGACTCACCAGTTCCTGCTTTGATGTGACATGTGACTCCCCAGAATACACCTTGCTTCTGTAGACCAGCTCCAACAGGATTCCATGGTAGCTGGGATGTTAGGGCTCAG by up to approximately 1500 nucleotides, approximately 1000 nucleotides, approximately 800 nucleotides, approximately 700 nucleotides, approximately 600 nucleotides, approximately 500 nucleotides, approximately 400 nucleotides, approximately 300 nucleotides, approximately 200 nucleotides, approximately 100 nucleotides, approximately 80 nucleotides, approximately 70 nucleotides, approximately 60 nucleotides, or approximately 50 nucleotides. In some cases, the target region of the precursor mRNA is located downstream of the sequence GCTCCATAAAAATACAGACTCACCAGTTCCTGCTTTGATGTGAC ATGTGACTCCCCAGAATACACCTTGCTTCTGTAGACCAGCTCCAACAGGATTCCATGGTAGCTGGGATGTTAGGGCTCAG, with a range of approximately 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides. In some cases, the target region of the precursor mRNA is located downstream of the genomic site GRCh38 / hg38:chrX 154092184, ranging from approximately 1500 nucleotides, 1000 nucleotides, 800 nucleotides, 700 nucleotides, 600 nucleotides, 500 nucleotides, 400 nucleotides, 300 nucleotides, 200 nucleotides, 100 nucleotides, 80 nucleotides, 70 nucleotides, 60 nucleotides, to 50 nucleotides.In some cases, the target region of the precursor mRNA is located downstream of the genomic site GRCh38 / hg38:chrX154092184, specifically within a range of at least approximately 1500 nucleotides, approximately 1000 nucleotides, approximately 800 nucleotides, approximately 700 nucleotides, approximately 600 nucleotides, approximately 500 nucleotides, approximately 400 nucleotides, approximately 300 nucleotides, approximately 200 nucleotides, approximately 100 nucleotides, approximately 80 nucleotides, approximately 70 nucleotides, approximately 60 nucleotides, or approximately 50 nucleotides. In other cases, the target region of the precursor mRNA is within the sequence GCTCCATAAAAATA CAGACTCACCAGTTCCTGCTTTGATGTGACATGTGACTCCCCAGAATACACCTTGCTTCTGTAGACCAGCTCCAACAGGATTCCATGGTAGCTGGGATGTTAGGGCTCAG. In some cases, the target region of the precursor mRNA lies within the sequence between a pair of genomic sites GRCh38 / hg38: chrX 154092307 and GRCh38 / hg38: chrX 154092184. In some cases, the therapeutic agent is an antisense oligomer complementary to the sequence GCTCCATAAAAATACAGACTCACCAGTTCCTGC TTTGATGTGACATGTGACTCCCCAGAATACACCTTGCTTCTGTAGACCAGCTCCAACAGGATTCCATGGTAGCTGGGATGTTAGGGCTCAG, which has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence GCTCCATAAAAATACAGACTCACCAGTTCCTGCTTTGATGAATACACCTTGCTTCTGTAGACCAGCTCCAACAGGATTCCATGGTAGCTGGGATGTTAGGGCTCAG. In some cases, the therapeutic agent is an antisense oligomer complementary to the sequence GCTCCATAAAAATACAGACTCACCAGTTCCTGCTTTGATGTGAC ATGTGACTCCCCAGAATACACCTTGCTTCTGTAGACCAGCTCCAACAGGATTCCATGGTAGCTGGGATGTTAGGGCTCAG, which has 100% sequence identity with the sequence GCTCCATAAAAATACAGACTCACCAGTTCCTGCTGATGTTAGGGCTCAG.In some cases, therapeutic agents increase the level of the first processed mRNA encoding the protein and decrease the level of the mRNA encoding the protein with the sequence MVAGMLGLREEKSEDQDLQGLKDKPLKFK KVKKDKKEEKEGKHEPVQPSAHHSAEPAEAGKAETSEGSGSAPAVPEASASPKQRRSIIRDRGPMYDDPTLPEGWTRKLKQRKSGRSAGKYDVYLINPQGKAFRSKVELIAYFEKVGDTSLDPNDFDFTVTGRGSPSRREQKPPKKPKSPKAPGTGRGRGRPKGSGTTRPKAATSEGVQVKRVLEKSPGKLLVKMPFQTSPGGKAEGGGATTSTQVMVIKRPGRKRKAEADPQAIPKK The level of the second processed mRNA of the protein. In some cases, the target region of the precursor mRNA is located up to approximately 1500 nucleotides, approximately 1000 nucleotides, approximately 800 nucleotides, approximately 700 nucleotides, approximately 600 nucleotides, approximately 500 nucleotides, approximately 400 nucleotides, approximately 300 nucleotides, approximately 200 nucleotides, approximately 100 nucleotides, approximately 80 nucleotides, approximately 70 nucleotides, approximately 60 nucleotides, or approximately 50 nucleotides upstream of the sequence GCTCCATAAAAAT ACAGACTCACCAGTTCCTGCTTTGATGTGACATGTGACTCCCCAGAATACACCTTGCTTCTGTAGACCAGCTCCAACAGGATTCCATGGTAGCTGGGATGTTAGGGCTCAG.In some cases, the target region of the precursor mRNA is located at least about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides upstream of the sequence GCTCCATAAA AATACAGACTCACCAGTTCCTGCTTTGATGTGACATGTGACTCCCCAGAATACACCTTGCTTCTGTAGACCAGCTCCAACAGGATTCCATGGTAGCTGGGATGTTAGGGCTCAG. In some cases, the target region of the precursor mRNA is located upstream of the genomic site GRCh38 / hg38:chrX 154092307, which may be up to approximately 1500 nucleotides, approximately 1000 nucleotides, approximately 800 nucleotides, approximately 700 nucleotides, approximately 600 nucleotides, approximately 500 nucleotides, approximately 400 nucleotides, approximately 300 nucleotides, approximately 200 nucleotides, approximately 100 nucleotides, approximately 80 nucleotides, approximately 70 nucleotides, approximately 60 nucleotides, or approximately 50 nucleotides. In some cases, the target region of the precursor mRNA is located upstream of the genomic site GRCh38 / hg38:chrX154092307, which is at least about 1500 nucleotides, about 1000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, or about 50 nucleotides. In some cases, the target region of the precursor mRNA is located downstream of the sequence GCTCCATAAAAATACAGACT CACCAGTTCCTGCTTTGATGTGACATGTGACTCCCCAGAATACACCTTGCTTCTGTAGACCAGCTCCAACAGGATTCCATGGTAGCTGGGATGTTAGGGCTCAG by up to approximately 1500 nucleotides, approximately 1000 nucleotides, approximately 800 nucleotides, approximately 700 nucleotides, approximately 600 nucleotides, approximately 500 nucleotides, approximately 400 nucleotides, approximately 300 nucleotides, approximately 200 nucleotides, approximately 100 nucleotides, approximately 80 nucleotides, approximately 70 nucleotides, approximately 60 nucleotides, or approximately 50 nucleotides.In some cases, the target region of the precursor mRNA is located downstream of the sequence GCTCCATAAAAATACAGACTCACCAGTTCCTGCTTTGATGTGACATGTGACTCCCCAGAATACACCTTGCTTCTGTAGACCAGCTCCAACAGGATTCCATGGTAGCTGGGATGTTAGGGCTCAG, with a range of approximately 1500, 1000, 800, 700, 600, 500, 400, 300, 200, 100, 80, 70, 60, or 50 nucleotides. In some cases, the target region of the precursor mRNA is located downstream of the genomic site GRCh38 / hg38:chrX 154092184, ranging from approximately 1500 nucleotides, 1000 nucleotides, 800 nucleotides, 700 nucleotides, 600 nucleotides, 500 nucleotides, 400 nucleotides, 300 nucleotides, 200 nucleotides, 100 nucleotides, 80 nucleotides, 70 nucleotides, 60 nucleotides, to 50 nucleotides. In some cases, the target region of the precursor mRNA is located downstream of the genomic site GRCh38 / hg38:chrX 154092184, specifically within a range of at least approximately 1500 nucleotides, approximately 1000 nucleotides, approximately 800 nucleotides, approximately 700 nucleotides, approximately 600 nucleotides, approximately 500 nucleotides, approximately 400 nucleotides, approximately 300 nucleotides, approximately 200 nucleotides, approximately 100 nucleotides, approximately 80 nucleotides, approximately 70 nucleotides, approximately 60 nucleotides, or approximately 50 nucleotides. In other cases, the target region of the precursor mRNA is within the sequence GCTCCATAAA AATACAGACTCACCAGTTCCTGCTTTGATGTGACATGTGACTCCCCAGAATACACCTTGCTTCTGTAGACCAGCTCCAACAGGATTCCATGGTAGCTGGGATGTTAGGGCTCAG. In some cases, the target region of the precursor mRNA lies within the sequence between a pair of genomic sites GRCh38 / hg38: chrX154092307 and GRCh38 / hg38: chrX 154092184.In some cases, the therapeutic agent is an antisense oligomer complementary to the sequence GCTCCATAAAAATACAGACTCACCAGTTC CTGCTTTGATGTGACATGTGACTCCCCAGAATACACCTTGCTTCTGTAGACCAGCTCCAACAGGATTCCATGGTAGCTGGGATGTTAGGGCTCAG, having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence GCTCCATAAAAATACAGACTCACCAGTTCCTGCTTTGATGT GACATGTGACTCCCCAGAATACACCTTGCTTCTGTAGACCAGCTCCAACAGGATTCCATGGTAGCTGGGATGTTAGGGCTCAG. In some cases, the therapeutic agent is an antisense oligomer having a sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 1-245. In some cases, the target peptide sequence is part of the MECP2 protein, and the method treats diseases or conditions including: intellectual disability; Rett syndrome, language-preserving variant; Lubs X-linked intellectual disability syndrome; severe neonatal encephalopathy due to MeCP2 mutation; intellectual disability with psychosis, pyramidal tract signs, and megaorchidism; intellectual disability, X-linked, with spasticity; trisomy Xq28; intellectual disability, X-linked 16; epileptic encephalopathy; intellectual disability, X-linked, syndrome 13; intellectual disability, X-linked 1; Rett syndrome, atypical; intellectual disability, X-linked 79; microcephaly; Ppm-X syndrome; Rett syndrome, Zappella variant; Rett syndrome; or autism susceptibility, X-linked 3.

[0266] In some cases, the therapeutic agents provided herein stably reduce full-length MeCP2 mRNA (i.e., the incorporated exon 2) in cells for a sustained period of time. In some embodiments, the therapeutic agents provided herein stably reduce full-length MeCP2 mRNA in cells for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, or at least 6 months. In some embodiments, the therapeutic agents provided herein stably reduce full-length MeCP2 mRNA in cells by about 0.9-fold or less (i.e., the expression level in said cells is 0.9-fold or less than the expression level in control cells) for at least 1 week, at least 2 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, or at least 4 months compared to the level of full-length MeCP2 mRNA in control cells. In some embodiments, the therapeutic agents described herein stably reduce full-length MeCP2 mRNA levels in cells by about 0.8-fold or less, for at least 1 week, at least 2 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, or at least 4 months, compared to the level of full-length MeCP2 mRNA in control cells. In some embodiments, the therapeutic agents described herein stably reduce full-length MeCP2 mRNA levels in cells by about 0.7-fold or less, for at least 1 week, at least 2 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, or at least 4 months, compared to the level of full-length MeCP2 mRNA in control cells. In some embodiments, the therapeutic agents described herein stably reduce full-length MeCP2 mRNA levels in cells by about 0.6-fold or less, for at least 1 week, at least 2 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, or at least 4 months, compared to the level of full-length MeCP2 mRNA in control cells. In some embodiments, the therapeutic agents described herein stably reduce the level of full-length MeCP2 mRNA in cells by about 0.5-fold or less, for at least 1 week, at least 2 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, or at least 4 months, compared to the level of full-length MeCP2 mRNA in control cells. In some embodiments, the therapeutic agents described herein stably reduce the level of full-length MeCP2 mRNA in cells by about 0.4-fold or less, for at least 1 week, at least 2 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, or at least 4 months, compared to the level of full-length MeCP2 mRNA in control cells. In some embodiments, the therapeutic agents described herein stably reduce the level of full-length MeCP2 mRNA in cells by about 0.3-fold or less, for at least 1 week, at least 2 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, or at least 4 months, compared to the level of full-length MeCP2 mRNA in control cells.In some embodiments, the therapeutic agents described herein stably reduce the level of full-length MeCP2 mRNA in cells by about 0.2-fold or less, for at least 1 week, at least 2 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, or at least 4 months, compared to the level of full-length MeCP2 mRNA in control cells. In some embodiments, the therapeutic agents described herein stably reduce the level of full-length MeCP2 mRNA in cells by about 0.1-fold or less, for at least 1 week, at least 2 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, or at least 4 months, compared to the level of full-length MeCP2 mRNA in control cells. In some cases, the cells are brain cells. In some cases, brain cells originate from the cortex.

[0267] Protein expression

[0268] In some embodiments, the methods described herein are used to increase the production of functional proteins, such as those having a target peptide sequence, such as the MeCP2 protein. As used herein, the term "functional" refers to the amount of activity or function of a protein necessary to eliminate any one or more symptoms of a treated symptom or disease, such as Rett syndrome. In some embodiments, the methods are used to increase the production of partially functional MeCP2 proteins. As used herein, the term "partially functional" refers to a protein having less than the amount of activity or function necessary to eliminate or prevent any one or more symptoms of a disease or symptom, such as Rett syndrome. In some embodiments, the partially functional protein will have at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the activity relative to the fully functional protein.

[0269] In some embodiments, the method is a means of increasing the expression of a target peptide sequence in the cells of a subject, the subject's cells having precursor mRNA encoding the target peptide sequence, wherein the subject suffers from a disease or symptom caused by insufficient activity of a functional target protein for which the target peptide sequence is at least functionally equivalent. In such embodiments, the subject has a first allele encoding a non-functional target protein. In another such embodiment, the subject has a first allele encoding a functional target protein and a second allele encoding a non-functional target protein. In yet another such embodiment, the subject has a first allele encoding a functional target protein and a second allele encoding a partially functional target protein. In some of these embodiments, an antisense oligomer binds to a target portion of the precursor mRNA transcribed from the second allele (encoding the target peptide sequence and containing an exon with an inefficient translation region), thereby inducing a hopping of the inefficient translation region from the precursor mRNA and causing an increase in the level of mature mRNA encoding the target peptide sequence, and an increase in the expression of the target peptide sequence in the subject's cells. In some of these embodiments, the antisense oligomer binds to the target portion of the precursor mRNA transcribed from the second allele (encoding the target peptide sequence and containing an exon with a PTC followed by a variable start codon), thereby inducing PTC and start codon skipping from the precursor mRNA and causing an increase in the level of the mature mRNA encoding the target peptide sequence, as well as an increase in the expression of the target peptide sequence in the subject's cells.

[0270] In some embodiments, the target peptide sequence as described herein may be a fully functional protein. In some embodiments, the target peptide sequence is a portion of a full-length protein, such as the N-terminal portion or the C-terminal portion of a full-length protein. In the case of MeCP2, the target peptide sequence may be the C-terminal portion of MeCP2. In some embodiments, the target peptide sequence is encoded by a portion of precursor mRNA downstream of an inefficient translation region or a variable start codon. In some embodiments, the target peptide sequence is encoded by a portion of precursor mRNA upstream of an inefficient translation region or a variable start codon. The target peptide sequence may comprise at least about 10 amino acids (aa), 20 aa, 30 aa, 40 aa, 50 aa, 60 aa, 70 aa, 80 aa, 90 aa, 100 aa, 120 aa, 150 aa, 200 aa, 300 aa, 400 aa, 500 aa, 600 aa, 700 aa, 800 aa, 100 aa, 1500 aa, or 2000 aa. The target peptide sequence may contain about 10 amino acids (aa), 20 aa, 30 aa, 40 aa, 50 aa, 60 aa, 70 aa, 80 aa, 90 aa, 100 aa, 120 aa, 150 aa, 200 aa, 300 aa, 400 aa, 500 aa, 600 aa, 700 aa, 800aa, 100aa, 1500aa or 2000aa.

[0271] In some embodiments, the method involves increasing the expression of a functional target protein (e.g., the MeCP2-e1 isoform encoded by exons 1, 3, and 4 of the MECP2 gene) in subject cells having a precursor mRNA encoding a target peptide sequence, wherein the subject suffers from a disease or symptom caused by insufficient activity of the MeCP2 protein. In such embodiments, the subject has an allele encoding a partially functional MeCP2, such as a sub-functional allele. In such embodiments, the subject has a first allele encoding a functional MeCP2 protein and a second allele encoding a partially functional MeCP2 protein. In some of these embodiments, an antisense oligomer binds to a target portion of the precursor mRNA transcribed from the second allele (encoding MeCP2 and containing exon 2 of MECP2), thereby inducing at least a portion of MECP2 exon 2 to jump from the precursor mRNA and increasing the level of mature mRNA encoding the MeCP2-e1 isoform, as well as increasing the expression of the MeCP2-e1 isoform in the subject cells. Without being bound by any particular theory, due to X inactivation, in subjects possessing a mutant allele containing the WT allele and encoding a partially functional MeCP2 allele, such as a sub-allele, there may be approximately 50% of cells expressing the WT allele but not the mutant allele, and another approximately 50% of cells expressing the mutant allele but not the WT allele. In some cases, the methods and compositions provided herein can increase the expression of functional target proteins (e.g., the MeCP2-e1 isotype encoded by exons 1, 3, and 4 of the MECP2 gene) with target peptide sequences derived from the sub-allele in cells containing precursor mRNA transcripts derived from the sub-allele. In some cases, the methods and compositions provided herein can increase the expression of the MeCP2-e1 isotype in approximately 50% of the cells possessing the sub-allele in subjects. In some cases, the methods and compositions restore the function of MeCP2 in approximately 50% of the cells of subjects with sub-effective mutant alleles, for example, to the functional level of WT MeCP2 protein with normal amounts.

[0272] In some embodiments, the method is a method for increasing the expression of a target protein in the cells of a subject, the subject's cells having a precursor mRNA encoding the target protein and containing an exon with an inefficient translation region or a PTC (followed by a variable start codon), wherein the subject suffers from a disease or symptom caused by insufficient quantity or activity of the target protein (caused by autosomal recessive inheritance). In some embodiments, the method is a method for increasing the expression of a target protein in the cells of a subject, the subject's cells having a precursor mRNA encoding the target protein and containing an exon with an inefficient translation region or a PTC (followed by a variable start codon), wherein the subject suffers from a disease or symptom caused by insufficient quantity or activity of the target protein (caused by autosomal dominant inheritance). In some embodiments, the disease or symptom is caused by or related to a haploid deficiency of the target gene encoding the target protein. In some embodiments, the method is a means of increasing the expression of a target protein in the cells of a subject, the subject's cells having a precursor mRNA encoding the target protein and containing an exon with an inefficient translation region or PTC (followed by a variable start codon), wherein the subject suffers from a disease or symptom caused by insufficient amount or activity of the target protein (caused by an X-linked dominant mutation). For example, the method may be a means of increasing the expression of the MeCP2-e1 isotype in the cells of a subject (e.g., brain cells, such as neurons or glial cells), the subject having a precursor mRNA encoding MeCP2 and containing exon 2, wherein the subject suffers from Rett syndrome caused by insufficient amount or activity of MeCP2 (caused by an X-linked dominant mutation).

[0273] In some embodiments, precursor mRNA transcripts encoding proteins that cause disease or symptoms are targeted by the ASO described herein. In some embodiments, precursor mRNA transcripts encoding proteins that do not cause disease are targeted by the ASO. For example, a disease caused by a mutation or deficiency of a first protein in a specific pathway can be improved by targeting precursor mRNA encoding a second protein (e.g., containing a target peptide sequence), thereby increasing the production of the second protein. In some embodiments, the function of the second protein is capable of compensating for the mutation or deficiency of the first protein (which causes the disease or symptoms).

[0274] In some embodiments, the methods and compositions are suitable for treating a subject who: (a) has a first mutant allele that produces a target protein at a reduced level compared to production from a wild-type allele, the target protein being produced in a form with reduced function compared to an equivalent wild-type protein, or not producing a target protein; and a second mutant allele that produces a target protein at a reduced level compared to production from a wild-type allele, the target protein being produced in a form with reduced function compared to an equivalent wild-type protein, or not producing a target protein.

[0275] In some embodiments, the level of a first processed mRNA encoding a first protein containing a target peptide sequence is increased in cells in contact with the therapeutic agent provided herein, compared to the level of a first processed mRNA in control cells that are otherwise identical but not in contact with the therapeutic agent. In some embodiments, the level of a first processed mRNA encoding a first protein containing a target peptide sequence is increased by about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about 10 times, compared to the level of the first processed mRNA in control cells. In some embodiments, compared to the level of a second processed mRNA in control cells that are otherwise identical but not exposed to the therapeutic agent, the level of a second processed mRNA containing an inefficiently translated region and encoding a second protein comprising a target peptide sequence is reduced in cells exposed to the therapeutic agent provided herein. In some embodiments, compared to the level of a second processed mRNA in control cells, the level of a second processed mRNA containing an inefficiently translated region and encoding a second protein comprising a target peptide sequence is reduced by approximately 1.1 to approximately 10-fold, approximately 1.5 to approximately 10-fold, approximately 2 to approximately 10-fold, approximately 3 to approximately 10-fold, approximately 4 to approximately 10-fold, approximately 1.1 to approximately 5-fold, approximately 1.1 to approximately 6-fold, approximately 1.1 to approximately 7-fold, and approximately 1.1... About 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about 10 times.

[0276] In some embodiments, the therapeutic agents provided herein increase the expression of target peptide sequences in cells. In some embodiments, the levels of target peptide sequences in cells exposed to the therapeutic agent are increased compared to the levels of target peptide sequences in control cells that are otherwise identical but not exposed to the therapeutic agent. In some embodiments, the level of the target peptide sequence in cells exposed to the therapeutic agent is increased by about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about 10 times compared to the level of the target peptide sequence in control cells.

[0277] In some embodiments, the therapeutic agents provided herein stably increase the expression of the target peptide in cells for a sustained period of time. In some embodiments, the therapeutic agents provided herein stably increase the expression of the target peptide in cells for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, or at least 6 months. In some embodiments, compared to the level of the target peptide in control cells, the therapeutic agents provided herein stably increase the expression of the target peptide in cells by at least 1.1-fold (i.e., the expression level in cells is 1.1-fold or more than the expression level in control cells), for at least 1 week, at least 2 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, or at least 4 months. In some embodiments, compared to the level of the target peptide in control cells, the therapeutic agents provided herein stably increase the expression of the target peptide in cells by at least 1.2-fold, for at least 1 week, at least 2 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, or at least 4 months. In some embodiments, the therapeutic agents provided herein stably increase the expression of the target peptide in cells by at least 1.3-fold compared to the level of the target peptide in control cells, for a duration of at least 1 week, at least 2 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, or at least 4 months. In some embodiments, the therapeutic agents provided herein stably increase the expression of the target peptide in cells by at least 1.4-fold compared to the level of the target peptide in control cells, for a duration of at least 1 week, at least 2 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, or at least 4 months. In some embodiments, the therapeutic agents provided herein stably increase the expression of the target peptide in cells by at least 1.5-fold compared to the level of the target peptide in control cells, for a duration of at least 1 week, at least 2 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, or at least 4 months. In some embodiments, the therapeutic agents provided herein stably increase the expression of the target peptide in cells by at least 1.6-fold compared to the level of the target peptide in control cells, for a duration of at least 1 week, at least 2 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, or at least 4 months. In some embodiments, the therapeutic agents provided herein stably increase the expression of the target peptide in cells by at least 1.7-fold compared to the level of the target peptide in control cells, for a duration of at least 1 week, at least 2 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, or at least 4 months. In some embodiments, the therapeutic agents provided herein stably increase the expression of the target peptide in cells by at least 1.8-fold compared to the level of the target peptide in control cells, for a duration of at least 1 week, at least 2 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, or at least 4 months.In some embodiments, the therapeutic agents provided herein stably increase the expression of the target peptide in cells by at least 1.9-fold compared to the level of the target peptide in control cells, for a duration of at least 1 week, at least 2 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, or at least 4 months. In some embodiments, the therapeutic agents provided herein stably increase the expression of the target peptide in cells by at least 2.0-fold compared to the level of the target peptide in control cells, for a duration of at least 1 week, at least 2 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, or at least 4 months. In some embodiments, the therapeutic agents provided herein stably increase the expression of the target peptide in cells by at least 2.1-fold compared to the level of the target peptide in control cells, for a duration of at least 1 week, at least 2 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, or at least 4 months. In some embodiments, the therapeutic agents provided herein stably increase the expression of the target peptide in cells by at least 2.2-fold compared to the level of the target peptide in control cells, for a duration of at least 1 week, at least 2 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, or at least 4 months. In some embodiments, the therapeutic agents provided herein stably increase the expression of the target peptide in cells by at least 2.3-fold compared to the level of the target peptide in control cells, for a duration of at least 1 week, at least 2 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, or at least 4 months. In some embodiments, the therapeutic agents provided herein stably increase the expression of the target peptide in cells by at least 2.4-fold compared to the level of the target peptide in control cells, for a duration of at least 1 week, at least 2 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, or at least 4 months. In some embodiments, the therapeutic agents provided herein stably increase the expression of the target peptide in cells by at least 2.5-fold compared to the level of the target peptide in control cells, for a duration of at least 1 week, at least 2 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, or at least 4 months. In some embodiments, the therapeutic agents provided herein stably increase the expression of the target peptide in cells by at least 3.0-fold compared to the level of the target peptide in control cells, for a duration of at least 1 week, at least 2 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, or at least 4 months. In some cases, the cell is a brain cell. In other cases, brain cells originate from the cortex. In some embodiments, the target peptide is the MeCP2 protein.

[0278] Inefficient translation area

[0279] As described herein, an inefficient translation region can refer to any region in an mRNA transcript that contributes to the inefficient translation of a target peptide sequence encoded by the mRNA transcript, in such a way that a corresponding mRNA transcript without an inefficient translation region would have higher translation efficiency to produce the target peptide sequence compared to an mRNA transcript with an inefficient translation region. An inefficient translation region can reduce the translation efficiency of the mRNA transcript of the target peptide sequence at the stages of translation initiation, elongation, termination, or any combination thereof. In some embodiments, the translation efficiency of an mRNA transcript with an inefficient translation region is approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% lower than that of a corresponding mRNA transcript without an inefficient translation region. In some embodiments, the translation efficiency of mRNA transcripts with inefficient translation regions is approximately 1.2, 1.5, 1.8, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 9, 10, 11, 12, 15, 18, 20, 22, 25, 28, 30, 35, 40, 50, 60, 70, 80, 90, 100, 120, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 5000, 10 4 10 5 This is several times or even more. In some embodiments, the translation efficiency of mRNA transcripts with inefficient translation regions is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% lower than that of corresponding mRNA transcripts without inefficient translation regions. In some embodiments, the translation efficiency of mRNA transcripts with inefficient translation regions is at least about 1.2, 1.5, 1.8, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 9, 10, 11, 12, 15, 18, 20, 22, 25, 28, 30, 35, 40, 50, 60, 70, 80, 90, 100, 120, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 5000, 10 4 Or 10 5 times.

[0280] In some embodiments, the inefficient translation region contains a PTC followed by a variable start codon. Motifs or exons containing a PTC and a variable start codon can contribute to inefficient translation of downstream sequences, as the close proximity of the variable start codon to the PTC can prevent nonsense-mediated decay induction, rather than restarting translation of the downstream open reading frame, even though the PTC induces premature termination of translation of the upstream open reading frame in the mRNA. In this respect, the translation initiation efficiency of the downstream open reading frame can be relatively lower than that of a canonical open reading frame starting from a canonical start codon (e.g., upstream of the downstream open reading frame). As illustrated in the case of MECP2 mRNA processing, removal of exon 2 containing a PTC and a variable start codon can increase the production of the MeCP2-e1 isotype; however, the relatively mature mRNA level can remain stable, indicating that the MECP2 e1 mRNA isotype lacking exon 2 (containing a PTC and a variable start codon) has higher translation efficiency compared to the MECP2 e2 mRNA isotype with exon 2. In some embodiments, the distance between the PTC in the inefficient translation region and the downstream start codon is short, for example, up to 75 nucleotides (nt), 70 nt, 60 nt, 55 nt, 50 nt, 45 nt, 40 nt, 35 nt, 30 nt, 27 nt, 24 nt, 21 nt, 18 nt, 15 nt, 12 nt, 10 nt, 9 nt, 6 nt, 3 nt, or 2 nt. In some embodiments, the distance between the PTC in the inefficient translation region and the downstream start codon is approximately 3 nt, 6 nt, 9 nt, 10 nt, 12 nt, 15 nt, 18 nt, 21 nt, 24 nt, 27 nt, 30 nt, 35 nt, 40 nt, 45 nt, 50 nt, 55 nt, 60 nt, 70 nt, or 75 nt. In some cases, the distance between the PTC in the inefficient translation region and the downstream start codon is at least 2 nt, 3 nt, 6 nt, 9 nt, 10 nt, 12 nt, 15 nt, 18 nt, 21 nt, 24 nt, 27 nt, 30 nt, 35 nt, 40 nt, 45 nt, 50 nt, or 55 nt. In other cases, the distance between the PTC in the inefficient translation region and the downstream start codon is 2 nt to 75 nt, 5 nt to 70 nt, 10 nt to 65 nt, 15 nt to 60 nt, 20 nt to 55 nt, 25 nt to 50 nt, 30 nt to 45 nt, 40 nt to 50 nt, 45 nt to 55 nt, 50 nt to 60 nt, 55 nt to 70 nt, or 60 nt to 75 nt.

[0281] In some embodiments, the inefficient translation region comprises a region encoding a proline-rich peptide sequence. Where it is not desired to be bound by any particular theory, an mRNA sequence encoding a proline-rich peptide sequence may result in low translation efficiency of the mRNA transcript containing said peptide sequence, for example, low translation elongation efficiency. In some embodiments, the inefficient translation region encodes a peptide sequence having at least about 5, 8, 10, 12, 15, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 95, or 100 prolines. In some embodiments, the inefficient translation region encodes a peptide sequence having a continuous peptide sequence consisting of at least about 5, 8, 10, 12, 15, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 95, or 100 prolines. In some embodiments, the inefficient translation region encodes a peptide sequence having at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% proline.

[0282] The inefficient translation region can have any length. In some embodiments, the inefficient translation region can have a length of 5 to 10 nucleotides, 10 to 15 nucleotides, 15 to 20 nucleotides, 20 to 25 nucleotides, 25 to 30 nucleotides, 30 to 35 nucleotides, 35 to 40 nucleotides, 40 to 45 nucleotides, 45 to 50 nucleotides, 50 to 55 nucleotides, 55 to 60 nucleotides, 60 to 65 nucleotides, 65 to 70 nucleotides, 70 to 75 nucleotides, 75 to 80 nucleotides, 80 to 85 nucleotides, 85 to 90 nucleotides, 90 to 95 nucleotides, or 95 to 100 nucleotides. In some embodiments, the inefficient translation region may have a length of at least 10 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least 70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, or at least 100 nucleotides. In some embodiments, the inefficient translation region may have a length of 100 to 200 nucleotides, 200 to 300 nucleotides, 300 to 400 nucleotides, 400 to 500 nucleotides, 500 to 600 nucleotides, 600 to 700 nucleotides, 700 to 800 nucleotides, 800 to 900 nucleotides, or 900 to 1,000 nucleotides. In some embodiments, the length of the inefficient translation region may be longer than 1,000 nucleotides.

[0283] Modulation of translation of various targets

[0284] In all respects, the methods, compositions, and kits described herein are suitable for regulating the translation of various targets, such as target peptide sequences whose translation is regulated by inefficient translation regions present in processed mRNA transcripts encoding target peptide sequences. In some cases, the inefficient translation region comprises at least a portion of an exon containing a PTC followed by a variable start codon. Table 1 below lists exemplary target genes and their corresponding precursor mRNA transcripts, which can be processed into mRNA transcripts containing exons containing a PTC followed by a variable start codon. The sequences of the exons (and SEQ ID NO) and their corresponding genomic coordinates are listed in the table. In some cases, the methods, compositions, and kits described herein are suitable for regulating the translation of target peptide sequences encoded by the target genes listed in Table 1.

[0285] Table 1. Exemplary genes and exons containing PTC and variable start codons.

[0286]

[0287] Therapeutic agents

[0288] In various embodiments of this disclosure, compositions and methods comprising therapeutic agents are provided to modulate protein expression levels. In some embodiments, compositions and methods are provided to modulate alternative splicing of precursor mRNA encoding a target peptide sequence. In some embodiments, compositions and methods are provided to induce exon skipping during splicing of precursor mRNA encoding a target peptide sequence. In other embodiments, therapeutic agents may be used to induce exon inclusion to reduce protein expression levels.

[0289] In some cases, the therapeutic agent comprises a polynucleotide polymer. In some cases, the therapeutic agent comprises a viral vector expressing the polynucleotide polymer, which binds to a target region of a precursor mRNA encoding a target peptide sequence. In some cases, the viral vector comprises an adenovirus vector, an adeno-associated virus (AAV) vector, a lentiviral vector, a herpes simplex virus (HSV) viral vector, a retroviral vector, or any suitable viral vector. In some cases, the therapeutic agent comprises a gene-editing tool configured to modify a gene encoding a target peptide sequence, resulting in the deletion of a gene region encoding an inefficiently translated region. In some cases, the gene-editing tool comprises a vector, such as a viral vector, for gene editing based on CRISPR-Cas9, TALEN, zinc finger, or other suitable technologies.

[0290] According to one aspect of this disclosure, a method is provided for treating a disease or symptom in a subject by modulating the expression of a target peptide sequence in the cells of a subject having a precursor mRNA encoding the target peptide sequence and containing an inefficiently translated region. The method comprises: contacting the subject's cells with a therapeutic agent that modulates splicing of the inefficiently translated region from the precursor mRNA encoding the target peptide sequence, wherein the therapeutic agent binds to a target region of the precursor mRNA, thereby modulating the splicing of the inefficiently translated region from the precursor mRNA, thereby modulating the level of a first processed mRNA lacking the inefficiently translated region and encoding the target peptide sequence, and thereby modulating the expression of the target peptide sequence in the subject's cells, wherein the first processed mRNA has higher translation efficiency for producing the target peptide sequence in the cells compared to a second processed mRNA containing the inefficiently translated region. In some cases, the second processed mRNA is otherwise identical to the first processed mRNA but contains the inefficiently translated region.

[0291] In some other respects, this article provides a method for treating a disease or symptom in a subject of need by modulating the expression of a target peptide sequence in the cells of a subject having a precursor mRNA encoding the target peptide sequence and comprising a first start codon, a second start codon, and a premature stop codon (PTC) located downstream of the first start codon and upstream of the second start codon. The method comprises: contacting the subject's cells with a therapeutic agent that modulates the splicing of the PTC and the second start codon from the precursor mRNA encoding the target peptide sequence, wherein the therapeutic agent binds to a target region of the precursor mRNA, thereby modulating the splicing of the PTC and the second start codon from the precursor mRNA, thereby modulating the level of a first processed mRNA lacking the PTC and the second start codon and encoding the target peptide sequence, and thereby modulating the expression of the target peptide sequence in the subject's cells.

[0292] When reducing the inclusion of inefficiently translated regions or PTCs (substituent regions) in mature mRNA, the reduction can be complete, for example, 100%, or it can be partial. The reduction can be clinically significant. The reduction / correction can be relative to the inclusion level of inefficiently translated regions or PTCs relative to untreated subjects, or relative to the inclusion amount of inefficiently translated regions or PTCs relative to a similar subject population. The reduction / correction relative to the average number of subjects or pre-treatment subjects can be a reduction of at least 10%. The reduction relative to the average number of subjects or pre-treatment subjects can be a reduction of at least 20%. The reduction relative to the average number of subjects or pre-treatment subjects can be a reduction of at least 40%. The reduction relative to the average number of subjects or pre-treatment subjects can be a reduction of at least 50%. The reduction relative to the average number of subjects or pre-treatment subjects can be a reduction of at least 60%. The reduction relative to the average number of subjects or pre-treatment subjects can be a reduction of at least 80%. The reduction relative to the average number of subjects or pre-treatment subjects can be a reduction of at least 90%.

[0293] When referring to an increase in MeCP2-e1 protein levels, the increase is likely to be clinically significant. The increase can be the level of MeCP2-e1 protein relative to untreated subjects, or the amount of active MeCP2-e1 protein relative to a similar subject population. The increase can be at least 10% more active MeCP2-e1 protein relative to the average subject or pre-treatment subjects. The increase can be at least 20% more active MeCP2-e1 protein relative to the average subject or pre-treatment subjects. The increase can be at least 40% more active MeCP2-e1 protein relative to the average subject or pre-treatment subjects. The increase can be at least 50% more active MeCP2-e1 protein relative to the average subject or pre-treatment subjects. The increase can be at least 80% more active MeCP2-e1 protein relative to the average subject or pre-treatment subjects. The increase can be at least 100% more active MeCP2-e1 protein relative to the average subject or pre-treatment subjects. The increase can be at least 200% more active MeCP2-e1 protein relative to the average subject or pre-treatment subjects. Compared to the average subject or the subject before treatment, the increase can be at least 500% more active MeCP2-e1 protein.

[0294] In embodiments where the pharmaceutical agent comprises a polynucleotide polymer, the length of the polynucleotide polymer can be about 50 nucleotides. The length of the polynucleotide polymer can be about 45 nucleotides. The length of the polynucleotide polymer can be about 40 nucleotides. The length of the polynucleotide polymer can be about 35 nucleotides. The length of the polynucleotide polymer can be about 30 nucleotides. The length of the polynucleotide polymer can be about 24 nucleotides. The length of the polynucleotide polymer can be about 25 nucleotides. The length of the polynucleotide polymer can be about 20 nucleotides. The length of the polynucleotide polymer can be about 19 nucleotides. The length of the polynucleotide polymer can be about 18 nucleotides. The length of the polynucleotide polymer can be about 17 nucleotides. The length of the polynucleotide polymer can be about 16 nucleotides. The length of the polynucleotide polymer can be about 15 nucleotides. The length of the polynucleotide polymer can be about 14 nucleotides. The length of the polynucleotide polymer can be about 13 nucleotides. The length of the polynucleotide polymer can be about 12 nucleotides. The length of the polynucleotide polymer can be about 11 nucleotides. The length of the polynucleotide polymer can be about 10 nucleotides. The length of the polynucleotide polymer can be from about 10 to about 50 nucleotides. Polynucleotide polymers can be approximately 10 to 45 nucleotides in length. Polynucleotide polymers can be approximately 10 to 40 nucleotides in length. Polynucleotide polymers can be approximately 10 to 35 nucleotides in length. Polynucleotide polymers can be approximately 10 to 30 nucleotides in length. Polynucleotide polymers can be approximately 10 to 25 nucleotides in length. Polynucleotide polymers can be approximately 10 to 20 nucleotides in length. Polynucleotide polymers can be approximately 15 to 25 nucleotides in length. Polynucleotide polymers can be approximately 15 to 30 nucleotides in length. Polynucleotide polymers can be approximately 12 to 30 nucleotides in length.

[0295] The sequence of the polynucleotide polymer can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% complementary to the target sequence of the mRNA transcript (e.g., a partially processed mRNA transcript). The sequence of the polynucleotide polymer can be 100% complementary to the target sequence of the precursor mRNA transcript.

[0296] The sequence of the polynucleotide polymer (PNP) may have 4 or fewer mismatches with the target sequence of the precursor mRNA transcript. The sequence of the PNP may have 3 or fewer mismatches with the target sequence of the precursor mRNA transcript. The sequence of the PNP may have 2 or fewer mismatches with the target sequence of the precursor mRNA transcript. The sequence of the PNP may have 1 or fewer mismatches with the target sequence of the precursor mRNA transcript. The sequence of the PNP may have no mismatches with the target sequence of the precursor mRNA transcript.

[0297] Polynucleotide polymers can specifically hybridize with the target sequence of precursor mRNA transcripts. For example, polynucleotide polymers can have 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence complementarity with the target sequence of precursor mRNA transcripts. Hybridization can occur under highly stringent hybridization conditions.

[0298] The polynucleotide polymer comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 1-245. The polynucleotide polymer may also comprise a sequence having 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 1-245. Table 2 below lists exemplary sequences that the polynucleotide polymer may comprise.

[0299] Table 2. Exemplary sequences of polynucleic acid polymers used for translation regulation.

[0300]

[0301] When referring to a polynucleotide polymer sequence, those skilled in the art will understand that one or more substitutions can be accepted, optionally two substitutions, such that it maintains its ability to hybridize with a target sequence; or, where the substitution is located within the target sequence, its ability to be identified as the target sequence. Reference to sequence identity can be determined by BLAST sequence alignment using standard / default parameters. For example, the sequence may have 99% identity and still function according to this disclosure. In other embodiments, the sequence may have 98% identity and still function according to this disclosure. In yet another embodiment, the sequence may have 95% identity and still function according to this disclosure. In yet another embodiment, the sequence may have 90% identity and still function according to this disclosure.

[0302] Antisense oligomers

[0303] This document provides compositions comprising antisense oligomers that induce exon skipping by binding to a target portion of a precursor mRNA containing exons with inefficient translation regions or PTCs (followed by a variable start codon). As used herein, the terms “ASO” and “antisense oligomer” are used interchangeably and refer to oligomers, such as polynucleotides, containing nucleobases that hybridize with a target nucleic acid (e.g., MECP2 precursor mRNA) sequence via Watson-Crick base pairing or swing base pairing (GU). An ASO may have an exact sequence complementary to or nearly complementary to the target sequence (e.g., sufficient complementarity to bind to the target sequence and enhance splicing at splice sites). An ASO is engineered to bind (hybridize) to the target nucleic acid (e.g., the target portion of the precursor mRNA transcript) and maintain hybridization under physiological conditions. Typically, if the ASO hybridizes to a site other than the intended (targeting) nucleic acid sequence, it hybridizes to a limited number of sequences that are not the target nucleic acid (hybridizing to several sites other than the target nucleic acid). The design of an ASO can take into account the presence of sufficiently similar nucleic acid sequences at other locations in the genome, cellular precursor mRNA transcript, or transcriptome, limiting the likelihood that the ASO will bind to other sites and cause "off-target" effects. Any antisense oligomers known in the art, such as any antisense oligomer in PCT application PCT / US2014 / 054151 entitled "Reducing Nonsense-Mediated mRNA Decay" which is incorporated herein by reference, can be used to practice the methods described herein.

[0304] In some embodiments, ASO "specifically hybridizes" with or is "specific" to the target nucleic acid or precursor mRNA. Typically, such hybridization occurs at a T5 temperature substantially greater than 37°C, preferably at least 50°C, and typically from 60°C to approximately 90°C. m This type of hybridization preferably corresponds to strict hybridization conditions. At a given ionic strength and pH, T... m It is the temperature at which 50% of the target sequence hybridizes with complementary oligonucleotides.

[0305] When hybridization occurs between two single-stranded polynucleotides in an antiparallel configuration, oligomers, such as oligonucleotides, are “complementary” to each other. A double-stranded polynucleotide can be “complementary” to another polynucleotide if hybridization can occur between one strand of the first polynucleotide and the second polynucleotide. Complementarity (the degree to which one polynucleotide is complementary to another) can be quantified according to generally accepted base pairing rules based on the proportion (e.g., percentage) of bases in the opposing strands that are expected to form hydrogen bonds with each other. The sequence of an antisense oligomer (ASO) does not need to be 100% complementary to the sequence of its target nucleic acid for hybridization. In some embodiments, the ASO may comprise at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the sequence complementary to the target region within the target nucleic acid sequence it targets. For example, an ASO in which 18 of the 20 nucleobases of the oligomer are complementary to the target region and will therefore specifically hybridize would represent 90% complementarity. In this example, the remaining non-complementary nucleobases may cluster together or be scattered with complementary nucleobases, and need not be continuous or adjacent to each other. The percentage of complementarity between the ASO and the target nucleic acid region can be routinely determined using the BLAST procedure (basic local alignment search tool) and the PowerBLAST procedure known in the art (Altschul et al., Journal of Molecular Biology, 1990, 215, 403-410; Zhang and Madden, Genome Research, 1997, 7, 649-656).

[0306] ASO does not need to hybridize with all nucleotides in the target sequence, and the nucleotides it hybridizes with can be continuous or discontinuous. ASO can hybridize on one or more segments of the precursor mRNA transcript such that intermediate or adjacent segments are not involved in hybridization events (e.g., loop structures or hairpin structures can be formed). In some embodiments, ASO hybridizes with discontinuous nucleotides in the target precursor mRNA transcript. For example, ASO can hybridize with nucleotides in the precursor mRNA transcript that are separated by one or more nucleotides to which ASO does not hybridize.

[0307] The ASO described herein comprises a nucleobase complementary to a nucleobase present in the target portion of the precursor mRNA. The term ASO refers to oligonucleotides and any other oligomers containing nucleobases capable of hybridizing to complementary nucleobases on the target mRNA but excluding a sugar moiety, such as peptide nucleic acids (PNAs). An ASO may comprise naturally occurring nucleotides, nucleotide analogs, modified nucleotides, or any combination of two or three of the foregoing. The term "naturally occurring nucleotide" includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotide" includes nucleotides having modified or substituted sugar groups and / or having a modified backbone. In some embodiments, all nucleotides in an ASO are modified nucleotides. Chemical modifications of ASOs or components thereof that are compatible with the methods and compositions described herein will be apparent to those skilled in the art and can be found, for example, in the following documents: U.S. Patent No. 8,258,109 B2, U.S. Patent No. 5,656,612, U.S. Patent Publication No. 2012 / 0190728, and Dias and Stein, Molecular Cancer Ther., 2002, 347-355, which are incorporated herein by reference in their entirety.

[0308] One or more nucleotides of an ASO can be any naturally occurring, unmodified nucleotide (such as adenine, guanine, cytosine, thymine, and uracil) or any synthetic or modified nucleotide sufficiently similar to an unmodified nucleotide to enable it to hydrogen-bond with a nucleotide present on the target precursor mRNA. Examples of modified nucleotides include, but are not limited to, hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, and 5-hydroxymethylcytosine.

[0309] The ASOs described herein also include the backbone structure linking the oligomer components. The terms "backbone structure" and "oligomeric bond" are used interchangeably and refer to the linkages between the monomers of the ASO. In naturally occurring oligonucleotides, the backbone contains 3'-5' phosphodiester bonds linking the sugar moieties of the oligomers. The backbone structure or oligomeric bonds of the ASOs described herein may include (but are not limited to) thiophosphates, dithiophosphates, selenophosphates, diselenophosphates, phosphoroanilothioate, phosphoraniladate, aminophosphates, etc. See, for example, LaPlanche et al., Nucleic Acid Research 14:9081 (1986); Stec et al., ACS Journal 106:6077 (1984); Stein et al., Nucleic Acid Research 16:3209 (1988); Zon et al., Anti-Cancer Drug Design 6:539 (1991); Zon et al., Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (edited by F. Eckstein, Oxford University Press, Oxford, England (1991)); Stec et al., U.S. Patent No. 5,151,510; Uhlmann and Peyman, Chemical Reviews 90:543 (1990). In some embodiments, the main chain structure of ASO does not contain phosphorus but contains peptide bonds, such as peptide nucleic acids (PNA), or linking groups including urethanes, amides, and linear and cyclic hydrocarbon groups. In some embodiments, the main chain modification is a thiophosphate bond. In some embodiments, the main chain modification is an aminophosphate bond.

[0310] In some embodiments, the stereochemistry at each phosphate nucleotide bond in the ASO backbone is random. In some embodiments, the stereochemistry at each phosphate nucleotide bond in the ASO backbone is controlled and not random. For example, U.S. Patent Application Publication No. 2014 / 0194610, "Methods for the Synthesis of Functionalized Nucleic Acids," describes a method for independently selecting the chiral orientation at each phosphorus atom in a nucleic acid oligomer. In some embodiments, the ASO used in the methods of this disclosure, including but not limited to any of the ASOs shown in Tables 5 and 6, comprises an ASO having non-random phosphate nucleotide bonds. In some embodiments, the compositions used in the methods of this disclosure comprise pure diastereomeric ASOs. In some embodiments, the compositions used in the methods of this disclosure contain ASO with a diastereomeric purity of at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, about 90% to about 100%, about 91% to about 100%, about 92% to about 100%, about 93% to about 100%, about 94% to about 100%, about 95% to about 100%, about 96% to about 100%, about 97% to about 100%, about 98% to about 100%, or about 99% to about 100%.

[0311] In some embodiments, ASO has a non-random mixture of Rp and Sp configurations at its phosphonucleotide internucleotide bond. For example, it has been proposed that a mixture of Rp and Sp is required in antisense oligonucleotides to achieve a balance between good activity and nuclease stability (Wan et al., 2014, “Synthesis, biophysical properties and biological activity of second generation antisense oligonucleotides containing chiralphosphorothioate linkages”, Nucleic Acid Research 42(22): 13456-13468, which is incorporated herein by reference). In some embodiments, the ASO used in the methods of this disclosure includes, but is not limited to, any of the ASOs shown in SEQ ID NO: 1-245, comprising about 5-100% Rp, at least about 5% Rp, at least about 10% Rp, at least about 15% Rp, at least about 20% Rp, at least about 25% Rp, at least about 30% Rp, at least about 35% Rp, at least about 40% Rp, at least about 45% Rp, at least about 50% Rp, at least about 55% Rp, at least about 60% Rp, at least about 65% Rp, at least about 70% Rp, at least about 75% Rp, at least about 80% Rp, at least about 85% Rp, at least about 90% Rp, or at least about 95% Rp, and the remaining portion Sp, or about 100% Rp.In some embodiments, the ASO used in the methods of this disclosure includes, but is not limited to, any of the ASOs described herein that contain a sequence complementary to a region containing at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity with any of the regions containing at least eight consecutive nucleic acids as described herein, comprising approximately 10% to 100% Rp, approximately 15% to 100% Rp, approximately 20% to 100% Rp, approximately 25% to 100% Rp, approximately 30% to 100% Rp, approximately 35% to 100% Rp, approximately 40% to 100% Rp, approximately 45% to 100% Rp, approximately 50% to 100% Rp, approximately 55% to 100% Rp, approximately 60% to 100% Rp, approximately 65% ​​to 100% Rp, approximately 70% to 100% Rp, and approximately 75% to 100% Rp. Rp, approximately 80% to approximately 100% Rp, approximately 85% to approximately 100% Rp, approximately 90% to approximately 100% Rp or approximately 95% to approximately 100% Rp, approximately 20% to approximately 80% Rp, approximately 25% to approximately 75% Rp, approximately 30% to approximately 70% Rp, approximately 40% to approximately 60% Rp or approximately 45% to approximately 55% Rp, and the remainder Sp.

[0312] In some embodiments, the ASO used in the methods of this disclosure includes, but is not limited to, any of the ASOs described herein that contain a sequence complementary to the sequence of at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity of a region containing at least 8 consecutive nucleic acids of any of SEQ ID NO: 1-245, containing about 5-100% Sp, at least about 5% Sp, at least about 10% Sp, at least about 15% Sp, at least about 20% Sp, at least about 25% Sp, at least about 30% Sp, at least about 35% Sp, at least about 40% Sp, at least about 45% Sp, at least about 50% Sp, at least about 55% Sp, at least about 60% Sp, at least about 65% Sp, at least about 70% Sp, at least about 75% Sp, at least about 80% Sp, at least about 85% Sp, at least about 90% Sp, or at least about 95% Sp, and the remaining portion Rp, or about 100% Sp. In embodiments, the ASO used in the methods of this disclosure includes, but is not limited to, any of the ASOs described herein that contain a sequence complementary to a region containing at least 80%, 85%, 90%, 95%, 97%, or 100% sequence identity with any of the regions containing at least eight consecutive nucleic acids as described herein, and containing approximately 10% to 100% Sp, approximately 15% to 100% Sp, approximately 20% to 100% Sp, approximately 25% to 100% Sp, approximately 30% to 100% Sp, approximately 35% to 100% Sp, approximately 40% to 100% Sp, approximately 45% to 100% Sp, approximately 50% to 100% Sp, approximately 55% to 100% Sp, approximately 60% to 100% Sp, approximately 65% ​​to 100% Sp, approximately 70% to 100% Sp, approximately 75% to 100% Sp, and approximately 80% to 100% Sp. Sp, approximately 85% to approximately 100% Sp, approximately 90% to approximately 100% Sp or approximately 95% to approximately 100% Sp, approximately 20% to approximately 80% Sp, approximately 25% to approximately 75% Sp, approximately 30% to approximately 70% Sp, approximately 40% to approximately 60% Sp or approximately 45% to approximately 55% Sp, and the remainder Rp.

[0313] Any of the ASOs described herein may contain a sugar moiety comprising ribose or deoxyribose, as found in naturally occurring nucleotides, or a modified sugar moiety or sugar analogue, including a morpholine ring. Non-limiting examples of modified sugar moieties include 2' substitutions such as 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'MOE), 2'-O-aminoethyl, 2'F;N3'->P5' aminophosphate, 2'dimethylaminooxyethoxy, 2'dimethylaminoethoxyethoxy, 2'-guanidine, 2'-O-guanidinylethyl, carbamate-modified sugars, and bicyclic-modified sugars. In some embodiments, the sugar moiety modification is selected from 2'-O-Me, 2'F, and 2'MOE. In some embodiments, the sugar moiety modification is an additional bridging bond, such as a locked nucleic acid (LNA). In some embodiments, the sugar analogue contains a morpholine ring, such as phosphodiamidomorpholino (PMO). In some embodiments, the sugar moiety comprises a ribofuranyl or 2'-deoxyribofuranyl modification. In some embodiments, the sugar moiety comprises a 2',4'-restricted 2'-O-methyloxyethyl (cMOE) modification. In some embodiments, the sugar moiety comprises a cEt2',4'-restricted 2'-O-ethyl BNA modification. In some embodiments, the sugar moiety comprises a tricyclic DNA (tcDNA) modification. In some embodiments, the sugar moiety comprises an ethylene nucleic acid (ENA) modification. In some embodiments, the sugar moiety comprises an MCE modification. Modifications are known in the art and described in the literature, for example, Jarver et al., 2014, “A Chemical View of Oligonucleotides for Exon Skipping and Related Drug Applications”, Nucleic Acid Therapeutics 24(1): 37-47, which is incorporated herein by reference for this purpose.

[0314] In some embodiments, each monomer of the ASO is modified in the same way, for example, each bond in the main chain of the ASO contains a thiophosphate bond, or each ribose moiety contains a 2'O-methyl modification. Such modifications present on each monomer component in the monomeric components of the ASO are referred to as "uniform modifications". In some instances, combinations of different modifications may be required; for example, the ASO may contain a combination of phosphodiamid bonds and sugar moieties containing morpholine rings (morpholino compounds). Combinations of different modifications to the ASO are referred to as "mixed modifications" or "mixed chemicals".

[0315] In some embodiments, the ASO comprises one or more backbone modifications. In some embodiments, the ASO comprises one or more sugar moieties. In some embodiments, the ASO comprises one or more backbone modifications and one or more sugar moieties. In some embodiments, the ASO comprises a 2'MOE modification and a thiophosphate backbone. In some embodiments, the ASO comprises phosphodiamidomorpholino (PMO). In some embodiments, the ASO comprises peptide nucleic acid (PNA). Any of the ASOs described herein or any component of an ASO (e.g., nucleobases, sugar moieties, backbone) may be modified to achieve the desired properties or activities of the ASO or to reduce the undesired properties or activities of the ASO. For example, an ASO or one or more components of any ASO may be modified to enhance binding affinity to target sequences on precursor mRNA transcripts; reduce binding to any non-target sequences; reduce degradation by cellular nucleases (i.e., RNase H); improve ASO uptake into cells and / or the cell nucleus; alter the pharmacokinetics or pharmacodynamics of the ASO; and / or regulate the half-life of the ASO.

[0316] In some embodiments, the ASO comprises a nucleotide modified with 2'-O-(2-methoxyethyl) (MOE) thiophosphate. ASOs containing such nucleotides are particularly well-suited to the methods disclosed herein; oligomers with such modifications have been shown to exhibit significantly enhanced resistance to nuclease degradation and increased bioavailability, making them suitable for oral delivery, for example, in some of the embodiments described herein. See, for example, Geary et al., *Journal of Pharmacology and Experimental Therapeutics*, 2001; 296(3):890-7; Geary et al., *Journal of Pharmacology and Experimental Therapeutics*, 2001; 296(3):898-904.

[0317] Methods for synthesizing ASO are known to those skilled in the art. Alternatively or additionally, ASO may be obtained from commercial sources.

[0318] Unless otherwise specified, the left-hand end of a single-stranded nucleic acid sequence (e.g., precursor mRNA transcripts, oligonucleotides, ASO, etc.) is the 5' end, and the left-hand direction of a single-stranded or double-stranded nucleic acid sequence is referred to as the 5' direction. Similarly, the right-hand end or direction of a nucleic acid sequence (single-stranded or double-stranded) is the 3' end or direction. Generally, the region or sequence 5' from the reference point in a nucleic acid is referred to as "upstream," and the region or sequence 3' from the reference point is referred to as "downstream." Typically, the 5' direction or end of mRNA is the location of the start or start codon, while the 3' end or direction is the location of the stop codon. In some respects, nucleotides upstream of the reference point in a nucleic acid can be specified by negative numbers, while nucleotides downstream of the reference point can be specified by positive numbers. For example, a reference point (e.g., an exon-exon junction in mRNA) can be designated as a "zero" site, and nucleotides directly adjacent to and upstream of the reference point are designated as "negative one", such as "-1", while nucleotides directly adjacent to and downstream of the reference point are designated as "positive one", such as "+1".

[0319] In some embodiments, the ASO is complementary to (and binds to) a target portion of a precursor mRNA comprising an exon containing an inefficient translation region or PTC (followed by a variable start codon), and the target portion is downstream of the 5' splice site of the exon (in the 3' direction). In some embodiments, the target portion is located in a region approximately +1 to approximately +500 nucleotides relative to the 5' splice site (or 3' end) of the exon. In some embodiments, the target portion is located in a region of +6 to +40,000 nucleotides relative to the 5' splice site (or 3' end) of the exon. In some respects, ASO is associated with the 5' splice site (or 3' end) of the exon at approximately +1 to approximately +40,000, approximately +1 to approximately +30,000, approximately +1 to approximately +20,000, approximately +1 to approximately +15,000, approximately +1 to approximately +10,000, approximately +1 to approximately +5,000, approximately +1 to approximately +4,000, approximately +1 to approximately +3,000, approximately +1 to approximately +2,000, approximately +1 to approximately +1,000, approximately +1 to approximately + 500, approximately +1 to approximately +490, approximately +1 to approximately +480, approximately +1 to approximately +470, approximately +1 to approximately +460, approximately +1 to approximately +450, approximately +1 to approximately +440, approximately +1 to approximately +430, approximately +1 to approximately +420, approximately +1 to approximately +410, approximately +1 to approximately +400, approximately +1 to approximately +390, approximately +1 to approximately +380, approximately +1 to approximately +370, approximately +1 to approximately +360, approximately +1 to approximately +350, approximately +1 to approximately +34 0, approximately +1 to approximately +330, approximately +1 to approximately +320, approximately +1 to approximately +310, approximately +1 to approximately +300, approximately +1 to approximately +290, approximately +1 to approximately +280, approximately +1 to approximately +270, approximately +1 to approximately +260, approximately +1 to approximately +250, approximately +1 to approximately +240, approximately +1 to approximately +230, approximately +1 to approximately +220, approximately +1 to approximately +210, approximately +1 to approximately +200, approximately +1 to approximately +190, approximately +1 to approximately +180, The target portion is complementary to the target portion located in the region of approximately +1 to approximately +170, approximately +1 to approximately +160, approximately +1 to approximately +150, approximately +1 to approximately +140, approximately +1 to approximately +130, approximately +1 to approximately +120, approximately +1 to approximately +110, approximately +1 to approximately +100, approximately +1 to approximately +90, approximately +1 to approximately +80, approximately +1 to approximately +70, approximately +1 to approximately +60, approximately +1 to approximately +50, approximately +1 to approximately +40, approximately +1 to approximately +30, or approximately +1 to approximately +20 relative to the 5' splice site (or 3' end) of the exon.

[0320] In some embodiments, the ASO is complementary to (and binds to) a targeting portion of a precursor mRNA comprising an exon containing an inefficient translation region or PTC (followed by a variable start codon), and the targeting portion is upstream (in the 5' direction) of the exon's 5' splice site (or 3' end). In some embodiments, the targeting portion is located in a region of approximately -4 to approximately -270 nucleotides relative to the exon's 5' splice site (or 3' end). In some embodiments, the targeting portion is located in a region of approximately -1 to approximately -40,000 nucleotides relative to the exon's 5' splice site (or 3' end). In some respects, ASO is located at approximately -1 to -40,000, approximately -1 to -30,000, approximately -1 to -20,000, approximately -1 to -15,000, approximately -1 to -10,000, approximately -1 to -5,000, approximately -1 to -4,000, approximately -1 to -3,000, approximately -1 to -2,000, approximately -1 to -1,000, approximately -1 to -- 500, approximately -1 to approximately -490, approximately -1 to approximately -480, approximately -1 to approximately -470, approximately -1 to approximately -460, approximately -1 to approximately -450, approximately -1 to approximately -440, approximately -1 to approximately -430, approximately -1 to approximately -420, approximately -1 to approximately -410, approximately -1 to approximately -400, approximately -1 to approximately -390, approximately -1 to approximately -380, approximately -1 to approximately -370, approximately -1 to approximately -360, approximately -1 to approximately -350, approximately -1 to approximately -34 0, approximately -1 to approximately -330, approximately -1 to approximately -320, approximately -1 to approximately -310, approximately -1 to approximately -300, approximately -1 to approximately -290, approximately -1 to approximately -280, approximately -1 to approximately -270, approximately -1 to approximately -260, approximately -1 to approximately -250, approximately -1 to approximately -240, approximately -1 to approximately -230, approximately -1 to approximately -220, approximately -1 to approximately -210, approximately -1 to approximately -200, approximately -1 to approximately -190, approximately -1 to approximately -180, The target portions within the regions of approximately -1 to approximately -170, approximately -1 to approximately -160, approximately -1 to approximately -150, approximately -1 to approximately -140, approximately -1 to approximately -130, approximately -1 to approximately -120, approximately -1 to approximately -110, approximately -1 to approximately -100, approximately -1 to approximately -90, approximately -1 to approximately -80, approximately -1 to approximately -70, approximately -1 to approximately -60, approximately -1 to approximately -50, approximately -1 to approximately -40, approximately -1 to approximately -30, or approximately -1 to approximately -20 are complementary.

[0321] In some embodiments, the ASO is complementary to (and binds to) a target portion of a precursor mRNA comprising an exon containing an inefficient translation region or PTC (followed by a variable start codon), and the target portion is upstream (in the 5' direction) of the exon's 3' splice site (or 5' end). In some embodiments, the target portion is located in a region of approximately -1 to approximately -500 relative to the exon's 3' splice site (or 5' end). In some embodiments, the ASO is complementary to a target portion located in a region of approximately -1 to approximately -40,000 relative to the 3' splice site of the incorporated exon. In some respects, ASO is associated with the 3' splice sites of the included exons at approximately -1 to -40,000, approximately -1 to -30,000, approximately -1 to -20,000, approximately -1 to -15,000, approximately -1 to -10,000, approximately -1 to -5,000, approximately -1 to -4,000, approximately -1 to -3,000, approximately -1 to -2,000, approximately -1 to -1,000, and approximately -1 to -50. 0, approximately -1 to approximately -490, approximately -1 to approximately -480, approximately -1 to approximately -470, approximately -1 to approximately -460, approximately -1 to approximately -450, approximately -1 to approximately -440, approximately -1 to approximately -430, approximately -1 to approximately -420, approximately -1 to approximately -410, approximately -1 to approximately -400, approximately -1 to approximately -390, approximately -1 to approximately -380, approximately -1 to approximately -370, approximately -1 to approximately -360, approximately -1 to approximately -350, approximately -1 to approximately -340, Approximately -1 to approximately -330, Approximately -1 to approximately -320, Approximately -1 to approximately -310, Approximately -1 to approximately -300, Approximately -1 to approximately -290, Approximately -1 to approximately -280, Approximately -1 to approximately -270, Approximately -1 to approximately -260, Approximately -1 to approximately -250, Approximately -1 to approximately -240, Approximately -1 to approximately -230, Approximately -1 to approximately -220, Approximately -1 to approximately -210, Approximately -1 to approximately -200, Approximately -1 to approximately -190, Approximately -1 to approximately -180, Approximately The ASO is complementary to the target portion located in the regions of approximately -1 to -170, approximately -1 to -160, approximately -1 to -150, approximately -1 to -140, approximately -1 to -130, approximately -1 to -120, approximately -1 to -110, approximately -1 to -100, approximately -1 to -90, approximately -1 to -80, approximately -1 to -70, approximately -1 to -60, approximately -1 to -50, approximately -1 to -40, approximately -1 to -30, or approximately -1 to -20 relative to the 3' splice site of the included exon. In some aspects, the ASO is complementary to the target portion located in the regions of approximately -1 to -100, approximately -100 to -200, approximately -200 to -300, approximately -300 to -400, or approximately -400 to -500 relative to the 3' splice site of the included exon.

[0322] In some embodiments, the ASO is complementary to (and binds to) a target portion of a precursor mRNA comprising an exon containing an inefficient translation region or PTC (followed by a variable start codon), and the target portion is downstream (in the 3' direction) of the exon's 3' splice site (5' end). In some embodiments, the ASO is complementary to a target portion located in a region approximately +1 to approximately +40,000 relative to the exon's 3' splice site. In some respects, ASO is associated with the 3' splice site relative to the exon at approximately +1 to approximately +40,000, approximately +1 to approximately +30,000, approximately +1 to approximately +20,000, approximately +1 to approximately +15,000, approximately +1 to approximately +10,000, approximately +1 to approximately +5,000, approximately +1 to approximately +4,000, approximately +1 to approximately +3,000, approximately +1 to approximately +2,000, approximately +1 to approximately +1,000, approximately +1 to approximately +500, approximately +1 Approximately +490, Approximately +1 to approximately +480, Approximately +1 to approximately +470, Approximately +1 to approximately +460, Approximately +1 to approximately +450, Approximately +1 to approximately +440, Approximately +1 to approximately +430, Approximately +1 to approximately +420, Approximately +1 to approximately +410, Approximately +1 to approximately +400, Approximately +1 to approximately +390, Approximately +1 to approximately +380, Approximately +1 to approximately +370, Approximately +1 to approximately +360, Approximately +1 to approximately +350, Approximately +1 to approximately +340, Approximately +1 to approximately + 330, approximately +1 to approximately +320, approximately +1 to approximately +310, approximately +1 to approximately +300, approximately +1 to approximately +290, approximately +1 to approximately +280, approximately +1 to approximately +270, approximately +1 to approximately +260, approximately +1 to approximately +250, approximately +1 to approximately +240, approximately +1 to approximately +230, approximately +1 to approximately +220, approximately +1 to approximately +210, approximately +1 to approximately +200, approximately +1 to approximately +190, approximately +1 to approximately +180, approximately +1 to approximately +170 The target portions within the regions of approximately +1 to approximately +160, approximately +1 to approximately +150, approximately +1 to approximately +140, approximately +1 to approximately +130, approximately +1 to approximately +120, approximately +1 to approximately +110, approximately +1 to approximately +100, approximately +1 to approximately +90, approximately +1 to approximately +80, approximately +1 to approximately +70, approximately +1 to approximately +60, approximately +1 to approximately +50, approximately +1 to approximately +40, approximately +1 to approximately +30, or approximately +1 to approximately +20, or approximately +1 to approximately +10 are complementary.

[0323] In some embodiments, the target portion is located within a region from 5' splice site (3' end) +100 to 3' splice site (5' end) -100 relative to the exon. In some embodiments, the target portion is within an exon containing an inefficient translation region or a PTC (followed by a variable start codon). In some embodiments, the target portion includes the exon and intron boundaries.

[0324] The ASO can have any length suitable for effective enhancement of specific binding and splicing. In some embodiments, the ASO consists of 8 to 50 nucleobases. For example, the length of the ASO can be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, or 50 nucleobases. In some embodiments, the ASO consists of more than 50 nucleobases. In some embodiments, the length of the ASO is 8 to 50 nucleotides, 8 to 40 nucleotides, 8 to 35 nucleotides, 8 to 30 nucleotides, 8 to 25 nucleotides, 8 to 20 nucleotides, 8 to 15 nucleotides, 9 to 50 nucleotides, 9 to 40 nucleotides, 9 to 35 nucleotides, 9 to 30 nucleotides, 9 to 25 nucleotides, 9 to 20 nucleotides, 9 to 15 nucleotides, 10 to 50 nucleotides, 10 Up to 40 nucleotides, 10 to 35 nucleotides, 10 to 30 nucleotides, 10 to 25 nucleotides, 10 to 20 nucleotides, 10 to 15 nucleotides, 11 to 50 nucleotides, 11 to 40 nucleotides, 11 to 35 nucleotides, 11 to 30 nucleotides, 11 to 25 nucleotides, 11 to 20 nucleotides, 11 to 15 nucleotides, 12 to 50 nucleotides, 12 to 40 nucleotides, 12 to 35 nucleotides 12 to 30 nucleotides, 12 to 25 nucleotides, 12 to 20 nucleotides, 12 to 15 nucleotides, 13 to 50 nucleotides, 13 to 40 nucleotides, 13 to 35 nucleotides, 13 to 30 nucleotides, 13 to 25 nucleotides, 13 to 20 nucleotides, 14 to 50 nucleotides, 14 to 40 nucleotides, 14 to 35 nucleotides, 14 to 30 nucleotides, 14 to 25 nucleotides, 14 to 2 0 nucleotides, 15 to 50 nucleotides, 15 to 40 nucleotides, 15 to 35 nucleotides, 15 to 30 nucleotides, 15 to 25 nucleotides, 15 to 20 nucleotides, 20 to 50 nucleotides, 20 to 40 nucleotides, 20 to 35 nucleotides, 20 to 30 nucleotides, 20 to 25 nucleotides, 25 to 50 nucleotides, 25 to 40 nucleotides, 25 to 35 nucleotides, or 25 to 30 nucleotides. In some embodiments, the ASO is 18 nucleotides long. In some embodiments, the ASO is 15 nucleotides long. In some embodiments, the ASO is 25 nucleotides long.

[0325] In some embodiments, the ASO comprises the following formula:

[0326] .

[0327] In some embodiments, the ASO comprises the following formula:

[0328] .

[0329] In some embodiments, two or more ASOs with different chemical substances but complementary to the same target region of the precursor mRNA are used.

[0330] In some embodiments, the antisense oligonucleotides of this disclosure are chemically linked to one or more portions or conjugates that enhance the activity or cellular uptake of the oligonucleotide, such as targeting portions or other conjugates. Such portions include, but are not limited to, lipid portions, such as cholesterol portions, cholesterol groups, aliphatic chains such as dodecyl glycol or undecyl residues, polyamines, polyethylene glycol chains, or adamantaneacetic acid. Oligonucleotides containing lipophilic portions and methods of preparation have been described in published literature. In embodiments, the antisense oligonucleotide is conjugated to portions including, but not limited to, debased nucleotides, polyethers, polyamines, polyamides, peptides, carbohydrates such as N-acetylgalactosamine (GalNAc), N-Ac-glucosamine (GluNAc), or mannose (e.g., mannose-6-phosphate), lipids, or polyhydrocarbon compounds. The conjugates may, for example, be linked to one or more nucleotides of any nucleotide containing the antisense oligonucleotide at any of several positions on the sugar, base, or phosphate group using a linker, as understood in the art and described in the literature. The linker may include a divalent or trivalent branched linker. In one embodiment, the conjugate is attached to the 3' end of the antisense oligonucleotide. In another embodiment, the conjugate is attached to the 5' end of the antisense oligonucleotide. Methods for preparing oligonucleotide conjugates are described, for example, in U.S. Patent No. 8,450,467, "Carbohydrate conjugates as delivery agents for oligonucleotides," which is incorporated herein by reference.

[0331] In some embodiments, the antisense oligonucleotides of this disclosure are chemically linked to a lipophilic group. Representative conjugation moieties may include lipophilic molecules (aromatic and non-aromatic), including sterols and steroid molecules. The lipophilic conjugation moieties can be used, for example, to counteract the hydrophilic properties of oligomeric compounds and enhance cell penetration. Lipophilic moieties include, for example, steroids and related compounds such as cholesterol (US Patent No. 4,958,013 and Letsinger et al., Proceedings of the National Academy of Sciences, 1989, 86, 6553), mercaptocholesterol (Oberhauser et al., Nucleic Acid Research, 1992, 20, 533), lanosterol, coprosterol, stigmasterol, ergosterol, calciferol, cholic acid, deoxycholic acid, estrone, estradiol, estriol, progesterone, diethylstilbestrol, testosterone, androstenedione, deoxycorticosterone, cortisone, 17-hydroxycorticosterone, and their derivatives. Other lipophilic conjugate moieties include aliphatic groups, such as straight-chain, branched, and cyclic alkyl, alkenyl, and alkynyl groups. The aliphatic group may have, for example, 5 to about 50, 6 to about 50, 8 to about 50, or 10 to about 50 carbon atoms. Exemplary aliphatic groups include undecyl, dodecyl, hexadecyl, heptadecanyl, octadecyl, nonadecanyl, terpenes, borneol, adamantyl, and derivatives thereof. In some embodiments, one or more carbon atoms in the aliphatic group may be replaced by heteroatoms such as O, S, or N (e.g., geranyloxyhexyl). Other suitable lipophilic conjugate moieties include aliphatic derivatives of glycerol, such as alkylglycerols, di(alkyl)glycerols, tri(alkyl)glycerols, monoglycerides, diglycerides, and triglycerides. In some embodiments, the lipophilic conjugate is di-hexyldecyl-rac-glycerol or 1,2-di-O-hexyldecyl-rac-glycerol (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucleic Acid Research, 1990, 18, 3777) or their phosphonates. Saturated and unsaturated aliphatic functional groups, such as fatty acids, fatty alcohols, fatty esters, and fatty amines, may also serve as the lipophilic conjugate moiety. In some embodiments, the aliphatic functional group may contain about 6 to about 30 or about 8 to about 22 carbons. Exemplary fatty acids include decanoic acid, caprylic acid, lauric acid, palmitic acid, myristic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidic acid, arachidonic acid, eicosenoic acid, etc. In other embodiments, the lipophilic conjugate group may be a polycyclic aromatic group having 6 to about 50, 10 to about 50, or 14 to about 40 carbon atoms. Exemplary polycyclic aromatic groups include pyrene, purine, acridine, xanthine, fluorene, phenanthrene, anthracene, quinoline, isoquinoline, naphthalene, and their derivatives.Other suitable lipophilic conjugate moieties include menthol, triphenylmethyl (e.g., dimethoxytriphenylmethyl (DMT)), phenoxazine, lipoic acid, phospholipids, ethers, thioethers (e.g., hexyl-S-triphenylmethylthiol), and their derivatives. The preparation of lipophilic conjugates of oligomers is well described in the art, as in, for example, Saison-Behmoaras et al., *European Organization for Molecular Biology Journal* (EMBOJ), 1991; Kabanov et al., *Federation of European Biochemical Societies Letters* (FEBSLett.), 1990, 259, 327; Svinarchuk et al., *Biochemistry* (Biochimie), 1993, 75, 49; Mishra et al., *Biochim. Biophys. Acta*, 1995, 1264, 229; and Manoharan et al., *Tetrahedral Communications*, 1995, 36, 3651.

[0332] Oligomeric compounds containing conjugated moieties with affinity for low-density lipoprotein (LDL) can help provide efficient targeted delivery systems. The high expression levels of LDL receptors on tumor cells make LDL an attractive carrier for selectively delivering drugs to these cells (Rump et al., *Bioconjugate Chem.*, 1998, 9, 341; Firestone, *Bioconjugate Chem.*, 1994, 5, 105; Mishra et al., *Acta Biochimica et Biophysica Sinica*, 1995, 1264, 229). The LDL-affinity moieties include many lipophilic groups, such as steroids (e.g., cholesterol), fatty acids, their derivatives, and combinations thereof. In some embodiments, the LDL-affinity conjugated moieties can be dioleenyl esters of cholic acids, such as chenodeoxycholic acid and lithocholic acid.

[0333] In some embodiments, the conjugation group is or may contain a lipophilic moiety, such as a sterol (e.g., cholesterol, cholesterol group, cholesterolanol, stigmasterol, bile acid, and ergosterol). In some embodiments, the conjugation is or may contain cholesterol. See, for example, Soutschek et al., Nature (2004) 432, 173; KrQtzfeldt Nature 2005, NAR 2007.

[0334] In some embodiments, the conjugate is or may comprise lipids, phospholipids, or lipophilic alcohols, such as cationic lipids, neutral lipids, sphingolipids, and fatty acids, such as stearic acid, oleic acid, trans-oleic acid, linoleic acid, trans-linoleic acid, linolenic acid, arachidic acid, and myristic acid. In some embodiments, the fatty acid comprises a C4-C30 saturated or unsaturated alkyl chain. The alkyl chain may be straight-chain or branched.

[0335] In some embodiments, the fatty acid is a long-chain fatty acid. The long-chain fatty acid may be palmitic acid. In some embodiments, palmitic acid is attached to the 5' end of the antisense oligonucleotide. In some embodiments, palmitic acid is attached to the 5' end of the antisense oligonucleotide via a linker. In some embodiments, palmitic acid is attached to the 3' end of the antisense oligonucleotide. In some embodiments, palmitic acid is attached to the 3' end of the antisense oligonucleotide via a linker. The long-chain fatty acid may be stearic acid. In some embodiments, stearic acid is attached to the 5' end of the antisense oligonucleotide. In some embodiments, stearic acid is attached to the 5' end of the antisense oligonucleotide via a linker. In some embodiments, stearic acid is attached to the 3' end of the antisense oligonucleotide. In some embodiments, stearic acid is attached to the 3' end of the antisense oligonucleotide via a linker.

[0336] In some embodiments, the linker connecting one or more portions or conjugates (such as lipid portions) to the antisense oligonucleotide is a glycerol-based linker. In some embodiments, the linker comprises a glycerol-based linker. In some embodiments, the glycerol-based linker is a glycerol-based linker attached to the 3' end of the antisense oligonucleotide. In some embodiments, the glycerol-based linker is a glycerol-based linker attached to the 5' end of the antisense oligonucleotide. In some embodiments, the conjugate comprising a glycerol-based linker attached to the 3' end of the antisense oligonucleotide comprises the following structure:

[0337]

[0338] In some embodiments, palmitic acid is linked to an antisense oligonucleotide via a linker. In some embodiments, the linker comprises a glycerol-based linker connecting palmitic acid and the antisense oligonucleotide. In some embodiments, the glycerol-based linker is a glycerol-based linker that connects to the 3' end of the antisense oligonucleotide and links the antisense oligonucleotide to palmitic acid. In some embodiments, the glycerol-based linker is a glycerol-based linker that connects to the 5' end of the antisense oligonucleotide and links the antisense oligonucleotide to palmitic acid. In some embodiments, a conjugate comprising palmitic acid linked to an antisense oligonucleotide via a glycerol-based linker comprises the following structure:

[0339]

[0340] In some embodiments, stearic acid is linked to an antisense oligonucleotide via a linker. In some embodiments, the linker comprises a glycerol-based linker connecting stearic acid and the antisense oligonucleotide. In some embodiments, the glycerol-based linker is a glycerol-based linker that connects to the 3' end of the antisense oligonucleotide and links the antisense oligonucleotide to stearic acid. In some embodiments, the glycerol-based linker is a glycerol-based linker that connects to the 5' end of the antisense oligonucleotide and links the antisense oligonucleotide to stearic acid. In some embodiments, a conjugate comprising stearic acid linked to an antisense oligonucleotide via a glycerol-based linker comprises the following structure:

[0341]

[0342] In some embodiments, eicosanoic acid is linked to an antisense oligonucleotide via a linker. In some embodiments, the linker comprises a glycerol-based linker connecting eicosanoic acid and the antisense oligonucleotide. In some embodiments, the glycerol-based linker is a glycerol-based linker that connects to the 3' end of the antisense oligonucleotide and links the antisense oligonucleotide to eicosanoic acid. In some embodiments, the glycerol-based linker is a glycerol-based linker that connects to the 5' end of the antisense oligonucleotide and links the antisense oligonucleotide to eicosanoic acid. In some embodiments, the conjugate comprising eicosanoic acid linked to the antisense oligonucleotide via a glycerol-based linker comprises the following structure:

[0343]

[0344] In some embodiments, the linker for attaching one or more portions or conjugates (such as lipid portions) to an antisense oligonucleotide is an aminohexyl linker. In some embodiments, the linker comprises an aminohexyl linker. In some embodiments, the aminohexyl linker is an aminohexyl linker attached to the 3' end of the antisense oligonucleotide. In some embodiments, a conjugate comprising an aminohexyl linker attached to the 3' end of the antisense oligonucleotide comprises the following structure:

[0345]

[0346] In some embodiments, the aminohexyl linker is an aminohexyl linker attached to the 5' end of an antisense oligonucleotide. In some embodiments, a conjugate comprising an aminohexyl linker attached to the 5' end of an antisense oligonucleotide comprises the following structure:

[0347]

[0348] In some embodiments, palmitic acid is linked to an antisense oligonucleotide via a linker. In some embodiments, the linker comprises an aminohexyl linker connecting palmitic acid and the antisense oligonucleotide. In some embodiments, the aminohexyl linker is an aminohexyl linker that is attached to the 3' end of the antisense oligonucleotide and links the antisense oligonucleotide to palmitic acid. In some embodiments, a conjugate comprising palmitic acid linked to the antisense oligonucleotide via an aminohexyl linker comprises the following structure:

[0349]

[0350] In some embodiments, stearic acid is linked to an antisense oligonucleotide via a linker. In some embodiments, the linker comprises an aminohexyl linker connecting stearic acid and the antisense oligonucleotide. In some embodiments, the aminohexyl linker is an aminohexyl linker that is attached to the 3' end of the antisense oligonucleotide and links the antisense oligonucleotide to stearic acid. In some embodiments, a conjugate comprising stearic acid linked to the antisense oligonucleotide via an aminohexyl linker comprises the following structure:

[0351]

[0352] In some embodiments, eicosanoic acid is linked to an antisense oligonucleotide via a linker. In some embodiments, the linker comprises an aminohexyl linker linking the eicosanoic acid and the antisense oligonucleotide. In some embodiments, the aminohexyl linker is an aminohexyl linker that is attached to the 3' end of the antisense oligonucleotide and links the antisense oligonucleotide to the eicosanoic acid. In some embodiments, a conjugate comprising eicosanoic acid linked to the antisense oligonucleotide via an aminohexyl linker comprises the following structure:

[0353]

[0354] In some embodiments, palmitic acid is linked to an antisense oligonucleotide via a linker. In some embodiments, the linker comprises an aminohexyl linker connecting palmitic acid and the antisense oligonucleotide. In some embodiments, the aminohexyl linker is an aminohexyl linker that is attached to the 5' end of the antisense oligonucleotide and links the antisense oligonucleotide to palmitic acid. In some embodiments, a conjugate comprising palmitic acid linked to the antisense oligonucleotide via an aminohexyl linker comprises the following structure:

[0355]

[0356] In some embodiments, stearic acid is linked to an antisense oligonucleotide via a linker. In some embodiments, the linker comprises an aminohexyl linker connecting stearic acid and the antisense oligonucleotide. In some embodiments, the aminohexyl linker is an aminohexyl linker that is attached to the 5' end of the antisense oligonucleotide and links the antisense oligonucleotide to stearic acid. In some embodiments, a conjugate comprising stearic acid linked to the antisense oligonucleotide via an aminohexyl linker comprises the following structure:

[0357]

[0358] In some embodiments, eicosanoic acid is linked to an antisense oligonucleotide via a linker. In some embodiments, the linker comprises an aminohexyl linker connecting eicosanoic acid and the antisense oligonucleotide. In some embodiments, the aminohexyl linker is an aminohexyl linker that is attached to the 5' end of the antisense oligonucleotide and links the antisense oligonucleotide to eicosanoic acid. In some embodiments, a conjugate comprising eicosanoic acid linked to the antisense oligonucleotide via an aminohexyl linker comprises the following structure:

[0359]

[0360] In some embodiments, the linker connecting one or more portions or conjugates (such as lipid portions) to the antisense oligonucleotide is a proline-based linker. In some embodiments, the linker comprises a proline-based linker. In some embodiments, the proline-based linker is a proline-based linker attached to the 3' end of the antisense oligonucleotide. In some embodiments, the proline-based linker is a proline-based linker attached to the 5' end of the antisense oligonucleotide. In some embodiments, the conjugate comprising a proline-based linker attached to the 3' end of the antisense oligonucleotide comprises the following structure:

[0361]

[0362] In some embodiments, palmitic acid is linked to an antisense oligonucleotide via a linker. In some embodiments, the linker comprises a proline-based linker connecting palmitic acid and the antisense oligonucleotide. In some embodiments, the proline-based linker is a proline-based linker that connects to the 3' end of the antisense oligonucleotide and links the antisense oligonucleotide to palmitic acid. In some embodiments, the proline-based linker is a proline-based linker that connects to the 5' end of the antisense oligonucleotide and links the antisense oligonucleotide to palmitic acid. In some embodiments, a conjugate comprising palmitic acid linked to an antisense oligonucleotide via a proline-based linker comprises the following structure:

[0363]

[0364] In some embodiments, stearic acid is linked to an antisense oligonucleotide via a linker. In some embodiments, the linker comprises a proline-based linker connecting stearic acid and the antisense oligonucleotide. In some embodiments, the proline-based linker is a proline-based linker that connects to the 3' end of the antisense oligonucleotide and links the antisense oligonucleotide to stearic acid. In some embodiments, the proline-based linker is a proline-based linker that connects to the 5' end of the antisense oligonucleotide and links the antisense oligonucleotide to stearic acid. In some embodiments, a conjugate comprising stearic acid linked to an antisense oligonucleotide via a proline-based linker comprises the following structure:

[0365]

[0366] In some embodiments, eicosanoic acid is linked to an antisense oligonucleotide via a linker. In some embodiments, the linker comprises a proline-based linker linking eicosanoic acid and the antisense oligonucleotide. In some embodiments, the proline-based linker is a proline-based linker that links the antisense oligonucleotide to the 3' end and the antisense oligonucleotide to eicosanoic acid. In some embodiments, the proline-based linker is a proline-based linker that links the antisense oligonucleotide to the 5' end and the antisense oligonucleotide to eicosanoic acid. In some embodiments, a conjugate comprising eicosanoic acid linked to the antisense oligonucleotide via a proline-based linker comprises the following structure:

[0367]

[0368] In some embodiments, the conjugate comprises the antisense oligonucleotide of compound number 8 (SEQ ID NO: 103) and palmitic acid at or near the 5' end of the antisense oligonucleotide. In some embodiments, the conjugate comprises the antisense oligonucleotide of compound number 8 (SEQ ID NO: 103) and palmitic acid at or near the 3' end of the antisense oligonucleotide. In some embodiments, the conjugate comprises the antisense oligonucleotide of compound number 8 (SEQ ID NO: 103) and stearic acid at or near the 5' end of the antisense oligonucleotide. In some embodiments, the conjugate comprises the antisense oligonucleotide of compound number 8 (SEQ ID NO: 103) and stearic acid at or near the 3' end of the antisense oligonucleotide. In some embodiments, the conjugate comprises the antisense oligonucleotide of compound number 8 (SEQ ID NO: 103) and arachidic acid at or near the 5' end of the antisense oligonucleotide. In some embodiments, the conjugate comprises the antisense oligonucleotide of compound number 8 (SEQ ID NO: 103) and arachidic acid at or near the 3' end of the antisense oligonucleotide.

[0369] In some embodiments, the conjugate comprises the antisense oligonucleotide of compound number 8-1 (SEQ ID NO: 211) and palmitic acid at or near the 5' end of the antisense oligonucleotide. In some embodiments, the conjugate comprises the antisense oligonucleotide of compound number 8-1 (SEQ ID NO: 211) and palmitic acid at or near the 3' end of the antisense oligonucleotide. In some embodiments, the conjugate comprises the antisense oligonucleotide of compound number 8-1 (SEQ ID NO: 211) and stearic acid at or near the 5' end of the antisense oligonucleotide. In some embodiments, the conjugate comprises the antisense oligonucleotide of compound number 8-1 (SEQ ID NO: 211) and stearic acid at or near the 3' end of the antisense oligonucleotide. In some embodiments, the conjugate comprises the antisense oligonucleotide of compound number 8-1 (SEQ ID NO: 211) and arachidic acid at or near the 5' end of the antisense oligonucleotide. In some embodiments, the conjugate comprises the antisense oligonucleotide of compound number 8-1 (SEQ ID NO: 211) and arachidic acid at or near the 3' end of the antisense oligonucleotide.

[0370] In some embodiments, the conjugate comprises the antisense oligonucleotide of compound number 8-4 (SEQ ID NO: 246) and palmitic acid at or near the 5' end of the antisense oligonucleotide. In some embodiments, the conjugate comprises the antisense oligonucleotide of compound number 8-4 (SEQ ID NO: 246) and palmitic acid at or near the 3' end of the antisense oligonucleotide. In some embodiments, the conjugate comprises the antisense oligonucleotide of compound number 8-4 (SEQ ID NO: 246) and stearic acid at or near the 5' end of the antisense oligonucleotide. In some embodiments, the conjugate comprises the antisense oligonucleotide of compound number 8-4 (SEQ ID NO: 246) and stearic acid at or near the 3' end of the antisense oligonucleotide. In some embodiments, the conjugate comprises the antisense oligonucleotide of compound number 8-4 (SEQ ID NO: 246) and arachidic acid at or near the 5' end of the antisense oligonucleotide. In some embodiments, the conjugate comprises the antisense oligonucleotide of compound number 8-4 (SEQ ID NO: 246) and arachidic acid at or near the 3' end of the antisense oligonucleotide.

[0371] In some embodiments, the conjugate comprises the antisense oligonucleotide of compounds 8-5 (SEQ ID NO: 247) and palmitic acid at or near the 5' end of the antisense oligonucleotide. In some embodiments, the conjugate comprises the antisense oligonucleotide of compounds 8-5 (SEQ ID NO: 247) and palmitic acid at or near the 3' end of the antisense oligonucleotide. In some embodiments, the conjugate comprises the antisense oligonucleotide of compounds 8-5 (SEQ ID NO: 247) and stearic acid at or near the 5' end of the antisense oligonucleotide. In some embodiments, the conjugate comprises the antisense oligonucleotide of compounds 8-5 (SEQ ID NO: 247) and stearic acid at or near the 3' end of the antisense oligonucleotide. In some embodiments, the conjugate comprises the antisense oligonucleotide of compounds 8-5 (SEQ ID NO: 247) and arachidic acid at or near the 5' end of the antisense oligonucleotide. In some embodiments, the conjugate comprises the antisense oligonucleotide of compounds 8-5 (SEQ ID NO: 247) and arachidic acid at or near the 3' end of the antisense oligonucleotide.

[0372] In some embodiments, the conjugate comprises the following formula:

[0373] .

[0374] In some embodiments, the conjugate comprises the following formula:

[0375] .

[0376] In some embodiments, the conjugate comprises the following formula:

[0377] .

[0378] In some embodiments, the conjugate comprises the following formula:

[0379] .

[0380] In some embodiments, the conjugate comprises the following formula:

[0381] .

[0382] In some embodiments, the conjugate comprises the following formula:

[0383] .

[0384] In some embodiments, the conjugate comprises the following formula:

[0385] .

[0386] In some embodiments, the conjugate comprises the following formula:

[0387] .

[0388] In some embodiments, the conjugate comprises the following formula:

[0389] .

[0390] In some embodiments, the conjugate comprises the following formula:

[0391] .

[0392] In some embodiments, the conjugate comprises the following formula:

[0393] .

[0394] In some embodiments, the conjugate comprises the following formula:

[0395] .

[0396] In some embodiments, the conjugate comprises the following formula:

[0397] .

[0398] The conjugates disclosed herein can be synthesized via oligonucleotide synthesis on a solid-phase support (UnyLinker™). The oligonucleotides can be synthesized on the solid-phase support using a free 5' end that can be used to form the conjugate. An exemplary synthesis process is shown in Figure 18. For example, the precursor used for conjugation can be an aminohexyl linker synthesized via solid-phase oligonucleotide synthesis (…). Figure 18A The synthesis of conjugates (lipidization) can be achieved through peptide coupling reactions between aminohexyl linkers and fatty acids (lipids). Figure 18B This is accomplished by [method / method]. HATU can be used as a coupling agent. [Example shown] Figure 18B The scheme described herein can be applied to the synthesis of any of the 5' fusions disclosed herein.

[0399] The conjugates disclosed herein can be synthesized via oligonucleotide synthesis on a glycerol support. The oligonucleotides can be synthesized on a glycerol support using a free 3' end that can be used to form the conjugate. An exemplary synthetic process is shown in Figure 19. For example, the 3' conjugate precursor ( Figure 19A The 3' conjugate can be an aminohexyl linker synthesized via solid-phase oligonucleotide synthesis. The synthesis (lipidization) of the 3' conjugate can be achieved through peptide coupling of the aminohexyl linker with fatty acids (lipids). Figure 19B This is accomplished by [method / method]. HATU can be used as a coupling agent. [Example shown] Figure 19BThe scheme described herein can be applied to the synthesis of any of the 3' fusions disclosed herein.

[0400] In some embodiments, the nucleic acid to be targeted by ASO is a precursor mRNA expressed in cells such as eukaryotic cells. In some embodiments, the term "cell" may refer to a population of cells. In some embodiments, the cells are in a subject. In some embodiments, the cells are isolated from a subject. In some embodiments, the cells are in vitro. In some embodiments, the cells are symptom- or disease-related cells or cell lines. In some embodiments, the cells are in vitro (e.g., in a cell culture).

[0401] Pharmaceutical Composition

[0402] Pharmaceutical formulations comprising the described compositions (e.g., antisense oligonucleotides) and pharmaceutical compositions or formulations used in any of the methods described herein may be prepared according to conventional techniques well known in the pharmaceutical industry and described in published literature. In examples, pharmaceutical compositions or formulations for treating a subject comprise an effective amount of any antisense oligomer as described herein, or a pharmaceutically acceptable salt, solvate, hydrate, or ester thereof. Pharmaceutical formulations comprising antisense oligomers may further comprise pharmaceutically acceptable excipients, diluents, or carriers.

[0403] Pharmaceutically acceptable salts are suitable for contact with tissues in humans and lower animals without excessive toxicity, irritation, allergic reactions, etc., and are proportionate to a reasonable benefit / risk ratio. (See, for example, SM Berge et al., Journal of Pharmaceutical Sciences, 66: 1-19 (1977), which is incorporated herein by reference for this purpose). Salts can be prepared in situ during the final isolation and purification of the compound, or prepared separately by reacting the free base form with a suitable organic acid. Examples of pharmaceutically acceptable, non-toxic acid addition salts are those formed by amino groups with acids or by other methods described in the literature, such as ion exchange: inorganic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid; or organic acids, such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucono-heptate, glyceryl phosphate, gluconate, hemisulfate, heptaate, hydroiodate, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, dihydroxynaphthalate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, neopentanoate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium. Where appropriate, other pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations that resist the formation of counterions, such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates.

[0404] In some embodiments, the composition is formulated into any of a number of possible dosage forms, such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. In embodiments, the composition is formulated into a suspension in an aqueous, non-aqueous, or mixed culture medium. Aqueous suspensions may further contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethyl cellulose, sorbitol, and / or dextran. The suspension may also contain stabilizers. In embodiments, the pharmaceutical formulations or compositions of this disclosure include, but are not limited to, solutions, emulsions, microemulsions, foams, or formulations containing liposomes (e.g., cationic or non-cationic liposomes).

[0405] The pharmaceutical compositions or formulations described herein may contain one or more suitable and well-known to those skilled in the art or described in published literature, such as permeation enhancers, carriers, excipients, or other active or inactive ingredients. In embodiments, liposomes may also include spatially stable liposomes, such as liposomes comprising one or more specialized lipids. These specialized lipids produce liposomes with enhanced cycle life. In embodiments, spatially stable liposomes comprise one or more glycolipids or are derived from one or more hydrophilic polymers, such as polyethylene glycol (PEG) moiety. In some embodiments, surfactants are included in the pharmaceutical formulation or composition. The use of surfactants in pharmaceuticals, formulations, and emulsions is well-known in the art. In embodiments, this disclosure employs permeation enhancers to achieve efficient delivery of antisense oligonucleotides, for example, to aid diffusion across cell membranes and / or enhance the permeability of lipophilic drugs. In some embodiments, the permeation enhancer is a surfactant, fatty acid, bile salt, chelating agent, or non-chelating non-surfactant.

[0406] In some embodiments, the pharmaceutical formulation comprises a plurality of antisense oligonucleotides. In some embodiments, the antisense oligonucleotides are administered in combination with another drug or therapeutic agent.

[0407] Combination therapy

[0408] In some embodiments, the ASO disclosed herein may be used in combination with one or more other therapeutic agents. In some embodiments, one or more other therapeutic agents may comprise small molecules. For example, one or more other therapeutic agents may comprise the small molecules described in WO2016128343A1, WO2017053982A1, WO2016196386A1, WO201428459A1, WO201524876 A2, WO2013119916A2, and WO2014209841A2, which are incorporated herein by reference in their entirety. In some embodiments, one or more other therapeutic agents comprise an ASO that can be used to correct intron retention.

[0409] Treatment of subjects

[0410] Any of the compositions provided herein can be administered to an individual. The term "individual" may be used interchangeably with "subject" or "patient." An individual can be a mammal, such as a human, or an animal, such as a non-human primate, rodent, rabbit, rat, mouse, horse, donkey, goat, cat, dog, cow, pig, or sheep. In some embodiments, the individual is a human. In some embodiments, the individual is a fetus, embryo, or child. In other embodiments, the individual can be another eukaryotic organism, such as a plant. In some embodiments, the compositions provided herein are administered ex vivo to cells.

[0411] In some embodiments, the compositions provided herein are administered to an individual as a method of treating a disease or condition. In some embodiments, the individual suffers from a genetic disease, such as any of the diseases described herein. In some embodiments, the individual is at risk of developing a disease, such as any of the diseases described herein. In some embodiments, the individual has an increased risk of developing a disease or condition caused by insufficient protein quantity or insufficient protein activity. If an individual has an “increased” risk of developing a disease or condition caused by insufficient protein quantity or insufficient protein activity, the method involves preventative or therapeutic treatment. For example, an individual may be at an increased risk of developing such a disease or condition due to a family history of the disease. Typically, individuals at an increased risk of developing such a disease or condition benefit from preventative treatment (e.g., by preventing or delaying the onset or progression of the disease or condition). In embodiments, the fetus is treated in utero, for example, by administering the ASO composition directly or indirectly (e.g., via the mother).

[0412] The appropriate pathway for administering the ASO of this disclosure can vary depending on the cell type for which the ASO is to be delivered. The ASO of this disclosure can be administered to patients parenterally, for example by intrathecal injection, intraventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.

[0413] In embodiments, the antisense oligonucleotide is administered together with one or more agents capable of facilitating the penetration of the subject antisense oligonucleotide across the blood-brain barrier by any method known in the art. For example, delivery of the agent by administering an adenoviral vector to motor neurons in muscle tissue is described in U.S. Patent No. 6,632,427, “Adenoviral-vector-mediated gene transfer into medullary motor neurons,” which is incorporated herein by reference. Direct delivery of vectors to the brain, such as the striatum, thalamus, hippocampus, or substantia nigra, is described, for example, in U.S. Patent No. 6,756,523, “Adenoviral vector for transferring exogenous genes to cells in the central nervous system, specifically the brain,” which is incorporated herein by reference.

[0414] In some embodiments, the antisense oligonucleotide is linked or conjugated to an agent that provides the desired pharmaceutical or pharmacodynamic properties. In embodiments, the antisense oligonucleotide is conjugated to a substance known in the art that promotes the penetration or transport of antibodies, such as those targeting transferrin receptors, across the blood-brain barrier. In embodiments, the antisense oligonucleotide is linked to a viral vector, such as one that makes the antisense compound more effective or increases transport across the blood-brain barrier. In embodiments, permeable blood-brain barrier disruption is assisted by the infusion of sugars, such as erythritol, xylitol, D(+) galactose, D(+) lactose, D(+) xylose, aspartate, inositol, L(-) fructose, D(-) mannitol, D(+) glucose, D(+) arabinose, D(-) arabinose, cellobiose, D(+) maltose, D(+) raffinose, L(+) rhamnose, D(+) melibiose, D(-) nucleoside, etc. Sugars, calendula alcohol, D(+) arabinitol, L(-) arabinitol, D(+) fucose, L(-) fucose, D(-) lysose, L(+) lysose, and L(-) lysose; or amino acids, such as glutamine, lysine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, leucine, methionine, phenylalanine, proline, serine, threonine, tyrosine, valine, and taurine. Methods and materials for enhancing blood-brain barrier penetration are described, for example, in U.S. Patent No. 9,193,969, “Compositions and methods for selective delivery of oligonucleotide molecules to specific neuron types”, U.S. Patent No. 4,866,042, “Method for the delivery of genetic material across the blood-brain barrier”, U.S. Patent No. 6,294,520, “Material for passage through the blood-brain barrier”, and U.S. Patent No. 6,936,589, “Parenteral delivery systems”, each of which is incorporated herein by reference.

[0415] In some embodiments, the ASO of this disclosure is coupled with dopamine reuptake inhibitors (DRIs), selective serotonin reuptake inhibitors (SSRIs), norepinephrine reuptake inhibitors (NRIs), norepinephrine-dopamine reuptake inhibitors (NDRIs), and serotonin-norepinephrine-dopamine reuptake inhibitors (SNDRIs) using the methods described below: for example, U.S. Patent No. 9,193,969, which is incorporated herein by reference.

[0416] In some embodiments, the improvement of symptoms in subjects treated with the methods and compositions is evaluated using any methods known and described in the art.

[0417] Example

[0418] This disclosure will be illustrated in more detail with reference to the following examples. However, it should be understood that this disclosure is not limited in any way to these examples.

[0419] Example 1: Confirmation of exon 2 inclusion event in MECP2 mRNA processing.

[0420] RT-PCR analysis using cytoplasmic RNA from RenCellsVM (neural progenitor cells) treated with DMSO, puromycin (Puro), or cycloheximide (CHX) in exons confirmed the presence of bands corresponding to exons undergoing alternative splicing during mRNA processing. Primers were designed to target exons 1 and 3 of the MECP2 gene. Band density analysis was performed to calculate the percentage of exons included in the total transcript. Treatment of cells with cycloheximide or puromycin did not result in significant changes in e2 mRNA isotype.

[0421] Example 2: ASO stepping in exon 2 region of MECP2.

[0422] ASO walking was performed on the exon 2 region of MECP2 precursor mRNA containing various ASO-targeting sequences, using an 18-mer 2'-MOE ASO and the PS backbone. These sequences included those immediately upstream of the 3' splice site, across the 3' splice site, exon 2, across the 5' splice site, and downstream of the 5' splice site. Figure 4 ASO is designed to cover these regions by shifting 5 nucleotides at a time.

[0423] Example 3: ASO walking as assessed by RT-PCR.

[0424] ASO walking sequences were assessed, for example, by RT-PCR. RT-PCR was performed using RNA from HEK293 cells transfected with 80 nM ASO for 24 hours. Primers for RT-PCR analysis were localized to exons 1 and 3. Quantification of RT-PCR products was plotted as the percentage of e2 isotypes (e2 / (e2+e1)*100) (N = 2).

[0425] Example 4: Exemplary ASO induces MECP2 exon 2 skipping and increases MeCP2 protein expression.

[0426] RT-PCR was performed using RNA from HEK293 cells transfected with 80 nM ASO for 24 hours or untransfected (mimic). HEK293 cells treated with MECP2-targeting ASO induced exon 2 inclusion or exon 2 skipping. ASO1 targets MECP2 but has no effect on exon 2 splicing. Bar plots represent mean ± SE. n = 3. Compared to mimics 72 hours post-transfection, cells treated with exon-skipping ASO showed isotype conversion and increased total MeCP2 protein by Western blotting, which examined whole-cell lysates of ASO-treated cells. Isotypes e1 and e2 were detected with anti-MeCP2 antibodies. Quantifications of bands corresponding to MeCP2 were normalized relative to TBP (TATA-binding protein) loading controls and plotted as fold changes relative to controls (mimics).

[0427] Example 5: Quantification of total MECP2 mRNA (e1+e2).

[0428] The RT-PCR products from Example 4 were quantified and plotted as the sum of e1 + e2. ASO treatment did not increase the total mRNA level.

[0429] Example 6. Testing of an exemplary ASO in mouse cells.

[0430] ASOs were screened in both human and mouse cell lines. The exon and flanking intron sequences of MECP2 exon 2 are almost completely conserved between humans and mice. Leader candidates targeting the conserved regions of human transcripts retained their potency in mouse cells (mouse embryonic fibroblasts, MEF). RT-PCR was performed using RNA from MEF cells transfected with 10, 30, or 80 nM ASO for 24 hours or untransfected (mimicry). MEF cells treated with MECP2-targeting ASO induced exon 2 incorporation or exon 2 skipping. The effect was dose-responsive.

[0431] ASO microstepping sequences were assessed by, for example, RT-PCR. RT-PCR was performed using RNA from ReNcell VM cells transfected with 80 nM ASO nuclei for 24 hours. Primers for RT-PCR analysis were localized to exons 1 and 3. Quantification of RT-PCR products was plotted as the percentage of e2 isotypes (e2 / (e2+e1)*100) (N = 2).

[0432] Example 7. Testing of an exemplary ASO in an animal model of Rett syndrome.

[0433] Selected MECP2-targeting ASOs were tested in several mouse models to test the effects of (a) increased MeCP2 expression in wild-type mice; (b) increased MeCP2 expression in heterozygous null mice; and (c) increased MeCP2 expression in mice carrying a partially functional MECP2 p.A140V variant.

[0434] ASO was delivered to newborn wild-type mice via intraventricular bolus (ICV) injection. The effects of ASO on Mecp2 exon 2 skipping and protein expression were assessed after 2–14 days. Once the duration and magnitude of the effect were determined, the lead ASO was tested in functional studies using a mouse model of Rett syndrome. There may have been concerns about the use of TANGO-ASO to induce MECP2 duplication syndrome; therefore, a set of neural and behavioral assessments were performed in wild-type and heterozygous nulligenous animals (JAX B6.129P2(C)-Mecp2). tm1.1Bird The effects of therapeutic ASO were compared in [J]. Studies using heterozygous nullipopulation animals can also determine whether an increase in MeCP2 in a subset of neurons is beneficial and can alleviate the phenotype. Mice lacking Mecp2 expression can reconstruct several disease phenotypes in Ritter patients, including myasthenia gravis and neurological phenotypes (PsychoGenics Inc.). Compared to wild-type littermates, 8- to 12-week-old heterozygous Mecp2-nullipopulation female mice can have significantly lower grip strength and waiting time when falling from a robin. 16-week-old heterozygous female mice may have respiratory abnormalities (respiratory variability and apnea duration) and show reduced expression of brain-derived neurotrophic factor (BNDF) compared to mice with two copies of Mecp2.

[0435] A mouse model still expressing MECP2 p.A140V (JAX B6N.129-Mecp2) tm1.1Vnar The effect of ASO-mediated increased MeCP2 expression was tested in [J]. Compared with wild-type controls, female Mecp2 expression was significantly increased. A140 / yHippocampal neurons in animals can be significantly smaller. Furthermore, mTOR signaling may be dysregulated, as evidenced by reduced expression of the mTORC2 subunit RICTOR and decreased phosphorylation of mTOR and 4E-BP1. The effects of ASO, which increases MeCP2 expression, on the morphology of hippocampal neurons and mTOR signaling in the brains of these mice were tested.

[0436] Example 8. In vivo data - Adult mouse cortex.

[0437] Figure 11 Results of treatment with compound 8 in adult WT and T158M / + mice are shown. Treatment in T158M / + mice showed similar pharmacological effects compared to WT mice. Two weeks of treatment with compound 8 resulted in a reduction in exon 2 inclusion in Mecp2 mRNA in both adult WT and T158M / + cortical samples. Figure 11 (Left inset). Compared to the PBS control, MeCP2 protein expression was increased in adult WT and MECP2 T158M / + mice treated with compound number 8. Figure 11 (See the small image on the right).

[0438] Figures 17A-17H Results are shown for newborn wild-type mice treated with compound number 8 or compound number 8-1. PBS was used as a control. Figures 17A-17D The percentage of full-length MECP2 mRNA (incorporated exon 2) in the cortex of mice treated with the selected compounds at PND 8, PND 15, PND 29, and PND 114 is shown. Compared with the control group, the two compounds caused a stable decrease in full-length MECP2 mRNA in mouse cortical cells for a period of time (from PND 8 to PND 114). Figures 17E-17H The expression levels of MeCP2 protein in the cortex of mice treated with the selected compounds at PND 8, PND 15, PND 29, and PND 114 are shown. Compared with the control group, both compounds induced a stable increase in MeCP2 protein in the mouse cortex for a period of time (from PND 8 to PND 114).

[0439] Example 9. The effect of ASO on MECP2 mRNA levels.

[0440] The effects of ASO length, nucleobase chemistry, bonds, and lipid conjugates of different lengths were analyzed by RT-PCR in transfected HEK293 cells to identify the effects of ASO length, nucleobase chemistry, bonds, and lipid conjugates on the reduction of non-productive MECP2 mRNA and the increase of productive MECP2 mRNA. Figure 5 , Figure 6 , Figure 7and Figure 8 The changes in full-length MECP2 mRNA in response to ASO treatment were depicted in an in vitro compound screening assay. Cells were treated with 20 µM of ASO and allowed free uptake of ASO for 72 hours. The bar graph indicates the percentage of full-length MECP2 mRNA in the ASO-treated samples as measured by RT-PCR. NTC: Untreated control. Figure 9 The results of in vitro conversion of MECP2 isotypes and increased MECP2 protein expression by the indicated compounds (numbers 8 and 50) are shown. HEK293 cells were treated with ASO via nuclear transfection or by free uptake at specified concentrations. After 3 days of treatment, RNA and protein were collected to measure target binding (full-length MECP2 mRNA%) by RT-PCR, isotype expression (fold change relative to a simulated control) by qPCR, and protein expression (fold change relative to a simulated treatment) by Western blotting. Figure 10A The results of RT-PCR for MECP2 exon 2 inclusion using RNA from HEK293 cells treated with free uptake of indicated compounds (numbered 8, 8-1, 8-2, and 8-3) at increasing concentrations are depicted. The calculated EC50 concentrations are indicated in µM. Figure 10B Describing the comparison from Figure 10A The result is shown in the graph. Figure 12 The results of RT-PCR for MECP2 exon 2 inclusion using RNA from HEK293 cells are depicted. Cells were nuclear transfected with 3 µM of lipid-modified ASO for 24 h, or treated with 30 μM of lipid-modified ASO and allowed free uptake of ASO for 72 h. Bars represent the percentage of full-length MECP2 mRNA isotype. The lipid conjugates had no effect on ASO activity compared to unconjugated parental compounds #8 or #8.1 (target binding was comparable to that of unconjugated parental compounds #8 and #8.1 in nuclear-transfected samples), and increased free uptake activity in HEK293 cells. Figure 13The results of RT-PCR for MECP2 exon 2 inclusion using RNA from HEK293 cells are depicted. Cells were nuclear transfected with stearic acid-conjugated ASO for 24 hours, or treated with stearic acid-conjugated ASO for 30 μM, and allowed free uptake of ASO for 72 hours. Bars represent the percentage of MECP2 exon 2 inclusion. The lipid conjugates had no effect on ASO activity compared to the unconjugated parental compounds No. 8 and No. 8.1 (target binding was comparable to that of the unconjugated parental compounds No. 8 and No. 8.1 in the nuclear-transfected samples), and increased free uptake activity in HEK293 cells. Figure 14 depicts the structure of the PN bond. The PN bond can be... Figure 20 The procedure shown is for synthesis. Specifically, the precursor of PN-linked ASO3 can be phosphite triester 1, formed during solid-phase oligonucleotide synthesis. Treatment of phosphite triester 1 with 2-azido-1,3-dimethylimidazoline hexafluorophosphate 2 (typically a 0.3 M solution, 20 equivalents) at room temperature may induce a Staudinger-type reaction that oxidizes trivalent phosphorus. Subsequent treatment with diethylamine (DEA) can induce the formation of PN-linked ASO.

[0441] Figure 15 The results of RT-PCR for MECP2 exon 2 inclusion using RNA from HEK293 cells are depicted. Cells were nuclear transfected with 1 µM of PN-modified ASO (indicated by the cell line) for 24 hours, or treated with 20 μM of PN-modified ASO (indicated by the cell line) and allowed free ASO uptake for 72 hours. The bars represent five full-length MECP2 mRNA isotypes. Figure 16 The results of RT-PCR for MECP2 exon 2 inclusion using RNA from HEK293 cells are depicted. Cells were nuclear transfected with 1 µM PN-modified ASO for 24 hours, or treated with 20 μM PN-modified ASO and allowed free ASO uptake for 72 hours. Bars represent the percentage of full-length MECP2 mRNA isotype.

[0442] Example 10. Lipidification of ASO.

[0443] Figure 18 provides a general procedure applicable to the synthesis of the 5' conjugates disclosed herein. Precursors used for conjugation ( Figure 18A The aminohexyl linker 1 is synthesized through solid-phase oligonucleotide synthesis. The synthesis (lipidization) of the conjugate involves peptide coupling of aminohexyl linker 1 with fatty acid 2 (lipid). Figure 18B HATU is used as a coupling agent. (Shown in...) Figure 18BThe schemes described herein are generally applicable to the synthesis of any of the 5' fusions disclosed herein.

[0444] Specifically, DIPEA (10 equivalents) was added to a mixture of fatty acid 2 (2 equivalents) and HATU (2.2 equivalents) in 1.5 mL of DMF or NMP. The reaction mixture was vigorously vortexed at room temperature for 5 minutes to form the “fatty acid-HATU” complex. A solution of aminohexyl linker 1 (35 mg in 1.5 mL of water, 1 equivalent) was added to the “fatty acid-HATU” complex. The reaction mixture was vigorously vortexed for 10 minutes and held at room temperature for 60 minutes, with occasional vortexing. The reaction mixture was diluted to 15 mL with water and purified by RP HPLC using water / acetonitrile containing 0.1 M NaOAc as the eluent. The purified fractions were combined and desalted using a C-18 cartridge (10 g) to obtain the pure product.

[0445] Figure 19 provides a general procedure for the synthesis of the 3' conjugates disclosed herein. Precursors for 3' conjugates ( Figure 19A The synthesis of the aminohexyl linker 4' 3' conjugate (lipidization) is achieved through solid-phase oligonucleotide synthesis. This is accomplished via peptide coupling of the aminohexyl linker 4 with fatty acid 2 (lipid). Figure 19B HATU is used as a coupling agent. (Shown in...) Figure 19B The scheme described in this paper is generally applicable to all 3' fusions reported in this paper.

[0446] Specifically, DIPEA (10 equivalents) was added to a mixture of fatty acid 2 (2 equivalents) and HATU (2.2 equivalents) in 1.5 mL of DMF or NMP. The reaction mixture was vigorously vortexed at room temperature for 5 minutes to form the “fatty acid-HATU” complex. A solution of aminohexyl linker 4 (35 mg in 1.5 mL of water, 1 equivalent) was added to the “fatty acid-HATU” complex. The reaction mixture was vigorously vortexed for 10 minutes and held at room temperature for 60 minutes, with occasional vortexing. The reaction mixture was diluted to 15 mL with water and purified by RP HPLC using water / acetonitrile containing 0.1 M NaOAc as the eluent. The purified fractions were combined and desalted using a C-18 cartridge (10 g) to obtain the pure product.

[0447] Example 11. Exemplary ASO and ASO sequences.

[0448] Table 3. Sequences of exemplary ASOs.

[0449]

[0450] Table 4. Sequences of exemplary ASOs.

[0451]

[0452] Table 5. Sequences of exemplary ASOs in microstepping.

[0453]

[0454] Table 6. Sequences of exemplary PN-bonded modified ASOs.

[0455]

[0456] Table 7. Sequences of exemplary lipid-conjugated ASOs.

[0457]

[0458] Example 12. Evaluation of ASO compounds in mouse cortical neurons (MCN).

[0459] Mouse cortical neurons (MCNs) are derived from the mouse cortex and are allowed free uptake of ASO from their culture medium. Each tested ASO has a sugar moiety of a mononucleotide modified with 2'-F (2'-fluorine moiety) at the second carbon of the sugar (such nucleotides are indicated in bold and italics in the sequence columns of Table 8). These 2'-fluorine modifications of the sugar moiety of the nucleotides are placed at various positions along the ASO sequence. Dose-response curves of the ASO compounds, expressed in nM and the Hill coefficient, are used to evaluate their half-maximum effective concentration (EC50). The EC50 ratio is calculated by dividing the EC50 measured in the presence of the reference ASO compound (8-1) by the EC50 measured in the presence of the test ASO compound (8-X). An EC50 ratio greater than 1.0 indicates that the activity of the test ASO compound (8-X) is greater than that of the reference ASO compound (8-1). An EC50 ratio equal to or less than 1.0 indicates that the activity of the test ASO compound (8-X) is the same as or worse than that of the reference compound (8-1). Except for the placement of the 2'-fluorine modification in its sugar moiety, the nucleotide sequences (5'-CTACAGAAGCAAGGTG-3') and other modifications of all compounds are identical (bold = 2' MOE, italic = 2' O-Me). UnderlinedC = 5-methylcytosine. Compounds 8-10, containing 2'-fluorine modification at the sugar moiety of the fifth nucleotide, have an EC50 of 368 nM and an EC50 ratio of 0.27. Compound 8-4, containing 2'-fluorine modification at the sugar moiety of the sixth nucleotide, has an EC50 of 70 nM and an EC50 ratio of 1.49. Compound 8-12, containing 2'-fluorine modification at the sugar moiety of the seventh nucleotide, has an EC50 of 142 nM and an EC50 ratio of 1.10. Compound 8-13, containing 2'-fluorine modification at the sugar moiety of the eighth nucleotide, has an EC50 of 232 nM and an EC50 ratio of 0.67. Compound 8-1, containing 2'-fluorine modification at the sugar moiety of the ninth nucleotide, has an EC50 of 102 nM and an EC50 ratio of 1.00. Compounds 8-14, containing 2'-fluorine modification at the sugar moiety of the tenth nucleotide, have an EC50 of 149 nM and an EC50 ratio of 1.05. Compounds 8-15, containing 2'-fluorine modification at the sugar moiety of the eleventh nucleotide, have an EC50 of 99 nM and an EC50 ratio of 1.01. Compounds 8-17, containing 2'-fluorine modification at the sugar moiety of the twelfth nucleotide, have an EC50 of 81 nM and an EC50 ratio of 0.63. The EC50 and Hill coefficients for compounds 8-1 and 8-4 in Table 8 are averages across the assay set. The EC50 ratio for compound 8-4 is an average across the assay set. A summary of the average EC50 values ​​and Hill coefficients is presented in Table 9. Low-dose curves and other multi-dose experiments that did not produce EC50 values ​​were excluded from the summary table.

[0460] Table 8. EC50 measurements of various compounds from the 2'-fluorine step.

[0461]

[0462] Table 9. Summary of mean EC50 values ​​and Hill coefficients

[0463]

[0464] Example 13. Evaluation of the extended dose range of four ASO compounds in human MeCP2 T158M neurons.

[0465] Four different ASO compounds (compounds 8, 8-1, 8-4, and 8-5) at low to high concentrations were examined in human MeCP2 T158M neurons. Human T158M neurons contain the T159M mutation, in which amino acid residue 158 of MECP2 is mutated from threonine to methionine, resulting in downregulation of MeCP2 protein expression. These human MeCP2 T158M neurons were differentiated from patient-derived induced pluripotent stem cells (iPSCs) and exposed to either an ASO compound or a control (mimic) for three days on day 3 post-differentiation, followed by culture medium replacement, and were harvested on day 22 post-differentiation. The endpoints assessed included: (1) ASO-mediated target binding and resulting MECP2 protein levels; (2) phenotypic responses assessed as dendritic branch length; and (3) phenotypic responses assessed as synaptic I-point counts. Experiments were performed on three independently differentiated assemblages of human neurons.

[0466] ASO-mediated target binding and resulting MeCP2 protein levels in patient-derived MeCP2 T158M neurons

[0467] Following treatment with various ASO compounds, exon 2 levels were measured by quantitative RT-PCR of exons 1–2. Protein levels were measured from three independently differentiated neuronal assemblies, each containing 3–4 biological replicas. MeCP2 protein (3–4 replicas from each differentiation) was measured using capillary immunoelectrophoresis and COMPASS analysis on ProteinSimple JESS as a loading control, with HDAC1 (histone deacetylase 1) as a loading control. Effective ASO-mediated target binding was measured as a decrease in exon 2 inclusion and an increase in MeCP2 protein levels.

[0468] Exon 2 assessment by qRT-PCR

[0469] The control group consisted of neurons expressing wild-type MeCP2 protein treated with water (Wt x mimic) and human MeCP2 T158M neurons treated with the mimic (mutant x mimic). Test conditions required treatment of various human MeCP2 T158M neurons with four different ASO compounds (compounds 8, 8-1, 8-4, and 8-5) at various concentrations (0.001 μM, 1.0 μM, 10.0 μM, and 20.0 μM). MeCP2 exon 2 expression under each condition was normalized relative to the mutant x mimic.

[0470] When MeCP2 T158M neurons were treated with 10.0 μM of each of the tested ASO compounds, exon 2 expression was reduced to approximately 0.65-fold compared to exon 2 expression in the mutant x mimic control. When MeCP2 T158M neurons were treated with 20.0 μM of each of the tested ASO compounds, exon 2 expression was almost halved relative to exon 2 expression in the mutant x mimic control across all tested compounds.

[0471] Exon 2 expression in MeCP2 T158M neurons treated with 0.001 μM ASO compound 8 was 1.00-fold lower than that in the mutant x mimic (i.e., no change). Exon 2 expression in MeCP2 T158M neurons treated with 1.0 μM ASO compound 8 was reduced to 0.89-fold lower than that in the mutant x mimic. Exon 2 expression in MeCP2 T158M neurons treated with 10.0 μM ASO compound 8 was reduced to 0.65-fold lower than that in the mutant x mimic. Exon 2 expression in MeCP2 T158M neurons treated with 20.0 μM ASO compound 8 was reduced to 0.56-fold lower than that in the mutant x mimic. Figure 21A ).

[0472] Exon 2 expression in MeCP2 T158M neurons treated with 0.001 μM ASO compound 8-1 was 1.20-fold lower than that in the mutant x mimic. Exon 2 expression in MeCP2 T158M neurons treated with 1.0 μM ASO compound 8-1 was reduced to 0.95-fold lower than that in the mutant x mimic. Exon 2 expression in MeCP2 T158M neurons treated with 10.0 μM ASO compound 8-1 was reduced to 0.68-fold lower than that in the mutant x mimic. Exon 2 expression in MeCP2 T158M neurons treated with 20.0 μM ASO compound 8-1 was reduced to 0.51-fold lower than that in the mutant x mimic. Figure 21B ).

[0473] Exon 2 expression in MeCP2 T158M neurons treated with 0.001 μM ASO compound 8-4 was 1.07-fold higher than that in the mutant x mimic. Exon 2 expression in MeCP2 T158M neurons treated with 1.0 μM ASO compound 8-4 was reduced to 0.87-fold higher than that in the mutant x mimic. Exon 2 expression in MeCP2 T158M neurons treated with 10.0 μM ASO compound 8-4 was reduced to 0.61-fold higher than that in the mutant x mimic. Exon 2 expression in MeCP2 T158M neurons treated with 20.0 μM ASO compound 8-4 was reduced to 0.51-fold higher than that in the mutant x mimic. Figure 21C ).

[0474] Exon 2 expression in MeCP2 T158M neurons treated with 0.001 μM ASO compound 8-4 was 1.03-fold higher than that in the mutant x mimic. Exon 2 expression in MeCP2 T158M neurons treated with 1.0 μM ASO compound 8-4 was reduced to 0.97-fold higher than that in the mutant x mimic. Exon 2 expression in MeCP2 T158M neurons treated with 10.0 μM ASO compound 8-4 was reduced to 0.63-fold higher than that in the mutant x mimic. Exon 2 expression in MeCP2 T158M neurons treated with 20.0 μM ASO compound 8-4 was reduced to 0.52-fold higher than that in the mutant x mimic. Figure 21D ).

[0475] MeCP2 protein expression level

[0476] MeCP2 protein expression under each test condition was normalized relative to the mutant x mimic. Treatment of MeCP2 T158M neurons with 10.0 μM ASO compounds 8, 8-1, and 8-5 increased protein expression to approximately 1.13-fold compared to the mutant x mimic control. Treatment of MeCP2 T158M neurons with 1.0 μM ASO compound 8-4 increased protein expression to approximately 1.14-fold compared to the mutant x mimic control. Treatment of MeCP2 T158M neurons with 10.0 μM ASO compound 8-4 increased protein expression by 1.32-fold compared to the mutant x mimic control.

[0477] Protein expression in MeCP2 T158M neurons treated with 0.001 μM ASO compound 8 was 0.98-fold higher than that in the mutant x mimic. Protein expression in MeCP2 T158M neurons treated with 1.0 μM ASO compound 8 was 1.08-fold higher than that in the mutant x mimic. Protein expression in MeCP2 T158M neurons treated with 10.0 μM ASO compound 8 increased to 1.12-fold higher than that in the mutant x mimic. Protein expression in MeCP2 T158M neurons treated with 20.0 μM ASO compound 8 increased to 1.16-fold higher than that in the mutant x mimic. Figure 22A ).

[0478] Protein expression in MeCP2 T158M neurons treated with 0.001 μM ASO compound 8-1 was 0.95-fold higher than that in the mutant x mimic. Protein expression in MeCP2 T158M neurons treated with 1.0 μM ASO compound 8-1 was 1.00-fold higher than that in the mutant x mimic. Protein expression in MeCP2 T158M neurons treated with 10.0 μM ASO compound 8-1 increased to 1.14-fold higher than that in the mutant x mimic. Protein expression in MeCP2 T158M neurons treated with 20.0 μM ASO compound 8-1 increased to 1.22-fold higher than that in the mutant x mimic. Figure 22B ).

[0479] Protein expression in MeCP2 T158M neurons treated with 0.001 μM ASO compound 8-4 was 0.99-fold higher than that in the mutant x mimic. Protein expression in MeCP2 T158M neurons treated with 1.0 μM ASO compound 8-4 increased to 1.16-fold higher than that in the mutant x mimic. Protein expression in MeCP2 T158M neurons treated with 10.0 μM ASO compound 8-4 increased to 1.32-fold higher than that in the mutant x mimic. Protein expression in MeCP2 T158M neurons treated with 20.0 μM ASO compound 8-4 increased to 1.29-fold higher than that in the mutant x mimic. Figure 22C ).

[0480] Protein expression in MeCP2 T158M neurons treated with 0.001 μM ASO compound 8-4 was 0.99-fold higher than that in the mutant x mimic. Protein expression in MeCP2 T158M neurons treated with 1.0 μM ASO compound 8-4 increased to 1.12-fold higher than that in the mutant x mimic. Protein expression in MeCP2 T158M neurons treated with 10.0 μM ASO compound 8-4 increased to 1.16-fold higher than that in the mutant x mimic. Protein expression in MeCP2 T158M neurons treated with 20.0 μM ASO compound 8-4 increased to 1.15-fold higher than that in the mutant x mimic. Figure 22D ).

[0481] Neuronal morphology: Phenotypic response to dendritic branch length in MeCP2 T158M neurons of patient origin.

[0482] Dendritic branching was measured in MeCP2 T158M neurons stained with microtubule-associated protein 2 (MAP2), a major cytoskeletal regulator within neuronal dendrites. Cells were fixed, permeabilized, and labeled: DAPI-labeled nuclei and MAP2 and NeuN (“neuronal nuclei”) were also labeled. Neu ronal N Neurons labeled with "uclei" were identified by staining and size. Cell bodies and neurites were identified by staining and size. Skeletalized dendritic branches originating from the cell body were measured.

[0483] Positive staining for MAP2 (denoted as MAP2+) was used as the basis for determining the dendritic length (μm / NeuN+ neuron / well) in µm for each neuron (denoted as NeuN+) that was also positively stained for NeuN in each well. Each data point is an average of 25 fields of view per well, normalized relative to neuron count (NeuN+) / well. Reduced dendritic length phenotype was observed in neurons from patients with decreased or absent MeCP2 protein levels (e.g., Ritter patients), and untreated human MeCP2 T158M neurons showed such shortened dendrites.

[0484] Dendritic branching was normalized relative to the mutant x mimicry under each condition. Dendritic branching generally increased at almost all concentrations of ASO compound in each test. When MeCP2 T158M neurons were treated with 1.0 μM ASO compound 8, dendritic branching increased to approximately 1.20-fold compared to the mutant x mimicry control, and to approximately 1.25-fold with 10.0 μM ASO compound 8. When MeCP2 T158M neurons were treated with 0.001 μM ASO compound 8-1, dendritic branching increased to approximately 1.18-fold compared to the mutant x mimicry control. When MeCP2 T158M neurons were treated with 10.0 μM ASO compound 8-5, dendritic branching increased to 1.13-fold relative to the mutant x mimicry control.

[0485] Dendritic branching in MeCP2 T158M neurons treated with 0.001 μM ASO compound 8 was 1.12 times that in the mutant x mimic. Dendritic branching in MeCP2 T158M neurons treated with 1.0 μM ASO compound 8 was 1.20 times that in the mutant x mimic. Dendritic branching in MeCP2 T158M neurons treated with 10.0 μM ASO compound 8 increased to 1.25 times that in the mutant x mimic. Dendritic branching in MeCP2 T158M neurons treated with 20.0 μM ASO compound 8 increased to 1.18 times that in the mutant x mimic. Figure 23A ).

[0486] Dendritic branching in MeCP2 T158M neurons treated with 0.001 μM ASO compound 8-1 was 1.18 times that in the mutant x mimic. Dendritic branching in MeCP2 T158M neurons treated with 1.0 μM ASO compound 8-1 was 1.16 times that in the mutant x mimic. Dendritic branching in MeCP2 T158M neurons treated with 10.0 μM ASO compound 8-1 increased to 1.11 times that in the mutant x mimic. Dendritic branching in MeCP2 T158M neurons treated with 20.0 μM ASO compound 8-1 increased to 1.13 times that in the mutant x mimic. Figure 23B ).

[0487] Dendritic branching in MeCP2 T158M neurons treated with 0.001 μM ASO compound 8-4 was 0.98 times that in the mutant x mimic. Dendritic branching in MeCP2 T158M neurons treated with 1.0 μM ASO compound 8-4 increased to 1.10 times that in the mutant x mimic. Dendritic branching in MeCP2 T158M neurons treated with 10.0 μM ASO compound 8-4 increased to 1.06 times that in the mutant x mimic. Dendritic branching in MeCP2 T158M neurons treated with 20.0 μM ASO compound 8-4 increased to 1.09 times that in the mutant x mimic. Figure 23C ).

[0488] Dendritic branching in MeCP2 T158M neurons treated with 0.001 μM ASO compound 8-4 was 0.99 times that in the mutant x mimic. Dendritic branching in MeCP2 T158M neurons treated with 1.0 μM ASO compound 8-4 increased to 1.06 times that in the mutant x mimic. Dendritic branching in MeCP2 T158M neurons treated with 10.0 μM ASO compound 8-4 increased to 1.13 times that in the mutant x mimic. Dendritic branching in MeCP2 T158M neurons treated with 20.0 μM ASO compound 8-4 increased to 1.68 times that in the mutant x mimic. Figure 23D ).

[0489] Neuronal synapse: Presynaptic protein I in patient-derived MeCP2 T158M neurons

[0490] As described above, MAP2 staining was used to quantify the number of synapses in MeCP2 T158M neurons, and MAP2-positive staining served as the basis for counting the number of synaptic I points per pore per neuron (synaptic I point / neuron / pore). Each data point was the sum of 25 fields of view per pore normalized relative to neuron count (NeuN+) / pore. A reduced MAP2 expression phenotype was observed in neurons from patients with decreased or absent MeCP2 protein levels (e.g., Ritter patients), and untreated human MeCP2 T158M neurons exhibited this reduced MAP2 expression.

[0491] Synaptic I-point counts under each condition were normalized relative to the mutant x mimic. Synaptic I-point counts generally increased with almost all concentrations of ASO compound in each test. When MeCP2 T158M neurons were treated with 10.0 μM ASO compound 8, the synaptic I-point count increased to approximately 1.28-fold compared to the mutant x mimic control; with 20.0 μM ASO compound 8, it increased to approximately 1.24-fold; with 1.0 μM compound 8-1, it increased to approximately 1.20-fold; with 1.0 μM compound 8-1, it increased to approximately 1.23-fold; with 20.0 μM compound 8-1, it increased to approximately 1.37-fold; with 1.0 μM compound 8-4, it increased to approximately 1.19-fold; and with 10.0 μM compound 8-4, it increased to approximately 1.20-fold.

[0492] The synaptic I-point count in MeCP2 T158M neurons treated with 0.001 μM ASO compound 8 was 1.06 times that in the mutant x mimic. The synaptic I-point count in MeCP2 T158M neurons treated with 1.0 μM ASO compound 8 was 1.17 times that in the mutant x mimic. The synaptic I-point count in MeCP2 T158M neurons treated with 10.0 μM ASO compound 8 increased to 1.23 times that in the mutant x mimic. The synaptic I-point count in MeCP2 T158M neurons treated with 20.0 μM ASO compound 8 increased to 1.24 times that in the mutant x mimic. Figure 24A ).

[0493] The synaptic I-point count in MeCP2 T158M neurons treated with 0.001 μM ASO compound 8-1 was 1.12 times that in the mutant x mimic. The synaptic I-point count in MeCP2 T158M neurons treated with 1.0 μM ASO compound 8-1 was 1.20 times that in the mutant x mimic. The synaptic I-point count in MeCP2 T158M neurons treated with 10.0 μM ASO compound 8-1 increased to 1.23 times that in the mutant x mimic. The synaptic I-point count in MeCP2 T158M neurons treated with 20.0 μM ASO compound 8-1 increased to 1.37 times that in the mutant x mimic. Figure 24B ).

[0494] The synaptic I-point count in MeCP2 T158M neurons treated with 0.001 μM ASO compound 8-4 was 1.07 times that in the mutant x mimic. The synaptic I-point count in MeCP2 T158M neurons treated with 1.0 μM ASO compound 8-4 increased to 1.19 times that in the mutant x mimic. The synaptic I-point count in MeCP2 T158M neurons treated with 10.0 μM ASO compound 8-4 increased to 1.20 times that in the mutant x mimic. The synaptic I-point count in MeCP2 T158M neurons treated with 20.0 μM ASO compound 8-4 increased to 1.18 times that in the mutant x mimic. Figure 24C ).

[0495] The synaptic I-point count in MeCP2 T158M neurons treated with 0.001 μM ASO compound 8-4 was 0.96 times that in the mutant x mimic. The synaptic I-point count in MeCP2 T158M neurons treated with 1.0 μM ASO compound 8-4 increased to 1.01 times that in the mutant x mimic. The synaptic I-point count in MeCP2 T158M neurons treated with 10.0 μM ASO compound 8-4 increased to 1.14 times that in the mutant x mimic. The synaptic I-point count in MeCP2 T158M neurons treated with 20.0 μM ASO compound 8-4 increased to 1.10 times that in the mutant x mimic. Figure 24D ).

[0496] While preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, modifications, and substitutions will occur to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments of the present disclosure described herein can be used to practice the present disclosure. The appended claims are intended to define the scope of the present disclosure and thereby cover the methods and structures within the scope of these claims and their equivalents.

Claims

1. A compound of formula (I): X A X N1 X N2 X N3 X5 X6 X7 X8 X9 X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X C1 X C2 X C3 X B , where X A for And B A The following conditions must be met: (i) When X N1、 X N2 and X N3 And when X5 does not exist, it is (ii) When X N1、 X N2 and X N3 When it does not exist and X5 exists, it is: (iii) When X N1 and X N2 It does not exist and X N3 When X5 exists, it is (iv) When X N1 It does not exist and X N2 and X N3 And when X5 exists, it is ; or (v) when X N1 X N2 and X N3 And when X5 exists, it is ;X N1 for Or it does not exist, where if X N1 If it exists, then X N2 and X N3 And X5 exists; X N2 for Or it does not exist, where if X N2 If it exists, then X N3 And X5 exists; X N3 for It does not exist, where if X N3 If it exists, then X5 exists; X5 is Or it may not exist; X6 is X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for or ;X 14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X 18 for ;X 19 for ;X 20 for Or it may not exist; X C1 for Or it does not exist, where if X C1 If it exists, then X 20 Existence; X C2 for Or it does not exist, where if X C2 If it exists, then X C1 and X 20 Existence; X C3 for Or it does not exist, where if X C3 If it exists, then X C1 and X C2 and X 20 Exists; and X B for And B B The following conditions must be met: (i) When X 20 and X C1 X C2 and X C3 When it exists, it is (ii) When X 20 and X C1 and X C2 Existence and X C3 When it does not exist, it is (iii) When X 20 and X C1 Existence and X C2 and X C3 When it does not exist, it is (iv) When X 20 Existence and X C1 X C2 and X C3 When it does not exist, it is ; or (v) when X 20 and X C1 X C2 and X C3 When it does not exist, it is .

2. The compound according to claim 1, wherein B A For (i) ;X N1 Does not exist; X N2 Does not exist; X N3 X5 does not exist; X6 is not present. X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for or ;X 14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X 18 for ;X 19 for ;X 20 Does not exist; X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (v) .

3. The compound according to claim 1, wherein B A For (iii) ;X N1 Does not exist; X N2 Does not exist; X N3 for X5 is X6 is X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for or ;X 14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X 18 for ;X 19 for ;X 20 Does not exist; X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (v) .

4. The compound according to claim 1, wherein B A For (iii) ;X N1 Does not exist; X N2 Does not exist; X N3 for X5 is X6 is X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for or ;X 14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X 18 for ;X 19 for ;X 20 for ;X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (iv) .

5. The compound according to claim 1, wherein B A For (iii) ;X N1 Does not exist; X N2 Does not exist; X N3 for X5 is X6 is X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for or ;X 14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X 18 for ;X 19 for ;X 20 for ;X C1 for ;X C2 Does not exist; X C3 It does not exist; and B B For (iii) .

6. The compound according to claim 1, wherein B A For (iv) ;X N1 Does not exist; X N2 for ;X N3 for X5 is X6 is X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for or ;X 14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X 18 for ;X 19 for ;X 20 Does not exist; X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (v) .

7. The compound according to claim 1, wherein B A For (iv) ;X N1 Does not exist; X N2 for ;X N3 for X5 is X6 is X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for or ;X 14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X 18 for ;X 19 for ;X 20 for ;X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (iv) .

8. The compound according to claim 1, wherein B A For (v) ;X N1 for ;X N2 for ;X N3 for X5 is X6 is X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for or ;X 14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X 18 for ;X 19 for ;X 20 Does not exist; X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (v) .

9. The compound according to claim 1, wherein B A For (ii) ;X N1 Does not exist; X N2 Does not exist; X N3 Does not exist; X5 is X6 is X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for or ;X 14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X 18 for ;X 19 for ;X 20 for ;X C1 for ;X C2 for ;X C3 It does not exist; and B B For (ii) .

10. The compound according to claim 1, wherein B A For (ii) ;X N1 Does not exist; X N2 Does not exist; X N3 Does not exist; X5 is X6 is X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for or ;X 14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X 18 for ;X 19 for ;X 20 for ;X C1 for ;X C2 Does not exist; X C3 It does not exist; and B B For (iii) .

11. The compound according to claim 1, wherein B A For (ii) ;X N1 Does not exist; X N2 Does not exist; X N3 Does not exist; X5 is X6 is X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for or ;X 14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X 18 for ;X 19 for ;X 20 for ;X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (iv) .

12. The compound according to claim 1, wherein B A For (ii) ;X N1 Does not exist; X N2 Does not exist; X N3 Does not exist; X5 is X6 is X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for or ;X 14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X 18 for ;X 19 for ;X 20 Does not exist; X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (v) .

13. The compound according to claim 1, wherein B A For (i) ;X N1 Does not exist; X N2 Does not exist; X N3 X5 does not exist; X6 is not present. X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for or ;X 14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X 18 for ;X 19 for ;X 20 for ;X C1 for ;X C2 for ;X C3 for And B B For (i) .

14. The compound according to claim 1, wherein B A For (i) ;X N1 Does not exist; X N2 Does not exist; X N3 X5 does not exist; X6 is not present. X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for or ;X 14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X 18 for ;X 19 for ;X 20 for ;X C1 for ;X C2 for ;X C3 It does not exist; and B B For (ii) .

15. The compound according to claim 1, wherein B A For (i) ;X N1 Does not exist; X N2 Does not exist; X N3 X5 does not exist; X6 is not present. X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for or ;X 14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X 18 for ;X 19 for ;X 20 for ;X C1 for ;X C2 Does not exist; X C3 It does not exist; and B B For (iii) .

16. The compound according to claim 1, wherein B A For (i) ;X N1 Does not exist; X N2 Does not exist; X N3 X5 does not exist; X6 is not present. X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for or ;X 14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X 18 for ;X 19 for ;X 20 for ;X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (iv) .

17. The compound according to claim 1, wherein B A For (i) ;X N1 Does not exist; X N2 Does not exist; X N3 X5 does not exist; X6 is not present. X7 is X8 is X9 is ;X 10 for ;X 11 for ;X 12 for ;X 13 for ;X 14 for ;X 15 for ;X 16 for ;X 17 for ;X 18 for ;X 19 for ;X 20 Does not exist; X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (v) .

18. The compound according to claim 1, wherein B A For (i) ;X N1 Does not exist; X N2 Does not exist; X N3 X5 does not exist; X6 is not present. X7 is X8 is X9 is ;X 10 for ;X 11 for ;X 12 for ;X 13 for ;X 14 for or ;X 15 for ;X 16 for ;X 17 for ;X 18 for ;X 19 for ;X 20 Does not exist; X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (v) .

19. The compound according to claim 1, wherein B A For (i) ;X N1 Does not exist; X N2 Does not exist; X N3 X5 does not exist; X6 is not present. X7 is X8 is X9 is ;X 10 for ;X 11 for ;X 12 for ;X 13 for ;X 14 for or ;X 15 for ;X 16 for ;X 17 for ;X 18 for ;X 19 for ;X 20 Does not exist; X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (v) .

20. The compound according to claim 1, wherein B A For (i) ;X N1 Does not exist; X N2 Does not exist; X N3 X5 does not exist; X6 is not present. X7 is X8 is X9 is ;X 10 for ;X 11 for ;X 12 for ;X 13 for ;X 14 for ;X 15 for ;X 16 for ;X 17 for ;X 18 for ;X 19 for ;X 20 Does not exist; X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (v) .

21. The compound according to claim 1, wherein B A For (i) ;X N1 Does not exist; X N2 Does not exist; X N3 X5 does not exist; X6 is not present. X7 is X8 is X9 is ;X 10 for ;X 11 for ;X 12 for ;X 13 for ;X 14 for ;X 15 for ;X 16 for ;X 17 for ;X 18 for ;X 19 for ;X 20 Does not exist; X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (v) .

22. The compound according to claim 1, wherein B A For (iii) ;X N1 Does not exist; X N2 Does not exist; X N3 for X5 is X6 is X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for or ;X 14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X 18 for ;X 19 for ;X 20 for ;X C1 for ;X C2 Does not exist; X C3 It does not exist; and B B For (iii) .

23. The compound according to claim 1, wherein B A For (ii) ;X N1 Does not exist; X N2 Does not exist; X N3 Does not exist; X5 is X6 is X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for or ;X 14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X 18 for ;X 19 for ;X 20 for ;X C1 for ;X C2 for ;X C3 It does not exist; and B B For (ii) .

24. The compound according to claim 1, wherein B A For (ii) ;X N1 Does not exist; X N2 Does not exist; X N3 Does not exist; X5 is X6 is X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for or ;X 14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X 18 for ;X 19 for ;X 20 for ;X C1 for ;X C2 Does not exist; X C3 It does not exist; and B B For (iii) .

25. The compound according to claim 1, wherein B A For (i) ;X N1 Does not exist; X N2 Does not exist; X N3 X5 does not exist; X6 is not present. X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for or ;X 14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X 18 for ;X 19 for ;X 20 for ;X C1 for ;X C2 for ;X C3 for And B B For (i) .

26. The compound according to claim 1, wherein B A For (i) ;X N1 Does not exist; X N2 Does not exist; X N3 X5 does not exist; X6 is not present. X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for or ;X 14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X 18 for ;X 19 for ;X 20 for ;X C1 for ;X C2 for ;X C3 It does not exist; and B B For (ii) .

27. The compound according to claim 1, wherein B A For (i) ;X N1 Does not exist; X N2 Does not exist; X N3 X5 does not exist; X6 is not present. X7 is or X8 is X9 is ;X 10 for or ;X 11 for ;X 12 for ;X 13 for or ;X 14 for , or ;X 15 for ;X 16 for ;X 17 for or ;X 18 for ;X 19 for ;X 20 for ;X C1 for ;X C2 Does not exist; X C3 It does not exist; and B B For (iii) .

28. A compound of formula (I): X A X N1 X N2 X N3 X5 X6 X7 X8 X9 X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X C1 X C2 X C3 X B , where X A for And B A The following conditions must be met: (i) When X N1、 X N2 and X N3 And when X5 does not exist, it is (ii) When X N1、 X N2 and X N3 When it does not exist and X5 exists, it is: (iii) When X N1 and X N2 It does not exist and X N3 When X5 exists, it is (iv) When X N1 It does not exist and X N2 and X N3 And when X5 exists, it is ; or (v) when X N1 X N2 and X N3 And when X5 exists, it is ;X N1 for Or it does not exist, where if X N1 If it exists, then X N2 and X N3 And X5 exists; X N2 for Or it does not exist, where if X N2 If it exists, then X N3 And X5 exists; X N3 for Or it does not exist, where if X N3 If it exists, then X5 exists; X5 is Or it may not exist; X6 is X7 is X8 is X9 is ;X 10 for ;X 11 for ;X 12 for ;X 13 for ;X 14 for ;X 15 for ;X 16 for ;X 17 for ;X 18 for ;X 19 for ;X 20 for Or it may not exist; X C1 for Or it does not exist, where if X C1 If it exists, then X 20 Existence; X C2 for Or it does not exist, where if X C2 If it exists, then X C1 and X 20 Existence; X C3 for Or it does not exist, where if X C3 If it exists, then X C1 and X C2 and X 20 Exists; and X B for And B B The following conditions must be met: (i) When X C1 X C2 and X C3 and X 20 When it exists, it is (ii) When X C1 and X C2 and X 20 Existence and X C3 When it does not exist, it is (iii) When X C1 and X 20 Existence and X C2 and X C3 When it does not exist, it is (iv) When X 20 Existence and X C1 X C2 and X C3 When it does not exist, it is ; or (v) when X 20 and X C1 X C2 and X C3 When it does not exist, it is .

29. The compound according to claim 28, wherein B A For (i) ;X N1 Does not exist; X N2 Does not exist; X N3 X5 does not exist; X6 is not present. X7 is X8 is X9 is ;X 10 for ;X 11 for ;X 12 for ;X 13 for ;X 14 for ;X 15 for ;X 16 for ;X 17 for ;X 18 for ;X 19 for ;X 20 Does not exist; X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (v) .

30. The compound according to claim 28, wherein B A For (i) ;X N1 Does not exist; X N2 Does not exist; X N3 X5 does not exist; X6 is not present. X7 is X8 is X9 is ;X 10 for ;X 11 for ;X 12 for ;X 13 for ;X 14 for ;X 15 for ;X 16 for ;X 17 for ;X 18 for ;X 19 for ;X 20 for ;X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (iv) .

31. The compound according to claim 28, wherein B A For (i) ;X N1 Does not exist; X N2 Does not exist; X N3 X5 does not exist; X6 is not present. X7 is X8 is X9 is ;X 10 for ;X 11 for ;X 12 for ;X 13 for ;X 14 for ;X 15 for ;X 16 for ;X 17 for ;X 18 for ;X 19 for ;X 20 for ;X C1 for ;X C2 Does not exist; X C3 It does not exist; and B B For (iii) .

32. The compound according to claim 28, wherein B A For (i) ;X N1 Does not exist; X N2 Does not exist; X N3 X5 does not exist; X6 is not present. X7 is X8 is X9 is ;X 10 for ;X 11 for ;X 12 for ;X 13 for ;X 14 for ;X 15 for ;X 16 for ;X 17 for ;X 18 for ;X 19 for ;X 20 for ;X C1 for ;X C2 for ;X C3 It does not exist; and B B For (ii) .

33. The compound according to claim 28, wherein B A For (i) ;X N1 Does not exist; X N2 Does not exist; X N3 X5 does not exist; X6 is not present. X7 is X8 is X9 is ;X 10 for ;X 11 for ;X 12 for ;X 13 for ;X 14 for ;X 15 for ;X 16 for ;X 17 for ;X 18 for ;X 19 for ;X 20 for ;X C1 for ;X C2 for ;X C3 for And B B For (i) .

34. The compound according to claim 28, wherein B A For (ii) ;X N1 Does not exist; X N2 Does not exist; X N3 Does not exist; X5 is X6 is X7 is X8 is X9 is ;X 10 for ;X 11 for ;X 12 for ;X 13 for ;X 14 for ;X 15 for ;X 16 for ;X 17 for ;X 18 for ;X 19 for ;X 20 Does not exist; X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B (v) .

35. The compound according to claim 28, wherein B A For (ii) ;X N1 Does not exist; X N2 Does not exist; X N3 Does not exist; X5 is X6 is X7 is X8 is X9 is ;X 10 for ;X 11 for ;X 12 for ;X 13 for ;X 14 for ;X 15 for ;X 16 for ;X 17 for ;X 18 for ;X 19 for ;X 20 for ;X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (iv) .

36. The compound according to claim 28, wherein B A For (ii) ;X N1 Does not exist; X N2 Does not exist; X N3 Does not exist; X5 is X6 is X7 is X8 is X9 is ;X 10 for ;X 11 for ;X 12 for ;X 13 for ;X 14 for ;X 15 for ;X 16 for ;X 17 for ;X 18 for ;X 19 for ;X 20 for ;X C1 for ;X C2 Does not exist; X C3 It does not exist; and B B For (iii) .

37. The compound according to claim 28, wherein B A For (iii) ;X N1 Does not exist; X N2 Does not exist; X N3 for X5 is X6 is X7 is X8 is X9 is ;X 10 for ;X 11 for ;X 12 for ;X 13 for ;X 14 for ;X 15 for ;X 16 for ;X 17 for ;X 18 for ;X 19 for ;X 20 Does not exist; X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (v) .

38. The compound according to claim 28, wherein B A For (iii) ;X N1 Does not exist; X N2 Does not exist; X N3 for X5 is X6 is X7 is X8 is X9 is ;X 10 for ;X 11 for ;X 12 for ;X 13 for ;X 14 for ;X 15 for ;X 16 for ;X 17 for ;X 18 for ;X 19 for ;X 20 for ;X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (iv) .

39. The compound according to claim 28, wherein B A For (iii) ;X N1 Does not exist; X N2 Does not exist; X N3 for X5 is X6 is X7 is X8 is X9 is ;X 10 for ;X 11 for ;X 12 for ;X 13 for ;X 14 for ;X 15 for ;X 16 for ;X 17 for ;X 18 for ;X 19 for ;X 20 for ;X C1 for ;X C2 Does not exist; X C3 It does not exist; and B B For (iii) .

40. The compound according to claim 28, wherein B A For (iv) ;X N1 Does not exist; X N2 for ;X N3 for X5 is X6 is X7 is X8 is X9 is ;X 10 for ;X 11 for ;X 12 for ;X 13 for ;X 14 for ;X 15 for ;X 16 for ;X 17 for ;X 18 for ;X 19 for ;X 20 Does not exist; X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (v) .

41. The compound according to claim 28, wherein B A For (iv) ;X N1 Does not exist; X N2 for ;X N3 for X5 is ssss X6 is X7 is X8 is X9 is ;X 10 for ;X 11 for ;X 12 for ;X 13 for ;X 14 for ;X 15 for ;X 16 for ;X 17 for ;X 18 for ;X 19 for ;X 20 for ;X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (iv) .

42. The compound according to claim 28, wherein B A For (v) ;X N1 for ;X N2 for ;X N3 for X5 is X6 is X7 is X8 is X9 is ;X 10 for ;X 11 for ;X 12 for ;X 13 for ;X 14 for ;X 15 for ;X 16 for ;X 17 for ;X 18 for ;X 19 for ;X 20 Does not exist; X C1 Does not exist; X C2 Does not exist; X C3 It does not exist; and B B For (v) .

43. A compound selected from the group consisting of: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 and 。 44. A compound that is an antisense oligonucleotide (ASO) having a sequence according to SEQ ID NO: 103 or 211, wherein the ASO comprises a main chain modification comprising phosphorylguanidine diester or a derivative thereof.

45. The compound of claim 44, wherein the ASO comprises CnTACAGAAGCAAGGTG, CTnACAGAAGCAAGGTG, CTnACAGAAGCAAGGTG, CTACnAGAAGCAAGGTG, CTACAnGAAGCAAGGTG, CTACAGnAAGCAAGGTG, CTACAGAnAGCAAGGTG, CTACAGAAnGCAAGGTG, CTACAGAAGnCAAGGTG, CTACAGAAGCnAAGGTG, CTACAGAAGCAnAGGTG, CTACAGAAGCAAnGGTG, CTACAGAAGCAAGnGTG, CTACAGAAGCAAGGnTG, CTACAGAAGCAAGGTnG, CnTnACAGAAGCAAGGTnG, CTnAnCAGAAGCAAGGTnG, CTAnCnAGAAGCAAGGTnG, CTACnAnGAAGCAAGG CnT nACnAGAAGCAAGGTG, CTnACnAnGAAGCAAGGTG, CTAnCAnGnAAGCAAGGTG, CTACnAGnAnAGCAAGGTG, CTACAnGAnAnGCAAGGTG, CnTACAGAAGCAAGGTnG, CTnACAGAAGCAAGGnTG, CTACAGAAGCAAGGnTG or CTACAGAAGCAAGGTnG, where n is a phosphorylguanidine diester or a derivative thereof.

46. ​​The compound of claim 44 or 45, wherein the ASO further comprises a main chain modification comprising a thiophosphate (PS) bond or an aminophosphate bond.

47. The compound according to any one of claims 44 to 46, wherein the phosphorylguanidine diester or its derivative comprises (1,3-dimethylimidazolidine-2-ylidene)aminophosphate; ((4-acetamidophenyl)sulfonyl)aminophosphate; (1,3-dimethyltetrahydropyrimidine-2(1H)-ylidene)aminophosphate; (1,3-dimethyl-1,3-diazacycloheptane-2-ylidene)aminophosphate; or (di(pyrrolidine-1-yl)methylene)aminophosphate.

48. The compound according to any one of claims 44 to 47, wherein the ASO comprises a 2'-O-methyl, 2'-fluoro, and / or a 2'-O-methoxyethyl moiety.

49. The compound according to any one of claims 44 to 48, wherein the ASO comprises at least one modified sugar moiety.

50. The compound according to any one of claims 1 to 49, wherein the compound is conjugated with a lipid.

51. A compound that is an antisense oligonucleotide (ASO) having a sequence according to SEQ ID NO: 103 or 211, wherein the ASO is conjugated to a lipid.

52. The compound according to claim 50 or 51, wherein the lipid is conjugated to the 5' or 3' end of the compound.

53. The compound according to any one of claims 50 to 52, wherein the lipid is conjugated to the compound by means of a phosphate ester, an aminophosphate ester, or a thiophosphate ester.

54. The compound according to any one of claims 50 to 53, wherein the lipid is conjugated to the compound via a connector.

55. The compound of claim 54, wherein the linker is selected from the group consisting of: proline-based linkers, aminohexyl linkers, and glycerol-based linkers.

56. The compound of claim 55, wherein the connector is selected from the group consisting of: , , and .

57. The compound according to any one of claims 50 to 56, wherein the lipid is selected from the group consisting of: stearic acid, oleic acid, trans oleic acid, linoleic acid, trans linoleic acid, linolenic acid, arachidic acid, myristic acid, capric acid, caprylic acid, lauric acid, palmitic acid, arachidonic acid, and eicosenoic acid.

58. The compound of claim 57, wherein the lipid is stearic acid.

59. The compound according to any one of claims 44 to 58, wherein the compound has a structure according to formula (I): lipid-connector-ASO.

60. The compound of claim 59, wherein the compound has a structure selected from the group consisting of: 。 61. The compound according to any one of claims 44 to 58, wherein the compound has a structure according to formula (II): ASO-connector-lipid.

62. The compound of claim 61, wherein the compound has a structure selected from the group consisting of: 。

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