Peptide-antisense oligonucleotides and uses thereof for the treatment of neurodegenerative disorders

Abstract

Novel conjugates of antisense oligonucleotides and cell-penetrating peptides that induce exon-2 skipping during pre-mRNA splicing and their use in the treatment of neurodegenerative diseases such as Alzheimer's disease are disclosed.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 420,027, filed October 27, 2022, which is incorporated herein by reference in its entirety.

[0002] Disclosed herein are novel peptide-antisense oligonucleotides comprising cell-penetrating peptides conjugated to antisense oligonucleotides ("CPP-ASOs") that can induce exon skipping during pre-mRNA splicing, pharmaceutical compositions comprising the same, and methods of use thereof. [Background technology]

[0003] Neurodegenerative disorders are a group of disorders characterized by the deterioration of the structure and function of the central and peripheral nervous systems. Neurodegenerative disorders exhibit heterogeneous symptoms but may share similar features. Alzheimer's disease, one of these neurodegenerative disorders, is characterized by the accumulation of amyloid-β plaques and neurofibrillary tangles. It is also a leading cause of dementia. While rare familial cases of Alzheimer's disease involve autosomal dominant mutations in the amyloid-β precursor protein, the majority of cases are late-onset Alzheimer's disease (LOAD), which does not follow a Mendelian inheritance pattern. While the mechanism of LOAD is not fully understood, genome-wide association studies have identified genetic risk factors for LOAD. Scientists have identified genes that can affect the production, accumulation, or clearance of amyloid-β plaques. One such gene is CD33, also known as Siglec-3. Griciuc et al., Alzheimer's Disease Risk Gene CD33 Inhibits Microglial Uptake of Amyloid Beta, 78 NEURON 631 (2013).

[0004] CD33 is expressed on myeloid-derived cells, including macrophages such as microglia, and encodes the CD33 protein. Microglia account for approximately 10% of cells in the brain and serve as the first line of immunological defense. Microglia regulate several important activities in the brain, including homeostasis, cognition, and neurogenesis. Augusto-Oliveira et al., What Do Microglia Really Do in a Healthy Adult Brain?, 8 CELLS 1293 (2019). Microglial cells are known to contribute to neurodegeneration by releasing pro-inflammatory substances in the central nervous system. Wojtera et al., Microglial Cells in Neurodegenerative Disorders, 43 FOLIA NEUROPATHOLOGY 311 (2005).

[0005] CD33 is a transmembrane receptor protein with an extracellular receptor that binds the ligand sialic acid. Its intracellular immunoreceptor tyrosine-inhibitory motif recruits phosphatases upon phosphorylation of its tyrosine residues, leading to the suppression of immune cell activity, including phagocytosis. CD33 has been shown to inhibit the uptake of amyloid beta protein by microglia, suggesting that CD33-targeted therapies may potentially be a therapeutic option for LOAD. Griciuc et al., Alzheimer's Disease Risk Gene CD33 Inhibits Microglial Uptake of Amyloid Beta, 78 NEURON 631 (2013).

[0006] Two single nucleotide polymorphisms (SNPs) in the promoter region of the CD33 gene, rs3826656 and rs3865444, are associated with LOAD. The rs3865444 SNP has two forms, rs3865444-C and rs3865444-A. The first form results in normal-length CD33 protein. The second form, rs3865444-A, modulates splicing of the CD33 pre-mRNA, resulting in exon 2 skipping, resulting in CD33 protein lacking the sialic acid-binding domain. Malik et al., CD33 Alzheimer's Risk—Altering Polymorphism, CD33 Expression, and Exon 2 Splicing, 33 J. Neuroscience 13320 (2013).

[0007] In eukaryotic genes, which contain coding (exon) and non-coding (intron) sequences, mRNA is formed by excision of the non-coding introns from the pre-mRNA transcript and splicing together the coding exons. If introns remain in the final mRNA transcript or exons are omitted, the mRNA reading frame can be disrupted during translation of the mRNA, resulting in a non-functional polypeptide sequence or a premature stop codon. The splicing process is further complicated by alternative splicing, in which the same pre-mRNA sequence can be spliced ​​to different combinations of exons, resulting in the formation of multiple mRNA sequences.

[0008] Splicing of pre-mRNA is an intricate process involving a multi-megadalton ribonucleoprotein complex called the spliceosome. The spliceosome recognizes specific sequences in the pre-mRNA to precisely excise introns and ligate exons. The spliceosome catalyzes intron excision in two transesterification reactions using three conserved RNA sequences: the 5' splice site, the 3' splice site, and the branch site. Will & Luhrmann, Spliceosome Structure and Function, 3 Cold Spring Harbour. Perspective. Biol. 1 (2011).

[0009] Splicing begins with nucleophilic attack, in which the 2' OH group of the branch site binds to the 5' splice site, leading to cleavage of the 5' exon at the 5' splice site and the formation of a lariat. The 3' OH group of the 5' exon then attacks the 3' exon at the 3' splice site, ligating the 5' and 3' exons and cleaving the intron lariat. (Will & Luhrmann, Spliceosome Structure and Function, 3 Cold Spring Harbour. Perspective. Biol. 1 (2011)). Because the splicing process involves the spliceosome recognition site, the 5' and 3' splice sites, and the branch site, mutations in any one of these sites can disrupt the splicing process.

[0010] ASOs are polynucleotides designed to specifically bind to target nucleotide sequences and thereby affect one or more aspects of gene expression, such as transcription, splicing, stability, and / or translation. ASOs can be directed against either RNA or DNA. RNA-directed ASOs bind to target mRNA sequences and affect mRNA stability or translation at the ribosome.

[0011] ASOs that bind to target sequences in pre-mRNA transcripts can affect the splicing process. In some cases, ASOs can be used to induce exon skipping during pre-mRNA splicing. For example, Duchenne muscular dystrophy (DMD) is caused by mutations that alter the reading frame of dystrophin mRNA during translation, resulting in a premature stop codon and a truncated dystrophin protein. ASOs can be used to induce exon skipping during splicing, correcting the reading frame. Removal of the correct number of exons can shorten the mRNA transcript but correct the reading frame. Because dystrophin RNA consists of 79 exons, skipping one or several exons during splicing can still result in a partially functional protein. (Echigoya et al., Multiple Exon Skipping in the Duchenne Muscular Dystrophy Hot Spots: Prospects and Challenges, 8 J. PERS. MED. 41 (2018)). The FDA approved an exon-skipping drug called Exondys 51 (eteplirsen) for the treatment of DMD in 2016. Dowling, Eteplirsen therapy for Duchenne muscular dystrophy: skipping to the front of the line, 12 NATURE REV. NEUROLOGY 675 (2016). Summary of the Invention [Problem to be solved by the invention]

[0012] In other cases, ASOs can be used to prevent or reduce exon skipping during pre-mRNA splicing. For example, the ASO drug nusinersen (Spinraza®) reduces exon 7 skipping during splicing of the SMN2 gene to treat spinal muscular atrophy. Son & Yokota, Recent Advances and Clinical Applications of Exon Inclusion for Spinal Muscular Atrophy, in EXON SKIPPING & INCLUSION THERAPIES, 57-68 (2018). The rs3865444-A variant, which induces exon 2 skipping of CD33, confers protection from LOAD. Malik et al., CD33 Alzheimer's Risk—Altering Polymorphism, CD33 Expression, and Exon 2 Splicing, 33 J. NEUROSCIENCE 13320 (2013). However, there remains a need for ASOs that successfully induce exon-2 skipping during CD33 pre-mRNA splicing and their use in the treatment of neurodegenerative diseases. [Means for solving the problem]

[0013] Disclosed herein are CPP-ASOs, methods of using such CPP-ASOs to induce exon skipping during pre-mRNA splicing, pharmaceutical compositions comprising such CPP-ASOs, and methods of using such compositions to treat neurodegenerative diseases.

[0014] In some embodiments, the present specification discloses a peptide-antisense oligonucleotide conjugate comprising a cell-penetrating peptide conjugated to an antisense oligonucleotide, wherein the antisense oligonucleotide is complementary to a portion of SEQ ID NO: 1, and the peptide-antisense oligonucleotide conjugate has a CD33 exon-2 skipping efficiency of 30% or greater according to a standard exon skipping efficiency assay for antisense oligonucleotides. In some embodiments, the peptide-antisense oligonucleotide conjugate comprises a cell-penetrating peptide conjugated to an antisense oligonucleotide, and the antisense oligonucleotide comprises all or a portion of SEQ ID NO: 2, SEQ ID NO: 12, or SEQ ID NO: 224. In some embodiments, the antisense oligonucleotide is 16-30 nucleotides long, 18-30 nucleotides long, 18-25 nucleotides long, 18-21 nucleotides long, 21-30 nucleotides long, 21-25 nucleotides long, or 25-30 nucleotides long. In some embodiments, the antisense oligonucleotide is 21-30 nucleotides long. In some embodiments, the antisense oligonucleotide is 21-25 nucleotides long. In some embodiments, the antisense oligonucleotide is 18 to 21 nucleotides in length. In some embodiments, the antisense oligonucleotide is 18 to 25 nucleotides in length. In some embodiments, the antisense oligonucleotide is 25 to 30 nucleotides in length. In some embodiments, the antisense oligonucleotide is 21 or 25 nucleotides in length. In some embodiments, the antisense oligonucleotide is 25 nucleotides in length.

[0015] In some embodiments, the present specification discloses peptide-antisense oligonucleotide conjugates, wherein the antisense oligonucleotide comprises one or more unnatural sugar moieties, one or more unnatural internucleotide linkages, or one or more unnatural sugar moieties and one or more unnatural internucleotide linkages. In some embodiments, the antisense oligonucleotide comprises one or more unnatural sugar moieties. In some embodiments, the antisense oligonucleotide comprises a phosphorodiamidate morpholino oligomer (PMO). In some embodiments, the antisense oligonucleotide has a CD33 exon-2 skipping efficiency of 30% or greater, as determined by a standard exon skipping efficiency assay for PMO ASOs. In some embodiments, the antisense oligonucleotide comprises a methoxyethyl ribose oligomer (MOE). In some embodiments, the antisense oligonucleotide has a CD33 exon-2 skipping efficiency of 30% or greater, as determined by a standard exon skipping efficiency assay for MOE ASOs. In some embodiments, the antisense oligonucleotide comprises one or more unnatural internucleotide linkages. In some embodiments, the one or more unnatural internucleotide linkages comprise one or more phosphorodiamidate linkages and / or one or more phosphorothioate linkages. In some embodiments, all of the one or more non-natural internucleotide linkages have Sp configuration. In some embodiments, all of the one or more non-natural internucleotide linkages have Rp configuration. In some embodiments, the antisense oligonucleotide comprises one or more non-natural internucleotide linkages with Sp configuration and one or more non-natural internucleotide linkages with Rp configuration. In some embodiments, the antisense oligonucleotide comprises one or more modified nucleobases.

[0016] In some embodiments, disclosed herein are compositions comprising a peptide-antisense oligonucleotide conjugate and, optionally, a pharmaceutically acceptable carrier or excipient.

[0017] In some embodiments, disclosed herein are peptide-antisense oligonucleotide conjugates comprising a cell-penetrating peptide conjugated to all or a portion of PMO-002 (SEQ ID NO: 2), MOE-012 (SEQ ID NO: 12), or PMO-424 (SEQ ID NO: 224). In some embodiments, the peptide-antisense oligonucleotide conjugate comprises a cell-penetrating peptide conjugated to an antisense oligonucleotide selected from the group consisting of PMO-002 (SEQ ID NO: 2), MOE-012 (SEQ ID NO: 12), and PMO-424 (SEQ ID NO: 224). In some embodiments, the peptide comprises at least one proteinogenic amino acid, at least one non-proteinogenic amino acid, or at least one proteinogenic amino acid and at least one non-proteinogenic amino acid. In some embodiments, the peptide comprises 5-25 amino acids. In some embodiments, the non-proteinogenic amino acid comprises a modified proline residue, a lipophilic group, and / or a lactam group. In some embodiments, the peptide is a linear peptide. In some embodiments, the linear peptide comprises at least a portion of a Pip6a, ApoE, and / or neurotensin-based peptide. In some embodiments, the peptide is a cyclic peptide. In some embodiments, the cyclic peptide is CPP9 [ka] , peptide 1 [ka] , peptide 2 [ka] , or peptide 3 [ka] is.

[0018] In some embodiments, the peptide comprises a lipoic acid group. In some embodiments, the lipoic acid group is an (R)-lipoic acid group. In some embodiments, the lipoic acid group is an (S)-lipoic acid group. In some embodiments, the peptide comprises an oxadiazole bond. In some embodiments, the peptide is directly conjugated to the antisense oligonucleotide. In some embodiments, the peptide is conjugated to the antisense oligonucleotide using a chemical reaction. In some embodiments, the chemical reaction is a strain-promoted azide-alkyne cycloaddition reaction, a strained alkene-tetrazine cycloaddition reaction, or an amide bond reaction.

[0019] In some embodiments, the peptide is indirectly conjugated to the antisense oligonucleotide, and a linker is conjugated between the peptide and the antisense oligonucleotide. In some embodiments, the peptide-antisense oligonucleotide conjugate further comprises one or more nuclear localization sequences, and the one or more nuclear localization sequences are independently conjugated to the peptide and / or the antisense oligonucleotide.

[0020] In some embodiments, the present specification discloses a method for inducing exon-2 skipping in the CD33 gene during pre-mRNA splicing, the method comprising introducing into a cell a peptide-antisense oligonucleotide conjugate, the peptide-antisense oligonucleotide conjugate comprising a cell-penetrating peptide conjugated to an antisense oligonucleotide, wherein the antisense oligonucleotide is complementary to a portion of SEQ ID NO: 1, and the peptide-antisense oligonucleotide conjugate has a CD33 exon-2 skipping efficiency of 30% or greater according to a standard exon skipping efficiency assay for antisense oligonucleotides. In some embodiments, the peptide-antisense oligonucleotide conjugate comprises a cell-penetrating peptide conjugated to an antisense oligonucleotide, wherein the antisense oligonucleotide comprises all or a portion of SEQ ID NO: 2, SEQ ID NO: 12, or SEQ ID NO: 224. In some embodiments, the antisense oligonucleotide is 16-30 nucleotides in length, 18-30 nucleotides in length, 18-25 nucleotides in length, 18-21 nucleotides in length, 21-30 nucleotides in length, 21-25 nucleotides in length, or 25-30 nucleotides in length. In some embodiments, the antisense oligonucleotide comprises one or more unnatural sugar moieties, one or more unnatural internucleotide linkages, or one or more unnatural sugar moieties and one or more unnatural internucleotide linkages. In some embodiments, the antisense oligonucleotide comprises one or more unnatural sugar moieties. In some embodiments, the antisense oligonucleotide comprises a phosphorodiamidate morpholino oligomer (PMO). In some embodiments, the peptide-antisense oligonucleotide has a CD33 exon-2 skipping efficiency of 30% or greater according to a standard exon skipping efficiency assay for PMO ASOs. In some embodiments, the antisense oligonucleotide comprises a methoxyethyl ribose oligomer (MOE).In some embodiments, the antisense oligonucleotide has a CD33 exon-2 skipping efficiency of 30% or greater according to a standard exon skipping efficiency assay for MOE ASO. In some embodiments, the antisense oligonucleotide comprises one or more unnatural internucleotide linkages. In some embodiments, all of the one or more unnatural internucleotide linkages have the Sp configuration. In some embodiments, all of the one or more unnatural internucleotide linkages have the Rp configuration. In some embodiments, the one or more unnatural internucleotide linkages comprise one or more unnatural internucleotide linkages having the Sp configuration and one or more unnatural internucleotide linkages having the Rp configuration. In some embodiments, the antisense oligonucleotide comprises one or more modified nucleobases. In some embodiments, the peptide-antisense oligonucleotide further comprises a pharmaceutically acceptable carrier or excipient.

[0021] In some embodiments, the present specification discloses a method for inducing exon-2 skipping in the CD33 gene during pre-mRNA splicing, the method comprising introducing into a cell a peptide-antisense oligonucleotide conjugate, the peptide-antisense oligonucleotide conjugate comprising an antisense oligonucleotide conjugated to a cell-penetrating peptide, the antisense oligonucleotide comprising all or a portion of PMO-002 (SEQ ID NO: 2), MOE-012 (SEQ ID NO: 12), or PMO-424 (SEQ ID NO: 224). In some embodiments, the cell is an animal cell. In some embodiments, the cell is a human cell.

[0022] In some embodiments, disclosed herein are methods of treating a subject having a neurodegenerative disease, comprising administering to the subject a therapeutically effective amount of a peptide-antisense oligonucleotide conjugate, wherein the peptide-antisense oligonucleotide conjugate comprises an antisense nucleotide conjugated to a cell-penetrating peptide, wherein the antisense oligonucleotide is complementary to a portion of SEQ ID NO: 1, and wherein the peptide-antisense oligonucleotide conjugate has a CD33 exon-2 skipping efficiency of 30% or greater according to a standard exon skipping efficiency assay for antisense oligonucleotides. In some embodiments, the antisense oligonucleotide is complementary to all or a portion of SEQ ID NO: 2, SEQ ID NO: 12, or SEQ ID NO: 224. In some embodiments, the antisense oligonucleotide is 16-30 nucleotides in length, 18-30 nucleotides in length, 18-25 nucleotides in length, 18-21 nucleotides in length, 21-30 nucleotides in length, 21-25 nucleotides in length, or 25-30 nucleotides in length. In some embodiments, the antisense oligonucleotide comprises one or more unnatural sugar moieties, one or more unnatural internucleotide linkages, or one or more unnatural sugar moieties and one or more unnatural internucleotide linkages. In some embodiments, the antisense oligonucleotide comprises one or more unnatural sugar moieties. In some embodiments, the antisense oligonucleotide comprises a phosphorodiamidate morpholino oligomer (PMO). In some embodiments, the peptide-antisense oligonucleotide has a CD33 exon-2 skipping efficiency of 30% or greater according to a standard exon skipping efficiency assay for PMO ASOs. In some embodiments, the antisense oligonucleotide comprises a methoxyethyl ribose oligomer (MOE). In some embodiments, the antisense oligonucleotide has a CD33 exon-2 skipping efficiency of 30% or greater according to a standard exon skipping efficiency assay for MOE ASOs. In some embodiments, the antisense oligonucleotide comprises one or more unnatural internucleotide linkages.In some embodiments, all of the one or more non-natural internucleotide linkages have the Sp configuration. In some embodiments, all of the one or more non-natural internucleotide linkages have the Rp configuration. In some embodiments, the one or more non-natural internucleotide linkages include one or more non-natural internucleotide linkages having the Sp configuration and one or more non-natural internucleotide linkages having the Rp configuration. In some embodiments, the antisense oligonucleotide comprises one or more modified nucleobases. In some embodiments, the antisense oligonucleotide further comprises a pharmaceutically acceptable carrier or excipient.

[0023] In some embodiments, disclosed herein is a method of treating a subject having a neurodegenerative disease, comprising administering to the subject a therapeutically effective amount of a peptide-antisense oligonucleotide conjugate, wherein the peptide-antisense oligonucleotide conjugate comprises an antisense oligonucleotide conjugated to a cell-penetrating peptide, and the antisense oligonucleotide comprises all or a portion of PMO-002 (SEQ ID NO: 2), MOE-012 (SEQ ID NO: 12), or PMO-424 (SEQ ID NO: 224).

[0024] In some embodiments, disclosed herein is a method of treating a subject having a neurodegenerative disease, comprising administering to the subject a therapeutically effective amount of a peptide-antisense oligonucleotide conjugate, wherein the peptide-antisense oligonucleotide conjugate comprises an antisense oligonucleotide conjugated to a cell-penetrating peptide, and the antisense oligonucleotide is selected from the group consisting of PMO-002 (SEQ ID NO: 2), MOE-012 (SEQ ID NO: 12), or PMO-424 (SEQ ID NO: 224). In some embodiments, the subject is a human subject. In some embodiments, the neurodegenerative disease is Alzheimer's disease.

[0025] In some embodiments, the present specification discloses the peptide-antisense oligonucleotide conjugate of claim 1 for use in a method for inducing exon-2 skipping in the CD33 gene during pre-mRNA splicing, comprising introducing into a cell the peptide-antisense oligonucleotide conjugate of claim 1, wherein the peptide-antisense oligonucleotide conjugate hybridizes to a target region of the CD33 gene and induces exon-2 skipping during pre-mRNA splicing of the CD33 gene.

[0026] In some embodiments, the present specification discloses a peptide-antisense oligonucleotide conjugate for use in a method for inducing exon-2 skipping in the CD33 gene during pre-mRNA splicing, comprising introducing the peptide-antisense oligonucleotide conjugate into a cell, wherein the peptide-antisense oligonucleotide conjugate hybridizes to a target region of the CD33 gene and induces exon-2 skipping during pre-mRNA splicing of the CD33 gene. In some embodiments, the antisense oligonucleotide is 16-30 nucleotides, 18-30 nucleotides, 18-25 nucleotides, 18-21 nucleotides, 21-30 nucleotides, 21-25 nucleotides, or 25-30 nucleotides in length. In some embodiments, the antisense oligonucleotide comprises one or more unnatural sugar moieties, one or more unnatural internucleotide linkages, or one or more unnatural sugar moieties and one or more unnatural internucleotide linkages. In some embodiments, the antisense oligonucleotide comprises one or more modified sugar moieties. In some embodiments, the antisense oligonucleotide comprises a phosphorodiamidate morpholino oligomer (PMO). In some embodiments, the peptide-antisense oligonucleotide has a CD33 exon-2 skipping efficiency of 30% or greater according to a standard exon skipping efficiency assay for PMO ASOs. In some embodiments, the antisense oligonucleotide comprises a methoxyethyl ribose oligomer (MOE). In some embodiments, the antisense oligonucleotide has a CD33 exon-2 skipping efficiency of 30% or greater according to a standard exon skipping efficiency assay for MOE ASOs. In some embodiments, the antisense oligonucleotide comprises one or more non-natural internucleotide linkages. In some embodiments, all of the one or more non-natural internucleotide linkages have the Sp configuration. In some embodiments, all of the one or more non-natural internucleotide linkages have the Rp configuration.In some embodiments, the one or more unnatural internucleotide linkages comprise one or more unnatural internucleotide linkages having an Sp configuration and one or more unnatural internucleotide linkages having an Rp configuration. In some embodiments, the antisense oligonucleotide comprises one or more modified nucleobases. In some embodiments, the antisense oligonucleotide further comprises a pharmaceutically acceptable carrier or excipient.

[0027] In some embodiments, the present specification discloses a peptide-antisense oligonucleotide conjugate for use in a method of inducing exon-2 skipping in the CD33 gene during pre-mRNA splicing, comprising introducing into a cell the peptide-antisense oligonucleotide conjugate, wherein the peptide-antisense oligonucleotide conjugate hybridizes to a target region of the CD33 gene and induces exon-2 skipping during pre-mRNA splicing of the CD33 gene.

[0028] In some embodiments, the present specification discloses a peptide-antisense oligonucleotide conjugate for use in a method for inducing exon-2 skipping in the CD33 gene during pre-mRNA splicing, the method comprising introducing the peptide-antisense oligonucleotide conjugate into a cell, the peptide-antisense oligonucleotide conjugate hybridizing to a target region of the CD33 gene and inducing exon-2 skipping during pre-mRNA splicing of the CD33 gene. In some embodiments, the cell is an animal cell. In some embodiments, the cell is a human cell.

[0029] In some embodiments, the present specification discloses a peptide-antisense oligonucleotide conjugate for use in a method for treating a subject with a neurodegenerative disease, comprising administering a therapeutically effective amount of the peptide-antisense oligonucleotide conjugate to the subject. In some embodiments, the antisense oligonucleotide is 16-30 nucleotides in length, 18-30 nucleotides in length, 18-25 nucleotides in length, 18-21 nucleotides in length, 21-30 nucleotides in length, 21-25 nucleotides in length, or 25-30 nucleotides in length. In some embodiments, the antisense oligonucleotide comprises one or more unnatural sugar moieties, one or more unnatural internucleotide linkages, or one or more unnatural sugar moieties and one or more unnatural internucleotide linkages. In some embodiments, the antisense oligonucleotide comprises one or more unnatural sugar moieties. In some embodiments, the antisense oligonucleotide comprises a phosphorodiamidate morpholino oligomer (PMO). In some embodiments, the peptide-antisense oligonucleotide conjugate has a CD33 exon-2 skipping efficiency of 30% or greater, according to a standard exon skipping efficiency assay for PMO ASOs. In some embodiments, the antisense oligonucleotide comprises a methoxyethyl ribose oligomer (MOE). In some embodiments, the antisense oligonucleotide has a CD33 exon-2 skipping efficiency of 30% or greater according to a standard exon skipping efficiency assay for MOE ASO. In some embodiments, the antisense oligonucleotide comprises one or more unnatural internucleotide linkages. In some embodiments, all of the one or more unnatural internucleotide linkages have the Sp configuration. In some embodiments, all of the one or more unnatural internucleotide linkages have the Rp configuration. In some embodiments, the one or more unnatural internucleotide linkages comprise one or more unnatural internucleotide linkages having the Sp configuration and one or more unnatural internucleotide linkages having the Rp configuration.In some embodiments, the antisense oligonucleotide comprises one or more modified nucleobases. In some embodiments, the peptide-antisense oligonucleotide further comprises a pharmaceutically acceptable carrier or excipient. In some embodiments, the neurodegenerative disease is Alzheimer's disease.

[0030] In some embodiments, the present specification discloses peptides comprising cyclic peptides comprising a lipoic acid group. In some embodiments, the lipoic acid group is an (R)-lipoic acid group. In some embodiments, the lipoic acid group is an (S)-lipoic acid group. In some embodiments, the cyclic peptide comprises 4-40 amino acids, optionally the cyclic peptide comprises 6-10 amino acids. In some embodiments, the cyclic peptide comprises 1-5 arginine residues, optionally the cyclic peptide comprises 2-4 arginine residues. In some embodiments, the cyclic peptide comprises 1-5 aromatic hydrophobic amino acids, optionally the cyclic peptide comprises 2-4 aromatic hydrophobic amino acids. In some embodiments, the cyclic peptide comprises [ka] is selected from.

[0031] In some embodiments, the cyclic peptide comprises two or more lipoic acid groups, which may include two or more (R)-lipoic acid groups, two or more (S)-lipoic acid groups, and / or one or more (R)-lipoic acid groups and one or more (S)-lipoic acid groups.

[0032] In some embodiments, disclosed herein are methods for making peptides, which are synthesized according to general procedure C.

[0033] In some embodiments, the present specification discloses a peptide comprising a cyclic lactam group. In some embodiments, the peptide is a cell-penetrating peptide. In some embodiments, the peptide is a cyclic peptide. In some embodiments, the peptide comprises 4 to 40 amino acids, optionally, the peptide comprises 6 to 10 amino acids. In some embodiments, at least one amino acid of the peptide comprises a cyclic lactam group. In some embodiments, the cyclic lactam group is an 8-, 9-, or 10-membered ring. In some embodiments, the cyclic lactam group is represented by Formula III: [ka] (In the formula, R 1 and R 2 are each independently selected from the group consisting of H, aryl groups, heteroaryl groups, alkylaryl groups, arylalkyl groups, straight chain alkyl groups, branched alkyl groups, and guanidine-containing groups, wherein R 1 and R 2 wherein each of R is optionally substituted with one or more substituents; and n is an integer from 1 to 3. In some embodiments, the cyclic lactam comprises at least one side group. In some embodiments, the side group is R 1 or R 2 In some embodiments, R 1 and / or R 2 independently comprise a substituted or unsubstituted aryl group. The side chain groups comprise natural or unnatural groups including aromatic groups, straight chain alkyl groups, branched alkyl groups, functionalized alkyl groups, guanidine groups, proline groups, lipophilic groups, or arginine groups. In some embodiments, the aryl group is selected from the group consisting of phenyl groups, benzyl groups, and naphthyl groups. In some embodiments, R 1 and / or R 2 independently comprises a substituted or unsubstituted guanidine-containing group. In some embodiments, the guanidine-containing group is —(CH 2 ) 2 CN 3 H 4 。In some embodiments, the peptide comprises 1-5 arginine residues, and optionally, the peptide comprises 2-4 arginine residues.

[0034] In some embodiments, provided herein are compounds of formula IV: [ka] (In the formula, R 1 contains an aryl group or a guanidine-containing group, where R 1 Disclosed are cyclic peptides comprising at least one amino acid having a structure according to the formula: (wherein: optionally substituted by one or more substituents). In some embodiments, the aryl group is selected from the group consisting of a benzyl group, a phenyl group, and a naphthyl group. In some embodiments, the guanidine-containing group is —(CH)—CNH.

[0035] In some embodiments, provided herein are compounds of formula V: [ka] Disclosed are cyclic peptides comprising at least one oxadiazole linkage having a structure according to the formula: wherein R comprises a substituted or unsubstituted aryl group. In some embodiments, the aryl group is selected from the group consisting of a phenyl group, a benzyl group, a naphthyl group, and a methylnaphthyl group.

[0036] The application file contains color drawings. Copies of this patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0037] [Figure 1] CD33 mRNA levels in the plasma and cerebrospinal fluid of patients are shown for the rs3865444 SNP. C=rs3865444-C, A=rs3865444-A. [Figure 2]Various cognitive outcomes are shown for patients with the rs3865444-A allele compared to patients with the rs201074739 indel frameshift allele. [Figure 3] Various physiological outcomes are shown comparing patients with the rs3865444-A allele with patients with the rs201074739 indel allele. [Figure 4] 1 shows CD33 mRNA levels in plasma and cerebrospinal fluid of patients for rs201074739 indel. [Figure 5] HPLC chromatogram and HRMS trace of PMO-424 are shown. [Figure 6] HPLC chromatogram and HRMS trace of PMO-324 are shown. [Figure 7] The Tm of PMO-324, PMO-424, and PMO-224 is shown. [Figure 8] HPLC chromatogram and HRMS trace of PMO-502 are shown. [Figure 9] HPLC chromatogram and HRMS trace of PMO-402 are shown. [Figure 10] The Tm of PMO-402, PMO-502, and PMO-002 are shown. [Figure 11] Chromatogram of PMO-424 (cleaved resin) with N3'-trityl group is shown. [Figure 12] The melting temperatures of MOE-012, MOE-277, and MOE-278 are shown. [Figure 13] HPLC elution profiles of stereo-pure ASOs MOE-288 to MOE-292 and stereo-random ASO MOE-252 are shown. [Figure 14A] Figure 1 shows the in vitro exon-2 skipping efficiency for several doses of peptide-ASO conjugates using mouse bone marrow-derived macrophages. [Figure 14B] Figure 1 shows the in vitro exon-2 skipping efficiency for several doses of peptide-ASO conjugates using mouse bone marrow-derived macrophages. [Figure 15] 1 shows the in vivo activity of a 30 μg dose of Compound 30 and 30 μg, 100 μg and 300 μg doses of PMO-002. [Figure 16] 1 shows the duration of in vivo skipping activity of Compound 30 following a single 30 μg ICV dose. [Figure 17] Brain concentrations of Compound 30 and naked PMO-002 after a single 30 μg ICV dose are shown. [Figure 18] 1 shows the in vivo skipping activity in the cortex of 3 μg, 10 μg, 30 μg and 60 μg of compound 31 and 30 μg and 100 μg ICV doses of Sp-PMO-424. [Figure 19] 1 shows the in vivo skipping activity in the cortex and hippocampus of a 10 μg ICV dose of lipoic acid-containing peptides Compound 32, Compound 33, and Compound 34, along with 10 μg Compound 30 for comparison. [Figure 20] Examples of cyclic lactam amino acids are shown below. [Figure 21] 1 shows examples of cell-penetrating peptides with lactam building blocks. [Figure 22] 1 shows an exemplary synthesis of an unsaturated amino acid. [Figure 23] An exemplary synthesis of an 8-membered lactam with phenyl and 2-naphthalene side chains is shown. [Figure 24] 1 shows an exemplary synthesis of a lactam with a guanidine side chain. [Figure 25] 1 shows an exemplary synthesis of lactams via sequential Claisen rearrangement. [Figure 26] Exemplary syntheses of 9- and 10-membered lactam amino acid rings are shown. [Figure 27] Exemplary syntheses of 9- and 10-membered lactam amino acid rings bearing guanidine side chains are shown. [Figure 28] 1 shows an example of a cell-penetrating peptide with modified proline residues. [Figure 29] 1 shows examples of cell-penetrating peptides containing 1,3,4-oxadiazole bonds. [Figure 30]1 shows in vitro cellular uptake data for Compound 160, Compound 161, and Compound 162. [Figure 31] 1 shows the in vivo activity of Compounds 166 / 167 and Compounds 168 / 169 at a 30 μg dose. [Figure 32] Pharmacodynamic data for Compound 31 and Compound 33 in a 90-day duration study in transgenic hCD33 mice are shown. [Figure 33] Pharmacokinetic data for Compound 31 and Compound 33 in a 90-day duration study in transgenic hCD33 mice are shown. [Figure 34] 1 shows efficacy data for compound 31 in 5XFAD hCD33 mice. DETAILED DESCRIPTION OF THE INVENTION

[0038] definition The term "oligonucleotide" is used herein to refer to a nucleotide sequence containing at least 10 DNA or RNA nucleotides.

[0039] The term "antisense oligonucleotide", abbreviated as "ASO", is used herein to refer to a nucleotide sequence that comprises an antisense sequence that is sufficiently complementary to a target nucleotide sequence to form a stable double-stranded hybrid with the target nucleotide sequence. In some embodiments, the target nucleotide sequence is an RNA nucleotide sequence. Unless otherwise specified, the ASOs presented herein are displayed in a 5' to 3' orientation.

[0040] The term "peptide" is used herein to refer to a compound comprising two or more proteinogenic or non-proteinogenic amino acids.

[0041] The term "amino acid" is used herein to refer to a chemical compound that contains an amine group and a carboxylic acid group in the same compound.

[0042] The term "proteinogenic amino acid" is used herein to refer to an amino acid that occurs naturally in peptides.

[0043] The term "non-proteinogenic amino acid" is used herein to refer to an amino acid that does not naturally occur in peptides. Non-proteinogenic amino acids include naturally occurring or synthetic amino acids.

[0044] The term "cell penetrating peptide" or "CPP" is used herein to refer to a peptide that is capable of penetrating a cell membrane, and a peptide that is capable of penetrating a cell membrane when conjugated to an antisense oligonucleotide.

[0045] The term "nucleobase" is used herein to refer to a base that is a component of a nucleoside. Exemplary nucleobases include adenine, guanine, thymine, cytosine, and uracil.

[0046] The term "nucleoside" is used herein to refer to a nucleobase covalently linked to a sugar. Examples of naturally occurring and unnatural nucleosides are described below.

[0047] The term "nucleotide" is used herein to refer to a nucleoside covalently linked to a phosphate group. Examples of naturally occurring nucleotides include adenosine, thymidine, uridine, cytidine, 5-methylcytidine, and guanosine. Descriptions and examples of non-naturally occurring nucleotides are provided below.

[0048] The term "pharmaceutically acceptable salt" is used herein to refer to acid addition salts or base addition salts of the compounds of the present disclosure. A pharmaceutically acceptable salt is any salt that retains the activity of the parent compound and does not impart undue harmful or undesirable effects to the subject to which it is administered and in the context in which it is administered. Pharmaceutically acceptable salts include, but are not limited to, metal complexes and salts of both inorganic acids and carboxylic acids. Pharmaceutically acceptable salts also include metal salts such as sodium, calcium, potassium, magnesium, aluminum, iron, manganese, and complex salts. Additionally, pharmaceutically acceptable salts include, but are not limited to, acetic acid, aspartic acid, alkylsulfonic acid, arylsulfonic acid, axetil, benzenesulfonic acid, benzoic acid, bicarbonate, bisulfate, bitartaric acid, butyric acid, calcium edetate, camsylic acid, carbonic acid, chlorobenzoic acid, citric acid, edetic acid, edisylic acid, estrus acid, esyl, esylic acid, formic acid, fumaric acid, gluceptic acid, gluconic acid, glutamic acid, glycolic acid, glycolarilsanilic acid, hexamic acid, hexylresorcylic acid, hydrabamic acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, hydroxynaphthoic acid Acid salts of isethionic acid, lactic acid, lactobionic acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, methyl nitrate, methyl sulfate, mucic acid, muconic acid, napsylic acid, nitric acid, oxalic acid, p-nitromethanesulfonic acid, pamoic acid, pantothenic acid, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, phthalic acid, polygalactouronic acid, propionic acid, salicylic acid, stearic acid, succinic acid, sulfamic acid, sulfanilic acid, sulfonic acid, sulfuric acid, tannic acid, tartaric acid, teoclic acid, and toluenesulfonic acid. A CPP-ASO that is a pharmaceutically acceptable salt may include a pharmaceutically acceptable salt of a CPP of the CPP-ASO, a pharmaceutically acceptable salt of an ASO of the CPP-ASO, and / or a pharmaceutically acceptable salt of any linker that can conjugate the CPP to the ASO of the CPP-ASO.

[0049] Within the ASO structure of CPP-ASO, the phosphate group is generally referred to as forming the "internucleotide linkage" of the ASO. The naturally occurring internucleotide linkage in RNA and DNA is a 3' to 5' phosphodiester bond. A "phosphoroamidate" group contains a phosphorus atom attached to three oxygen atoms and one nitrogen atom, while a "phosphorodiamidate" group contains a phosphorus atom attached to two oxygen atoms and two nitrogen atoms. A "phosphorotriamidate" group (or phosphate triamide group) contains a phosphorus atom attached to one oxygen atom and three nitrogen atoms. In the uncharged or cationic internucleotide linkage of the morpholino-based ASO described herein, one nitrogen is always pendant to the linking chain. The second nitrogen of the phosphorodiamidate linkage is typically the ring nitrogen of the morpholino ring structure.

[0050] The term "non-natural" is used herein to refer to a molecule that has an artificial modification compared to its naturally occurring counterpart. In some embodiments, "non-natural" can refer to one or more nucleotide subunits having at least one modification selected from (i) a modified internucleotide linkage, e.g., an internucleotide linkage other than the standard phosphodiester linkage found in naturally occurring oligonucleotides, (ii) a modified sugar moiety, e.g., a moiety other than a ribose or deoxyribose moiety found in naturally occurring oligonucleotides, (iii) a modified nucleobase, e.g., a base other than those found in naturally occurring oligonucleotides, or (iv) any combination of the foregoing. In some embodiments, the ASO of the CPP-ASO is selected from ASOs that do not have a phosphorus atom in the internucleotide linkage (backbone). In some embodiments, the ASO has a phosphorodiamidate or phosphorothioate modified internucleotide linkage (backbone).

[0051] The term "morpholino" is used herein to refer to a nucleotide that has a morpholinyl ring in place of a ribose.

[0052] The term "morpholino-based ASO" is used herein to refer to an ASO that contains at least one nucleotide that has a morpholinyl ring in place of ribose.

[0053] The term "sterically controlled" is used herein to describe when a nucleotide and / or oligonucleotide is designed or selected to have a specific stereochemistry. In some embodiments, the nucleobase portion of a nucleotide or oligonucleotide is sterically controlled, including any and all non-natural modifications. In some embodiments, the nucleoside portion of a nucleotide or oligonucleotide is sterically controlled, including any and all non-natural modifications. In some embodiments, the internucleotide linkage portion of a nucleotide or oligonucleotide is sterically controlled, including any and all non-natural modifications. In some embodiments, a nucleotide can contain one or a combination of these sterically controlled moieties. In some embodiments, an oligonucleotide can contain a combination of nucleotides, including a combination of sterically controlled nucleotides. In some embodiments, an oligonucleotide can contain a combination of sterically controlled and non-sterically controlled nucleotides. In some embodiments, the ratio of sterically controlled nucleotides is 15% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 50% to 90%, 50% to 95%, 60% to 100%, 60% to 90%, 60% to 95%, 70% to 100%, 70% to 90%, 70% to 95%, 80% to 100%, 80% to 90%, 80% to 95%, 90% to 100%, 90% to 95%, 90% to 96%, 90% to 97%, 90% to 98%, 90% to 99%, 95% to 98%, A range of 10% to 100% nucleotides, such as 95% to 99%, 95 to 100%, 50% to 90%, or 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0054] The term "sterically pure," as applied to nucleotides, is used herein to describe when at least 90% of the nucleotides in an oligonucleotide are stereorestricted. In some embodiments, the proportion of stereorestricted nucleotides in a stereorestricted CPP-ASO is in the range of 90-100%, 95-100%, 90-95%, 90-96%, 90-97%, 90-98%, 90-99%, 95-98%, 95-99%, or 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the nucleotides. In some embodiments, all or a portion of the nucleotides in an oligonucleotide are stereocontrolled so that they are similarly stereochemically pure, i.e., all or a portion of the nucleotides are stereocontrolled and are designed or selected to have the same stereochemistry. In some embodiments, all or a portion of the nucleotides in an oligonucleotide are stereocontrolled so that they are not similarly stereochemically pure, i.e., all or a portion of the nucleotides are stereocontrolled, but are designed or selected to have different stereochemistry. When applied to the internucleotide linkage portion of an oligonucleotide, the term "sterically pure" is used to describe when at least 90% of the internucleotide linkages are stereocontrolled. In some embodiments, the proportion of sterically controlled internucleotide linkages in the sterically pure CPP-ASO is in the range of 90-100%, 95-100%, 90%-95%, 90%-96%, 90%-97%, 90%-98%, 90%-99%, 95%-98%, 95%-99%, or 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% internucleotide linkages.In some embodiments, all or a portion of the internucleotide linkages in an oligonucleotide are stereocontrolled so that they are similarly sterically pure, i.e., all or a portion of the internucleotide linkages are stereocontrolled and are designed or selected to have the same stereochemistry. In some embodiments, all or a portion of the internucleotide linkages in an oligonucleotide are stereocontrolled so that they are not similarly sterically pure, i.e., all or a portion of the internucleotide linkages are stereocontrolled, but are designed or selected to have different stereochemistry. In some embodiments, the internucleotide linkages are phosphorodiamidate linkages. In some embodiments, the internucleotide linkages are phosphorothioate linkages.

[0055] The stereochemistry of the (Rp, Sp) and phosphate (PO) internucleotide linkages is depicted as follows: [ka] The stereochemistry of the Rp, Sp, and PO internucleotide linkages is also designated as follows: S=Sp, R=Rp, O=phosphate.

[0056] For example, the stereochemistry of the internucleotide bond of MOE-298 can be depicted using either of the following notations: [ka] or (5'-CCGAAAGAAGTATGAACC-3') (SEQ ID NO: 252); stereo pattern: SOSSSRSSSRSSSOSSS.

[0057] When applied to nucleotides, the term "sterically random" is used herein to describe when the nucleotides in an oligonucleotide are not sterically controlled. When applied to internucleotide linkages, the term "sterically random" is used herein to describe when the internucleotide linkages in an oligonucleotide are not sterically controlled. In some embodiments, the internucleotide linkages are phosphorodiamidate linkages. In some embodiments, the internucleotide linkages are phosphorothioate linkages.

[0058] The term "complementary" is used herein to denote when corresponding portions of at least two nucleotide sequences are occupied by nucleotides that are capable of hydrogen bonding with each other.

[0059] The term "hybridize" is used herein to refer to the binding of two complementary nucleotide sequences to form a double-stranded molecule. When a sufficient number of corresponding nucleotides in the two sequences can hydrogen bond with each other, i.e., when they are sufficiently complementary, they can form a stable hybrid. It is understood in the art that 100% complementarity is not necessarily required for a CPP-ASO to hybridize with a target sequence.

[0060] The term "sufficient complementarity" is used herein to refer to a level of complementarity sufficient for the ASO of a CPP-ASO to bind to its target sequence and form a stable hybrid. In some embodiments, the complementarity between the ASO and the target sequence is at least 99%, or 98%, or 97%, or 96%, or 95%, or 94%, or 93%, or 92%, or 91%, or 90%, or 89%, or 88%, or 87%, or 86%, or 85%, or 84%, or 83%, or 82%, or 81%, or 80%, or 79%, or 78%, or 77%, or 76%, or 75%, or 74%, or 73%, or 72%, or 71%, or 70%.

[0061] The term "sequence similarity" is used herein to describe the similarity between two CPP-ASOs. Sequence similarity is expressed as the percentage of nucleotides shared between the two CPP-ASOs. Identical sequences are understood to have 100% sequence similarity.

[0062] The terms "target region" and "target sequence" are used interchangeably herein to designate the nucleotide sequence to which the ASO of a CPP-ASO will hybridize under physiological conditions. It is not necessary for the ASO and the target region to be 100% complementary, as long as there is sufficient complementarity for the ASO to hybridize to the target sequence and form a stable hybrid. The ASO may hybridize to all or a portion of the target sequence.

[0063] The terms "treat," "treating," or "treatment" are used herein to refer to ameliorating a disease or disorder (i.e., slowing or arresting or reducing the onset of a disease or at least one of its clinical symptoms). These terms also refer to alleviating or improving at least one physical parameter, including those that may not be discernible to the patient. These terms also refer to either physically modulating a disease or disorder (e.g., through stabilization of discernible symptoms), physiologically modulating a disease or disorder (e.g., through stabilization of a physical parameter), or both.

[0064] The terms "prevent," "preventing," or "prevention" are used herein to refer to inhibiting or delaying the onset of a disease or disorder.

[0065] The term "therapeutically effective amount" is used herein to refer to an amount of a therapeutic agent or composition effective in preventing or treating a disorder or disease. In some embodiments, this includes an amount of a therapeutic agent or composition effective in preventing or treating a neurodegenerative disease.

[0066] The term "pharmaceutically acceptable" is used herein to refer to a molecular entity or composition that is pharmaceutically useful and that is not biologically or otherwise undesirable.

[0067] The term "carrier" is used herein to refer to a diluent, adjuvant, excipient, or vehicle with which the compound is administered.

[0068] The term "excipient" as used herein refers to any ingredient, other than the active ingredient, that is in a pharmaceutical composition.

[0069] As used herein, the "skipping efficiency" of an oligonucleotide is calculated using the following formula:

number

[0070] A standard exon skipping efficiency assay for CPP-ASOs involves using murine bone marrow-derived macrophage (mBMDM) cells cultured and maintained in the appropriate medium (Dulbecco's modified Eagle's medium containing 10% fetal bovine serum) as suggested in the vendor's protocol. The assay is performed in a 96-well plate format, seeding approximately 50,000 cells per well and treating with PMO CPP-ASOs at a concentration of 0.5 μM without additional transfection reagent. The cells are incubated at 37°C in a cell culture incubator for 48 hours, after which total RNA is isolated. Total RNA was isolated and converted to cDNA according to the supplier's protocol, and then Taqman gene expression assays were used to quantify exon-2-skipped CD33 (forward primer: CGCTGCTGCTACTGCTG (SEQ ID NO: 207); reverse primer: TTCTAGAGTGCCAGGGATGA (SEQ ID NO: 208); and probe: TGTGGGCAGACTTGACCCACAG (SEQ ID NO: 209)) and non-skipped CD33 (forward primer: GGATGGAGAGAGGAAGTA (SEQ ID NO: 210) or TTCGGATGGAGAGAGGAAGTA (SEQ ID NO: 291); reverse primer: GTGCCAGGGATGAGGATTT (SEQ ID NO: 211); and probe: TGCATGTGACAGACTTGACCCACA (SEQ ID NO: 212)) mRNA transcripts. Target transcript expression was normalized using the expression of the mouse housekeeping gene HPRT1 (Assay ID: Hs02800695_m1; ThermoFisher Scientific).

[0071] The term "alkyl" is used herein to refer to saturated hydrocarbons having one or more carbon atoms, including straight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, etc.), branched-chain alkyl groups (e.g., isopropyl, tert-butyl, sec-butyl, isobutyl, etc.), cyclic alkyl groups (also referred to as "cycloalkyl" groups), and alkyl-substituted alkyl groups (e.g., alkyl-substituted cycloalkyl and cycloalkyl-substituted alkyl groups). The terms "alkenyl" and "alkynyl" refer to unsaturated aliphatic groups analogous to alkyl, but which contain at least one double or triple carbon-carbon bond, respectively.

[0072] The term "alkoxy" is used herein to refer to an alkyl group linked to the remainder of the molecule via an oxygen atom. Examples of alkoxy groups include methoxy, ethoxy, isopropyloxy, propoxy, butoxy, and pentoxy groups. Alkoxy groups can be straight-chained or branched.

[0073] The term "alkoxyalkyl" is used herein to refer to an alkyl group substituted with an alkoxy group.

[0074] The term "arylalkyl" is used herein to refer to an alkyl group substituted with an aryl group (eg, phenylmethyl (ie, benzyl)).

[0075] The term "alkylaryl" is used herein to refer to an aryl group substituted with an alkyl group (e.g., p-methylphenyl (ie, p-tolyl)).

[0076] The terms "carbocycle" and "carbocyclic" are used herein to refer to closed ring hydrocarbon structures (e.g., monocyclic, polycyclic ring structures). Carbocyclic groups can be saturated or unsaturated. In addition, carbocyclic groups can be aromatic or non-aromatic.

[0077] The terms "heterocycle" and "heterocyclic" are used herein to refer to closed ring structures (e.g., monocyclic, polycyclic ring structures) in which one or more of the atoms in the closed ring structure is a heteroatom (i.e., an atom other than carbon). Specific exemplary heteroatoms include nitrogen, oxygen, and sulfur. Heterocyclic groups can be saturated or unsaturated. In addition, heterocyclic groups can be aromatic or non-aromatic.

[0078] Unless otherwise defined, all other scientific and technical terms have the same meaning as commonly understood by those skilled in the art. Such scientific and technical terms are explained in the literature, for example, in J. Sambrook, E. Fritsch, and T. Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press (1989); Martin, Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co. (1990); Glover, DNA Cloning: A Practical Approach, Volumes I and II, MRL Press, Ltd. (1985); and Ausubel, F. et al., Current Protocols in Molecular Biology, Greene Publishing Associates / Wiley Intersciences (2002).

[0079] Disclosed herein are novel CPP-ASOs. In some embodiments, the CPP-ASOs are directed against a target sequence in CD33 pre-mRNA. In some embodiments, the CPP-ASOs are directed against a target sequence in CD33 pre-mRNA. [ka] The CD33 pre-mRNA is complementary to all or a portion of the target sequence shown in SEQ ID NO: 1. SEQ ID NO: 1 includes exon 2 and a portion of the adjacent intron of the CD33 gene. This target sequence is involved in exon 2 skipping, which also occurs when the CD33 mRNA contains the rs3865444-A SNP. When exon 2 skipping occurs, the pre-mRNA containing this SNP is spliced ​​such that exon 2 is not included in the final transcript.

[0080] In some embodiments, the CPP-ASO has a CD33 exon-2 skipping efficiency of at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99%, according to a standard exon skipping efficiency assay for CPP-ASOs. In some embodiments, the CPP-ASO has a CD33 exon-2 skipping efficiency in the range of 25% to 99%, 30% to 99%, 35% to 99%, 40% to 99%, 50% to 99%, 60% to 99%, 70% to 99%, 80% to 99%, or 90% to 99%, according to a standard exon skipping efficiency assay for CPP-ASOs. In some embodiments, the CPP-ASO has a CD33 exon-2 skipping efficiency of 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% according to a standard exon skipping efficiency assay for CPP-ASOs.

[0081] In some embodiments, the CPP-ASO ASO is 16 to 30 nucleotides in length. In some embodiments, the CPP-ASO ASO is 20 to 30 nucleotides in length. In some embodiments, the CPP-ASO ASO is 25 to 30 nucleotides in length. In some embodiments, the CPP-ASO ASO is 21 to 30 nucleotides in length. In some embodiments, the CPP-ASO ASO is 21 to 25 nucleotides in length. In some embodiments, the CPP-ASO ASO is 18 to 21 nucleotides in length. In some embodiments, the CPP-ASO ASO is 18 to 25 nucleotides in length. In some embodiments, the CPP-ASO ASO is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0082] In some embodiments, the ASO of the CPP-ASO comprises 16 to 30 nucleotides, such as 18 to 30 nucleotides. In some embodiments, the ASO of the CPP-ASO consists of 16 to 30 nucleotides, such as 18 to 30 nucleotides.

[0083] Also disclosed herein are novel CPP-ASOs that are complementary to all or a portion of a 10-16 nucleotide target sequence in the CD33 pre-mRNA, as set forth in SEQ ID NO: 1, which includes portions of exon 2 and adjacent introns of the CD33 gene. In some embodiments, the CPP-ASO ASO is 10-14 nucleotides in length. In some embodiments, the CPP-ASO ASO is 10, 11, 12, 13, 14, 15, or 16 nucleotides in length.

[0084] In some embodiments, the CPP-ASO is complementary to all or a portion of a 16-30 nucleotide target sequence in the CD33 pre-RNA, is sufficiently complementary to form a stable hybrid with the target sequence, and is 16-30 nucleotides in length. In some embodiments, the ASO portion of the CPP-ASO is sufficiently complementary to all or a portion of a 25 nucleotide target sequence in the CD33 pre-RNA.

[0085] In some embodiments, the ASO of the CPP-ASO has one of the specific sequences disclosed in Table 1 or Table 2.

[0086] [Table 1]

[0087] [Table 2]

[0088] [Table 3]

[0089] [Table 4]

[0090] [Table 5]

[0091] [Table 6]

[0092] [Table 7]

[0093] [Table 8]

[0094] In some embodiments, PMOs and MOE ASOs were designed to cover CD33 exon-2 and the surrounding intron (SEQ ID NO: 1) in 20-25 nucleotide stretches, shifted 5 nucleotides at a time from the 5' to the 3' end of SEQ ID NO: 1. Regions that exhibited increased exon-2 skipping activity were identified where two or more consecutive PMOs or MOE ASOs complementary to a stretch of SEQ ID NO: 1 exhibited increased exon-2 skipping activity. These regions are identified below and in Table 3:

[0095] Region 1: (SEQ ID NO: 213) (See, e.g., PMO-002 and PMO-003)

[0096] Region 2: (SEQ ID NO: 214) (see, e.g., PMO-036, PMO-037, PMO-004, PMO-038, PMO-039, and PMO-005)

[0097] Region 3: (SEQ ID NO: 215) (see, e.g., PMO-082, PMO-083, and PMO-006)

[0098] Region 4: (SEQ ID NO: 216) (see, e.g., PMO-096, PMO-007, and PMO-097)

[0099] Region 5: (SEQ ID NO: 217) (see, e.g., MOE-009, MOE-128, and MOE-010)

[0100] Region 6: (SEQ ID NO: 218) (see, e.g., MOE-135, MOE-011, and MOE-012)

[0101] Region 7: (SEQ ID NO: 219) (see, e.g., MOE-015, MOE-183, and MOE-184)

[0102] Region 8: (SEQ ID NO: 220) (see, e.g., MOE-196 and MOE-197).

[0103] [Table 9]

[0104] In some embodiments, the ASO of the CPP-ASO is complementary to a region of SEQ ID NO: 1 that exhibits increased exon-2 skipping activity, including but not limited to regions 1, and 2, 3, 4, 5, 6, 7, and 8. In some embodiments, the ASO of the CPP-ASO is complementary to at least a portion of SEQ ID NO: 213; SEQ ID NO: 214; SEQ ID NO: 215; SEQ ID NO: 216; SEQ ID NO: 217; SEQ ID NO: 218; SEQ ID NO: 219; and / or SEQ ID NO: 220.

[0105] In some embodiments, the ASO of the CPP-ASO is complementary to at least a portion of region 1 of SEQ ID NO:1.

[0106] In some embodiments, the ASO of the CPP-ASO has a sequence disclosed in Table 4.

[0107] [Table 10]

[0108] In some embodiments, the ASO of the CPP-ASO is complementary to at least a portion of region 2 of SEQ ID NO:1.

[0109] In some embodiments, the ASO of the CPP-ASO has a sequence disclosed in Table 5.

[0110] [Table 11]

[0111] In some embodiments, the ASO of the CPP-ASO is complementary to at least a portion of region 6 of SEQ ID NO:1 (SEQ ID NO:218).

[0112] In some embodiments, the ASO of the CPP-ASO has a sequence disclosed in Table 6.

[0113] [Table 12]

[0114] In some embodiments, the CPP-ASO may share sequence similarity with one of the CPP-ASOs disclosed in Table 1, Table 2, and Tables 4-6. In some embodiments, the CPP-ASO shares at least 99%, or 98%, or 97%, or 96%, or 95%, or 94%, or 93%, or 92%, or 91%, or 90%, or 89%, or 88%, or 87%, or 86%, or 85%, or 84%, or 83%, or 82%, or 81%, or 80%, or 79%, or 78%, or 77%, or 76%, or 75%, or 74%, or 73%, or 72%, or 71%, or 70% sequence similarity with one of the CPP-ASOs disclosed in Table 1, Table 2, and Tables 4-6.

[0115] In some embodiments, one or more nucleobases of the ASO of the CPP-ASO comprise uracil. In some embodiments, one or more nucleobases of the ASO of the CPP-ASO comprise thymine. In some embodiments, one or more nucleosides of the ASO of the CPP-ASO comprise a ribose sugar moiety. In some embodiments, one or more nucleosides of the ASO of the CPP-ASO comprise a deoxyribose sugar moiety.

[0116] In some embodiments, the ASO of the CPP-ASO comprises at least one chemically modified nucleotide. In some embodiments, the at least one chemical modification of the nucleotide is selected from the group consisting of a chemical modification of at least one nucleobase, a chemical modification of at least one sugar moiety, a chemical modification of at least one phosphate, and any combination of these modifications. In some embodiments, the at least one chemical modification improves the ability of the nucleotide to resist nuclease degradation.

[0117] Specific exemplary chemical modifications useful in the present disclosure include chemical modifications of the phosphate backbone and one or more non-natural internucleoside linkages of the ASO. In some embodiments, the ASO of the CPP-ASO is selected from ASOs having a chemically modified phosphate backbone. In some embodiments, the chemically modified phosphate backbone contains one or more nitrogen atoms and / or one or more sulfur atoms. In some embodiments, for example, one or more non-bridging oxygen atoms in the phosphate backbone (e.g., in a phosphodiester bond) may be replaced with a nitrogen or sulfur atom. In some embodiments, the ASO of the CPP-ASO has a phosphoramidate, phosphorodiamidate, phosphorodithioate, or phosphorothioate modified backbone. In some embodiments, the ASO of the CPP-ASO is selected from ASOs that do not have a phosphorus atom in the backbone. In some embodiments, the modified backbone is stereocontrolled.

[0118] Certain exemplary chemical modifications useful in the present disclosure include chemical modification of at least one sugar moiety in the ASO. In some embodiments, the ASO of the CPP-ASO comprises at least one chemically modified (e.g., unnatural) sugar moiety.

[0119] In some embodiments, the ASO of the CPP-ASO comprises at least one chemically modified (e.g., unnatural) sugar moiety selected from a sugar moiety substituted at at least one position. In some embodiments, the at least one chemically modified (e.g., unnatural) sugar moiety is substituted at at least one position on the sugar selected from the 2', 3', and 5' positions. In some embodiments, at least one substituent on at least one substituted sugar moiety of the ASO is selected from hydroxyl; fluoro; alkoxy; amino; and substituted or unsubstituted linear or branched C1-C 10 Alkyl groups, substituted or unsubstituted, linear or branched, C2-C 10 Alkenyl groups, substituted or unsubstituted, linear or branched, C2-C 10 Alkynyl group, substituted or unsubstituted, straight or branched, C7-C 17Alkylaryl groups, substituted or unsubstituted, linear or branched, C3-C 10 Allyl group, substituted or unsubstituted, straight or branched C7-C 17 Aryl alkyl groups and substituted or unsubstituted straight or branched C2-C 10 In some embodiments, at least one substituent on at least one substituted sugar moiety of the ASO of the CPP-ASO comprises methoxy, methoxyethyl, ethoxy, propoxy, aminopropoxy, methoxyethoxy, dimethylaminoethoxy, or dimethylaminoethoxyethoxy. In some embodiments, one or more sugar moieties of the ASO of the CPP-ASO are selected from pyranoses, derivatives of pyranoses, deoxypyranoses, derivatives of deoxypyranoses, ribose, derivatives of ribose, deoxyribose, and derivatives of deoxyribose. In some embodiments, the sugar moiety is stereocontrolled.

[0120] In some embodiments, the ASO of the CPP-ASO comprises at least one sugar moiety that is modified in a manner to create a bicyclic sugar moiety. In some embodiments, the bicyclic sugar moiety is formed with a bridging modification between the 4' and 2' furanose ring atoms. In some embodiments, the bridging modification comprises at least one group that forms a bridge between the 4' and 2' furanose ring atoms. In some embodiments, at least one nucleotide in a given ASO of the CPP-ASO has a bridging modification. In some embodiments, at least one nucleotide in the ASO of the CPP-ASO is a locked nucleic acid (LNA).

[0121] In some embodiments, the ASO of the CPP-ASO comprises at least one sugar moiety containing fewer than five ring atoms, such as four ring atoms. In some embodiments, the ASO of the CPP-ASO comprises at least one sugar moiety containing more than five ring atoms, such as six ring atoms. In some embodiments, the ASO of the CPP-ASO comprises at least one sugar moiety containing a morpholinyl ring. In some embodiments, the ASO of the CPP-ASO is a morpholino-based ASO. A morpholino-based ASO refers to an ASO containing morpholino subunits in which a morpholinyl ring replaces the ribose moiety. Specific exemplary internucleotide linkages for such morpholino-based ASOs include, for example, phosphoramidate or phosphorodiamidate internucleotide linkages connecting the morpholinyl ring nitrogen of one morpholino subunit to the 4' exocyclic carbon of an adjacent morpholino subunit. Each morpholino subunit comprises a purine or pyrimidine nucleobase, which can bind to a nucleobase in a target sequence by base-specific hydrogen bonding. In some embodiments, morpholino-based ASOs may include at least one further modification.

[0122] In some embodiments, the ASO of the CPP-ASO is a phosphorodiamidate morpholino oligomer (PMO). In some embodiments, the ASO of the CPP-ASO has the structure of Formula I: [ka] wherein B is any nucleobase described herein, and n is an integer ranging from 1 to 19, 1 to 23, 1 to 28, 1 to 30, 8 to 19, 8 to 23, 8 to 28, 8 to 30, 14 to 19, 14 to 23, 14 to 28, 14 to 30, 16 to 19, 16 to 23, 16 to 28, or 16 to 30.

[0123] In some embodiments, the ASO of the CPP-ASO is a methoxyethyl ribose oligomer (MOE). In some embodiments, the ASO of the CPP-ASO has the structure of Formula II: [ka] wherein B is any nucleobase described herein, and m is an integer ranging from 1 to 19, 1 to 23, 1 to 28, 1 to 30, 8 to 19, 8 to 23, 8 to 28, 8 to 30, 14 to 19, 14 to 23, 14 to 28, 14 to 30, 16 to 19, 16 to 23, 16 to 28, or 16 to 30.

[0124] In some embodiments, both the sugar moiety and the linkage between the nucleobase and the sugar moiety of at least one nucleotide unit in the ASO of the CPP-ASO are replaced with non-natural groups. In some embodiments, the nucleobase unit is maintained for hybridization with an appropriate nucleic acid target compound. In some embodiments, the ASO of the CPP-ASO is selected from peptide nucleic acids (PNAs). In some embodiments, the sugar backbone of at least one oligonucleotide in the PNA is replaced with an amide-containing backbone, such as an aminoethylglycine backbone. In some embodiments, the nucleobase is retained and is directly or indirectly bound to an aza nitrogen atom of the amide portion of the backbone.

[0125] In some embodiments, the ASO of a CPP-ASO comprises at least one unnatural nucleobase (often referred to as a "base") (e.g., a nucleobase containing one or more modifications or substitutions). Examples of unnatural nucleobases include 5-substituted pyrimidines (e.g., 5-methylcytosine, 5-propynylcytosine, 5-propynyluracil), 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including, but not limited to, 2-aminopropyladenine. Certain unnatural nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the present disclosure. For example, 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6 to 1.2°C. In some embodiments, the modified nucleobase is stereocontrolled.

[0126] Not all positions in a given ASO need be uniformly modified; in fact, more than one of the aforementioned modifications may be incorporated into a single nucleotide within the ASO. In some embodiments, the ASO of a CPP-ASO may contain one or more chemically modified nucleotides and one or more unmodified nucleotides. The ASO may contain at least one region in which the nucleotide is modified to confer increased resistance to nuclease degradation, increased cellular uptake, and / or an additional region for increased binding affinity to the target nucleic acid.

[0127] Certain exemplary ASOs containing multiple modifications to the nucleobase, sugar moiety, and / or internucleotide linkage include ASOs having the sequences shown in Table 7, where each nucleotide has a 2'-MOE ribose sugar moiety, each C represents a 5-methylcytosine, each lowercase letter represents a locked nucleic acid, each (-) represents a phosphodiester (PO) linkage, each fX represents a 2'-fluororibonucleotide, and each mX represents a 2'-OMe ribonucleotide.

[0128] [Table 13]

[0129] Due to potential three-dimensional variations in the sugar moiety, nucleobase, and internucleotide linkage, some nucleotides may share the same molecular formula but have different spatial configurations, i.e., some nucleotides may be stereoisomers. In some embodiments, for example, modifying a phosphodiester internucleotide linkage by replacing one or more oxygen atoms with one or more nitrogen and / or sulfur atoms can make the phosphorus atom of the linkage a chiral center. In some embodiments, the ASO of the CPP-ASO contains one or more P-chiral internucleotide linkages in the Sp or Rp configuration. As illustrative, non-limiting examples, the Sp and Rp configurations of exemplary phosphorothioate linkages are shown below: [ka]

[0130] In some embodiments, the stereochemistry of nucleotides within a given ASO of a CPP-ASO is not controlled, resulting in a sterically random ASO. In some embodiments, the nucleotides within a given ASO of a CPP-ASO are sterically controlled. In some embodiments, one or more nucleotides within a given ASO are sterically controlled, resulting in a sterically pure ASO of a CPP-ASO. In some embodiments, a given ASO of a CPP-ASO is a combination of sterically controlled nucleotides and sterically random nucleotides.

[0131] In some embodiments, the ratio of sterically controlled nucleotides in the ASO of the CPP-ASO is 10% to 100%, 15% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 50% to 90%, 50% to 95%, 60% to 100%, 60% to 90%, 60% to 95%, 70% to 100%, 70% to 90%, 70% to 95%, 80% to 100%, 80% to 90%, 80% to 95%, 90% to 100%, 90% to 95%, 90% to 96%, 90% to 97%, 90% to 98%, 90%-99%, 95%-98%, 95%-99%, 95-100%, 50%-90%, or 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0132] In some embodiments, the proportion of Sp internucleotide linkages in the ASO of the CPP-ASO is at least 80%, 85%, 90%, 95%, 98%, or 99%. In some embodiments, the proportion of Sp internucleotide linkages in the ASO of the CPP-ASO is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0133] In some embodiments, the ratio of Rp internucleotide binding in the ASO of the CPP-ASO is at least 80%, 85%, 90%, 95%, 98%, or 99%. In some embodiments, the ratio of Rp internucleotide binding in the ASO of the CPP-ASO is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0134] In some embodiments, the ASO of the CPP-ASO is a stereochemically pure PMO ASO having a sequence disclosed in Table 8.

[0135] [Table 14]

[0136] In some embodiments, the ASO of the CPP-ASO is a stereomerically pure MOE ASO having a sequence disclosed in Table 9.

[0137] [Table 15]

[0138] [Table 16]

[0139] [Table 17]

[0140] In some ASOs of CPP-ASOs, certain modifications to sugar moieties, nucleobases, internucleotide linkages, and / or sterically restricted nucleotides can be arranged in regions that create a unique motif for that ASO. In some embodiments, the ASO of a CPP-ASO contains at least two regions. In some embodiments, the ASO of a CPP-ASO contains three regions: one region near the 5' end of the ASO, one region near the 3' end of the ASO, and a gap region between the two other regions. This type of arrangement is known as a gapmer motif. The length of each motif may be equal to the length of other motifs within the ASO of a CPP-ASO, or the length of each motif may be independent of the length of other motifs within the ASO. In some embodiments, one or more sugar moieties in the ASO of a CPP-ASO are modified so that a block of sugar moieties in one region of the ASO is different from a block of sugar moieties in a different region of the ASO. In some embodiments, the ASO of a CPP-ASO contains modified sugar moieties arranged in a gapmer motif. In some embodiments, one or more nucleobases in an ASO of a CPP-ASO are modified such that a block of nucleobases in one region of the ASO is different from a block of nucleobases in a different region of the ASO. In some embodiments, an ASO of a CPP-ASO comprises modified nucleobases arranged in a gapmer motif. In some embodiments, one or more internucleotide linkages in an ASO of a CPP-ASO are modified such that a block of internucleotide linkages in one region of the ASO is different from a block of internucleotide linkages in a different region of the ASO. In some embodiments, a given ASO of a CPP-ASO comprises modified internucleotide linkages arranged in a gapmer motif. In some embodiments, one or more sterically restricted nucleotides in an ASO of a CPP-ASO are modified such that a block of sterically restricted nucleotides in one region of the ASO is different from a block of sterically restricted nucleotides in a different region of the ASO. In some embodiments, an ASO of a CPP-ASO comprises sterically restricted nucleotides arranged in a gapmer motif.In some embodiments, the ASO has two or more motifs. In some embodiments, the ASO has two or more motifs that are independent of each other.

[0141] The CPP-ASO conjugates described herein comprise a CPP conjugated to an ASO. Because the CPP-ASO conjugate has the ability to penetrate cell membranes, the CPP-ASO conjugate enters the cytosol of cells. In some embodiments, two or more CPPs are conjugated to an ASO. In some embodiments, the CPP-ASO conjugate comprises two, three, four, or five CPPs conjugated to an ASO. In some embodiments, the two or more CPPs comprise at least two different CPPs. In some embodiments, the two or more CPPs are two or more of the same CPP. In some embodiments, conjugating two or more CPPs to an ASO increases ASO activity. In some embodiments, the CPP comprises 4 to 40 amino acids. In some embodiments, the CPP has 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23, or 24, or 25, or 26, or 27, or 28, or 29, or 30, or 31, or 32, or 33, or 34, or 35, or 36, or 37, or 38, or 39, or 40 amino acids.

[0142] In some embodiments, the CPP is conjugated directly or indirectly to the ASO. In some embodiments, the CPP is conjugated at the 5' end of the ASO. In some embodiments, the CPP is conjugated at the 3' end of the ASO. In some embodiments, the CPP is conjugated at any nucleotide of the ASO. Specific methods for conjugating a CPP are known in the art. In some embodiments, the CPP is conjugated to the ASO at the C-terminus. In some embodiments, the CPP is conjugated to the ASO at the N-terminus. In some embodiments, the CPP is conjugated to the ASO via the side chain of any amino acid of the CPP. In some embodiments, the CPP is covalently linked to the ASO. In some embodiments, the CPP is chemically conjugated to the ASO. In some embodiments, the CPP is non-covalently linked to the ASO.

[0143] In some embodiments, the CPP comprises one or more proteinogenic and / or non-proteinogenic amino acids. Specific exemplary amino acids include alanine, β-alanine, alloisoleucine, arginine, asparagine, aspartic acid, cysteine, cyclohexylalanine, 2,3-diaminopropionic acid, 4-fluorophenylalanine, glutamic acid, glutamine, glycine, histidine, homoproline, isoleucine, leucine, lysine, methionine, naphthylalanine, norleucine, phenylalanine, phenylglycine, 4-(phosphonodifluoromethyl)phenylalanine, proline, sarcosine, selenocysteine, serine, threonine, tyrosine, tryptophan, valine, tert-butyl-alanine, penicillamine, homoarginine, nicotinyl-lysine, triflouroacetyl-lysine, methyl-leucine, 3-(3-benzothienyl)-alanine, 6-aminohexanoic acid, and 5-aminopentanoic acid. In some embodiments, the CPP contains at least one non-proteinogenic amino acid. Specific exemplary non-proteinogenic amino acids include alloisoleucine, β-alanine, cyclohexylalanine, 2,3-diaminopropionic acid, 4-fluorophenylalanine, homoproline naphthylalanine, norleucine, phenylglycine, 4-(phosphonodifluoromethyl)phenylalanine, sarcosine, selenocysteine, tert-butylalanine, penicillamine, homoarginine, nicotinyl-lysine, trifluoroacetyl-lysine, methyl-leucine, 3-(3-benzothienyl)-alanine, 6-aminohexanoic acid, and 5-aminopentanoic acid. In some embodiments, the at least one non-proteinogenic amino acid comprises a modified proline (e.g., a substituted proline). In some embodiments, the CPP contains at least one proteinogenic amino acid.Specific exemplary proteinogenic amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, tryptophan, and valine. In some embodiments, the CPP contains an L- or D-amino acid. In some embodiments, at least one proteinogenic or non-proteinogenic amino acid is substituted with one or more substituents. Specific exemplary suitable substituents include halogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, acyl, alkylcarbamoyl, alkylcarboxamidyl, alkoxycarbonyl, alkylthio, and arylthiol groups. In some embodiments, the CPP contains a synthetic amino acid mimic, e.g., a replacement of a peptide amide bond. In some embodiments, the CPP contains non-natural structures, eg, non-peptide groups that do not contain amino acids.

[0144] In some embodiments, a CPP comprises one or more modified or unmodified arginine residues. In some embodiments, a CPP comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20 arginine residues. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the amino acids of the CPP comprise modified or unmodified arginine residues.

[0145] In some embodiments, the CPP comprises one or more hydrophobic amino acids. Examples of hydrophobic amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, proline, and tryptophan. In some embodiments, at least one of the one or more hydrophobic amino acids may be substituted with one or more substituents. In some embodiments, the CPP comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20 hydrophobic amino acids. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the amino acids of the CPP comprise hydrophobic amino acids. In some embodiments, the one or more hydrophobic amino acids comprise one or more aromatic hydrophobic amino acids. Examples of aromatic hydrophobic amino acids include phenylalanine, tryptophan, tyrosine, naphthylalanine, 3-(3-benzothienyl)-alanine, phenylglycine, and homophenylalanine. In some embodiments, at least one of the one or more aromatic hydrophobic amino acids may be substituted with one or more substituents. In some embodiments, the CPP comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20 aromatic hydrophobic amino acids. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the amino acids of the CPP comprise aromatic hydrophobic amino acids. In some embodiments, the CPP comprises one or more arginine residues and one or more hydrophobic amino acids. In some embodiments, the CPP comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 arginine residues and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 hydrophobic amino acids. In some embodiments, the CPP comprises one or more arginine residues and one or more aromatic hydrophobic amino acids. In some embodiments, the CPP comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 arginine residues and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 aromatic hydrophobic amino acids.

[0146] In some embodiments, the CPP comprises one or more modified or unmodified lysine residues, hi some embodiments, the CPP comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20 lysine residues.

[0147] In some embodiments, the CPP is linear. Specific exemplary linear CPPs include Pip6a peptides (Wood M.JA et al, Mol Therapy-Nucleic Acids, 2012,1,e38), ApoE peptides (Gait,MJ et al, Nucleic Acid Therapeutics, 2017,27,130), neurotensin-based peptides (Prakash,TP et al, J.Med.Chem. 2020,63,8471), polyarginine, antennapedia sequences, HIV-TAT, penetratin, Antp-3A, buforin II transportan, MAP (model amphipathic peptide), K-FGF, Ku70, prion, pVEC, Pep-1, SynB1, Pep-7, HN-1, BGSC (bis-guanidinium-spermidine-cholesterol), and BGTC (bis-guanidinium-tren-cholesterol). In some embodiments, the CPP is a linear peptide having a sequence disclosed in Table 10. In Table 10, B is β-alanine and X is 6-aminohexanoic acid.

[0148] [Table 18]

[0149] In some embodiments, the CPP is a cyclic cell-penetrating peptide ("cCPP"). Specific exemplary cCPPs include CPP9, CPP12 (Pei, D. et al. Biochemistry, 2016, 55, 2601), and others outlined in Tiwari, K. et al. Mol. Pharmaceutics 2019, 16, 9, 3727. In some embodiments, the cyclic CPP of the CPP-ASO has one of the following structures: [ka]

[0150] In some embodiments, CPPs are synthesized using solid-phase and / or liquid-phase methods. In some embodiments, CPPs are synthesized using both solid-phase and liquid-phase techniques, where part of the synthesis is performed using the solid phase and another part of the synthesis is performed using the liquid phase. Further information regarding the synthesis of particular CPPs according to some embodiments is included in the Examples below.

[0151] In some embodiments, the CPP comprises a lipoic acid group. In some embodiments, the lipoic acid moiety is an (R)-lipoic acid group. In some embodiments, the lipoic acid moiety is an (S)-lipoic acid group. In some embodiments, the lipoic acid group is conjugated to a lysine residue of the CPP. In some embodiments, the lipoic acid group is conjugated to a non-lysine residue (e.g., 5-aminopentanoic acid) of the CPP. In some embodiments, the CPP comprises 1 to 5 lipoic acid groups. In some embodiments, the CPP comprises 1, 2, 3, 4, or 5 lipoic acid groups.

[0152] In some embodiments, the cyclic CPP of the CPP-ASO has the following structure: [ka] It has one of the following.

[0153] In some embodiments, CPPs containing lipoic acid groups may be synthesized according to the exemplary synthetic schemes described in the Examples below.

[0154] In some embodiments, the CPP comprises one or more lactam amino acids. In some embodiments, the CPP comprises 1, 2, 3, 4, or 5 lactam amino acids. In some embodiments, each lactam amino acid is independently an 8-, 9-, or 10-membered ring.

[0155] In some embodiments, the one or more lactam amino acids each independently have the formula III: [ka] (In the formula, R 1 and R 2 are each independently selected from the group consisting of H, an aryl group, a heteroaryl group, an alkylaryl group, an arylalkyl group, a linear or branched alkyl group, and a guanidine-containing group, each of which is independently optionally substituted with one or more substituents, and wherein n is an integer from 1 to 3.

[0156] In some embodiments, R 1 and / or R 2 comprises an aryl group. In some embodiments, R 1 and R 2 Each independently comprises an aryl group. The aryl group can be monocyclic or polycyclic. Specific exemplary monocyclic aryl groups include phenyl and benzyl groups. An example of a polycyclic aryl group is a naphthyl group. In some embodiments, R 1 and / or R 2 In some embodiments, R 1 and R 2 each independently comprises a guanidine-containing group. An example of a guanidine-containing group is (CH2)2CN3H4.

[0157] In some embodiments, R 1 and R 2 is H. In some embodiments, R 1 is H and R 2 is phenyl. In some embodiments, R 1 is phenyl and R 2 is H. In some embodiments, R 1 and R 2 is phenyl. In some embodiments, R 1 is 2-naphthyl, and R 2is H. In some embodiments, R 1 is H and R 2 is 2-naphthyl. In some embodiments, R 1 and R 2 is 2-naphthyl. In some embodiments, R 1 is 2-naphthyl, and R 2 is phenyl. In some embodiments, R 1 is (CH2)2CN3H4, and R 2 is H. In some embodiments, R 1 is H and R 2 is (CH2)2CN3H4. In some embodiments, R 1 is (CH2)2CN3H4, and R 2 is 2-naphthyl. In some embodiments, R 1 and R 2 is (CH2)2CN3H4.

[0158] In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0159] In some embodiments, the CPP comprising a lactam amino acid has the structure shown below: [ka] It has.

[0160] In some embodiments, CPPs containing lactam amino acids may be synthesized according to the exemplary synthetic schemes shown in Figures 22-27 and in the Examples below.

[0161] In some embodiments, the CPP comprises one or more modified proline residues. In some embodiments, the one or more modified proline residues comprise one or more substituents. Specific exemplary suitable substituents include aryl groups and guanidine-containing groups. Examples of suitable aryl groups include phenyl, benzyl, and naphthyl groups. Examples of suitable guanidine-containing groups include, but are not limited to, —(CH 2 ) 2 —CN 3 H 4 .

[0162] In some embodiments, the one or more modified proline residues of the CPP each independently represent Formula IV: [ka] (In the formula, R 1 is an aryl group or a guanidine-containing group. 1 is a guanidine-containing group. In some embodiments, R 1 is —(CH)—CNH. In some embodiments, R 1 is an aryl group. In some embodiments, R 1 is benzyl. In some embodiments, R 1 is phenyl. In some embodiments, R 1 is naphthyl.

[0163] In some embodiments, the CPP comprising a modified proline residue has the structure shown below: [ka] It has.

[0164] In some embodiments, CPPs containing modified proline residues may be synthesized according to the exemplary synthetic schemes shown in the Examples below.

[0165] In some embodiments, the CPP comprises one or more oxadiazole linkages. In some embodiments, the one or more oxadiazole linkages are represented by Formula V: [ka] where R is a substituted or unsubstituted aryl group. Certain exemplary suitable aryl groups include phenyl, benzyl, naphthyl, and methylnaphthyl.

[0166] In some embodiments, the CPP containing an oxadiazole linkage has formula VI: [ka] wherein R is a substituted or unsubstituted aryl group. In some embodiments, R is phenyl, benzyl, naphthyl, or methylnaphthyl.

[0167] In some embodiments, CPPs containing oxadiazole linkages may be synthesized according to the exemplary synthetic schemes shown in the Examples below.

[0168] In some embodiments, the CPP-ASO conjugate further comprises a nuclear localization sequence (NLS). A nuclear localization sequence generally refers to an amino acid sequence that facilitates transport of a molecule containing that sequence into the nucleus of a eukaryotic cell. The nuclear localization sequence may be a simple or bipartite nuclear localization sequence. Examples of suitable nuclear localization sequences include sequences that include all or a portion of one or more of the following sequences: PKKKRKV (SEQ ID NO: 273), PKLKRQ (SEQ ID NO: 274), RPRK (SEQ ID NO: 275), RRARRPRG (SEQ ID NO: 276) from Simian Virus 40 (SV40), KRPAATKKAGQAKKKK (SEQ ID NO: 277) from nucleoplasmin, PAAKRVKLD (SEQ ID NO: 278) and RQRRNELKRSP (SEQ ID NO: 279) from c-myc, RMRKFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 280) from the IBB domain of importin alpha, VSRKRPRP (SEQ ID NO: 281) and PPKKARED (SEQ ID NO: 282) from polyomavirus large T, PQPKKKPL (SEQ ID NO: 283) from human p53, mouse c-abl SALIKKKKKMAP (SEQ ID NO: 284) from IV, DRLRR (SEQ ID NO: 285) and PKQKKRK (SEQ ID NO: 286) from influenza virus NS1, RKLKKKIKKL (SEQ ID NO: 287) from hepatitis virus delta antigen, REKKKFLKRR (SEQ ID NO: 288) from mouse Mxl protein, KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 289) from human poly(ADP-ribose) polymerase, and RKCLQAGMNLEARKTKK (SEQ ID NO: 290) from steroid hormone receptor (human) glucocorticoid. Further examples of nuclear localization sequences are described in Lu, J. et al., Types of nuclear localization signals and mechanisms of protein import into the nucleus. Cell Commun Signal 19, 60 (2021), which is incorporated herein by reference in its entirety.

[0169] In some embodiments, the NLS is covalently or non-covalently coupled to the CPP, ASO, and / or linker of the CPP-ASO. In some embodiments, the NLS is covalently or non-covalently coupled to the CPP of the CPP-ASO. In some examples, the CPP is a linear peptide. In some examples, the CPP is a cyclic peptide. In some embodiments, the NLS is covalently or non-covalently coupled to the ASO of the CPP-ASO. In some embodiments, the NLS is covalently or non-covalently coupled to the linker that couples the CPP and ASO.

[0170] Antisense oligonucleotide production The antisense molecules used in the present disclosure may be produced by well-known solid phase synthesis methods. Equipment for such synthesis is available from several suppliers, including, for example, Applied Biosystems (Foster City, Calif.). One method for synthesizing oligonucleotides on modified solid supports is described in U.S. Patent No. 4,458,066.

[0171] Additionally or alternatively, any other method of such synthesis known in the art can be used.It is well known to use techniques similar to those used to prepare oligonucleotides, such as phosphorothioates and alkylated derivatives.In one such automated embodiment, diethyl phosphoramidite is used as starting material, and can be synthesized as described by Beaucage, et al., Tetrahedron Letters, 22:1859-1862 (1981).

[0172] In some embodiments, the ASO is synthesized in a manner such that all nucleotides of the ASO are stereochemically pure.

[0173] In some embodiments, the ASO is synthesized in vitro and does not include antisense compositions of biological origin. In some embodiments, the ASO may also be mixed with, encapsulated in, conjugated to, or otherwise associated with other molecules, molecular structures, or mixtures of compounds, such as, for example, liposomes, lipids, receptor-targeting molecules, etc., to aid in uptake, distribution, and / or absorption.

[0174] Conjugating cell-penetrating peptides to antisense oligonucleotides In some embodiments, antisense oligonucleotides are conjugated to cell-penetrating peptides using known chemical reactions. Examples can be found in Gait, MJ et al. Curr. Pharm. Des. 2005, 11, 3639; Prescher, JA et al. Nat. Rev. Chem. 2020, 4, 476; Jeon, J. et al. Molecules, 2019, 24, 3567; Stetsenko, D. Molecules, 2021, 26, 5420.

[0175] In some embodiments, the CPP is conjugated to the ASO via strain-promoted azide-alkyne cycloaddition ("click chemistry"). In some embodiments, the CPP is conjugated to the ASO via strained alkene-tetrazine cycloaddition. In some embodiments, the CPP is conjugated to the ASO via an amide bond. In some embodiments, the CPP is conjugated to the ASO using one of the bonds in the following image: [ka]

[0176] In some embodiments, the CPP is directly conjugated to the ASO. In some embodiments, the CPP is indirectly conjugated to the ASO with a linker between the CPP and the ASO. In some embodiments, the linker comprises an alkyl group, a carbocyclic group, a heterocyclic group, polyethylene glycol, or one or more of these groups. In some embodiments, the linker comprises one or more proteinogenic or non-proteinogenic amino acids. In some embodiments, the one or more proteinogenic or non-proteinogenic amino acids comprise sarcosine. In some embodiments, the linker is a cleavable linker. Certain exemplary suitable cleavable linkers include linkers comprising valine-citrulline ("Val-Cit"), valine-alanine ("Val-Ala"), glutamic acid-valine-citrulline ("Glu-Val-Cit"), and / or alanine-alanine-asparagine ("Ala-Ala-Asn").

[0177] A method to induce exon-2 skipping during pre-mRNA splicing In some embodiments, a CPP-ASO is used to induce exon-2 skipping during processing of CD33 pre-mRNA. In some embodiments, at least one CPP-ASO disclosed herein is used to induce exon-2 skipping in CD33 pre-mRNA during pre-mRNA splicing. In some embodiments, at least one CPP-ASO is introduced into a cell, wherein the at least one CPP-ASO is complementary to all or a portion of SEQ ID NO: 1, and the CPP-ASO hybridizes to a target region of the CD33 gene, and the CPP-ASO induces exon-2 skipping during pre-mRNA splicing of the CD33 gene. In some embodiments, the CPP-ASO administered to induce exon-2 skipping during pre-mRNA splicing comprises one of SEQ ID NOs: 2-10. In some embodiments, the CPP-ASO administered to induce exon-2 skipping during pre-mRNA splicing comprises one of SEQ ID NOs: 2-15, 36-39, 82, 83, 96, 97, 128, 132, 135, 136, 183, 184, 190, 196, 197, 202, 224, or 252. In some embodiments, the CPP-ASO administered to induce exon-2 skipping during pre-mRNA splicing comprises one of SEQ ID NOs: 2, 12, 224, or 252.

[0178] In some embodiments, CPP-ASOs can be introduced by transfection in conjunction with one or more transfection agents. In some embodiments, the excipient or transfection agent is capable of forming complexes, nanoparticles, micelles, vesicles, and / or liposomes that can aid in the delivery of each CPP-ASO complexed or entrapped in the vesicles or liposomes through the cell membrane. Many of these excipients are known in the art. Suitable excipients or transfection agents include LipofectAMINE™ 2000 (Invitrogen), Endo-Porter peptides, polyethyleneimine (PEI; ExGen500 (MBI Fermentas)), or derivatives thereof, or similar cationic polymers and derivatives including polypropyleneimine or polyethyleneimine copolymers (PEC), synthetic amphiphiles (SAINT-18), Lipofectin™, DOTAP, and / or viral capsid proteins capable of self-assembling into particles that can be used to deliver CPP-ASOs into cells. Its high transfection capacity is combined with a predicted low to moderate toxicity in terms of overall cell viability. The ease of structural modification may allow for further modification and analysis of its (in vivo) nucleic acid transfer properties and toxicity.

[0179] treatment method Disclosed herein are methods of treating a subject with a neurodegenerative disease, the methods comprising administering at least one CPP-ASO disclosed herein. In some embodiments, the methods comprise administering a therapeutically effective amount of at least one CPP-ASO disclosed herein. In some embodiments, the methods comprise administering a therapeutically effective amount of at least one CPP-ASO that hybridizes to all or a portion of SEQ ID NO: 1. In some embodiments, the methods comprise administering a therapeutically effective amount of at least one CPP-ASO comprising one of SEQ ID NOs: 2-10. In some embodiments, the methods comprise administering a therapeutically effective amount of at least one CPP-ASO comprising one of SEQ ID NOs: 2-15, 36-39, 82, 83, 96, 97, 128, 132, 135, 136, 183, 184, 190, 196, 197, 202, 224, or 252. In some embodiments, the method comprises administering a therapeutically effective amount of at least one CPP-ASO comprising one of SEQ ID NOs: 2, 12, 224, or 252. In some embodiments, the neurodegenerative disease is characterized by a mutation in the CD33 gene. In some embodiments, the neurodegenerative disease is characterized by an abnormal microglial phenotype. In some embodiments, the neurodegenerative disease is Alzheimer's disease, microfibromyalgia, or multiple sclerosis.

[0180] In some embodiments, CPP-ASO administered to a subject with a neurodegenerative disease may be administered in a pharmaceutical composition. In some embodiments, the amount of CPP-ASO administered in a pharmaceutical composition may depend on the subject under treatment, the subject's weight, the method of administration, and the judgment of the prescribing physician. For example, in some embodiments, a dosing schedule may involve once-daily or twice-daily administration of a pharmaceutical composition at a prescribed dosage of about 1 μg to about 1000 mg. In some embodiments, intermittent administration of a dose of a pharmaceutical composition on a regular basis, such as once weekly, once monthly, quarterly, or once yearly, may be used. Following standard dosing regimens, in some embodiments, a physician will readily determine the optimal dosage and will be able to readily modify the administration to achieve such dosage.

[0181] The therapeutically effective amount of a compound or composition disclosed herein can be determined by the therapeutic efficacy of the compound. In some embodiments, however, dosage may vary depending on the patient's requirements, the severity of the condition being treated, and the compound used. In some embodiments, the therapeutically effective amount of a disclosed compound is sufficient to establish a maximum plasma concentration. In some embodiments, preliminary dosing, for example, as determined by animal testing, and scaling of dosages for human administration are performed according to art-recognized practices.

[0182] In some embodiments, toxicity and therapeutic efficacy are measured in cell culture or experimental animals, e.g., by LD 50 (lethal dose for 50% of the population) and ED 50 The LD (the dose therapeutically effective in 50% of the population) can be determined by standard pharmaceutical procedures for determination of the therapeutic index. The dose ratio between toxic and therapeutic effects is the therapeutic index, and the LD 50 / ED 50 It can be expressed as a ratio: In some embodiments, compositions that exhibit large therapeutic indices are desirable.

[0183] In some embodiments, data obtained from cell culture assays or animal studies can be used in formulating a range of dosages for use in humans. In some embodiments, a therapeutically effective dosage achieved in one animal model can be converted for use in other animals, including humans, using conversion factors known in the art (see, e.g., Freireich et al., Cancer Chemother. Reports 50(4):219244 (1966)).

[0184] The CPP-ASO herein may be administered in a pharmaceutical composition comprising a therapeutically effective amount of CPP-ASO together with pharmaceutically acceptable excipients, diluents, preservatives, solubilizers, emulsifiers, auxiliary agents, and / or carriers. In some embodiments, such compositions include diluents of various buffer contents (e.g., Tris-HCl, acetate, phosphate), pH, and ionic strength, as well as additives such as surfactants and solubilizers (e.g., Tween 80, polysorbate 80), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., thimersol, benzyl alcohol), and bulking agents (e.g., lactose, mannitol). In some embodiments, the material may be formulated into a particulate preparation of a polymeric compound such as polylactic acid or polyglycolic acid, or formulated into a liposome. In some embodiments, hyaluronic acid may also be used. Such compositions may influence the physical state, stability, rate of in vivo release, and / or rate of in vivo clearance of the CPP-ASO and derivatives. In some embodiments, the compositions may be prepared in liquid form or may be in a dry powder form, such as lyophilized form.

[0185] Administration In some embodiments, pharmaceutical compositions comprising CPP-ASO and a pharmaceutically acceptable carrier or excipient may be prepared for administration by techniques well known in the pharmaceutical industry, which in some embodiments include combining CPP-ASO with one or more carriers and / or excipients to form an association in a unit dosage form.

[0186] In some embodiments, compositions suitable for oral administration may be provided in discrete units, such as capsules, cachets, lozenges, or tablets, each containing a predetermined amount of a compound of the present disclosure as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion. In some embodiments, such formulations may be prepared by any suitable method including the step of bringing into association at least one embodiment of the present disclosure as an active compound with at least one carrier or excipient (which may constitute one or more accessory ingredients). In some embodiments, the at least one carrier is acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient. In some embodiments, the carrier may be solid or liquid, or both, and may be formulated as a unit-dose formulation, e.g., a tablet, which may contain from about 0.05% to about 95% by weight of at least one active compound, along with at least one compound described herein as the active compound. In some embodiments, other pharmacologically active agents, including other compounds, may also be present. In some embodiments, the formulations of the present disclosure may be prepared by any of the well-known techniques of pharmacy which consist essentially of mixing the ingredients.

[0187] For solid compositions, in some embodiments, conventional non-toxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like. In some embodiments, liquid pharmacologically administrable compositions may be prepared, for example, by dissolving or dispersing at least one active compound of the present disclosure as described herein and optional pharmaceutical adjuvants in an excipient, such as water, saline, aqueous dextrose, glycerol, ethanol, or the like, to form a solution or suspension. Generally, in some embodiments, suitable formulations may be prepared by uniformly and intimately admixing at least one active compound of the present disclosure with a liquid or finely divided solid carrier, or both, and then, if necessary, shaping the product. For example, in some embodiments, a tablet may be prepared by compressing or molding a powder or granules of at least one embodiment of the present disclosure, optionally combined with one or more accessory ingredients. In some embodiments, compressed tablets may be prepared by compressing in a suitable machine at least one embodiment of the present disclosure in a free-flowing form, such as a powder or granules, optionally mixed with one or more binders, lubricants, inert diluents, and / or surfactants / dispersants. In some embodiments, molded tablets may be made by molding in a suitable machine, in which at least one embodiment of the present disclosure in powdered form is moistened with an inert liquid diluent.

[0188] In some embodiments, formulations suitable for buccal (sublingual) administration include lozenges comprising at least one embodiment of the present disclosure in a flavored base, such as sucrose and acacia or tragacanth, and pastilles comprising at least one compound in an inert base, such as gelatin and glycerin or sucrose and acacia.

[0189] In some embodiments, formulations suitable for parenteral administration include sterile aqueous solution preparations of at least one embodiment of the present disclosure that are approximately isotonic with the blood of the intended recipient. In some embodiments, these preparations are administered intravenously, but administration may also be achieved by subcutaneous, intramuscular, intraperitoneal, intracerebroventricular, or intradermal injection. In some embodiments, these preparations are administered using an osmotic pump. In some embodiments, such preparations may conveniently be prepared by mixing at least one embodiment described herein with water and rendering the resulting solution sterile and isotonic with blood. In some embodiments, injectable compositions according to the present disclosure may contain from about 0.1 to about 5% w / w of the active compound.

[0190] In some embodiments, formulations suitable for rectal administration are provided in unit-dose suppositories, which may be prepared by mixing at least one embodiment as described herein with one or more conventional solid carriers, such as cocoa butter, and then shaping the resulting mixture.

[0191] In some embodiments, formulations suitable for topical application to the skin may take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. In some embodiments, carriers and excipients that may be used include petrolatum, lanolin, polyethylene glycols, alcohols, and combinations of two or more thereof. In some embodiments, the CPP-ASO is generally provided at a concentration of about 0.1% to about 15%, e.g., about 0.5 to about 2%, w / w of the composition. [Example]

[0192] The following examples are provided to further illustrate the present invention. The examples are intended for illustrative purposes and are not intended to limit the invention in any way.

[0193] Abbreviation ASO: antisense oligonucleotide CPP-ASO: cell-penetrating peptide conjugated to antisense oligonucleotide DNA: deoxyribonucleic acid CPP: cell-penetrating peptide cDNA: complementary deoxyribonucleic acid RNA: ribonucleic acid mRNA: messenger ribonucleic acid PMO: phosphorodiamidate morpholino oligomer MOE: methoxyethyl LOAD: Late-onset Alzheimer's disease SNP: Single Nucleotide Polymorphism PNA: peptide nucleic acid DOTAP: 1,2 dioleoyl 3 trimethylammoniopropane PEI: Polyethyleneimine PEC: Polyethyleneimine copolymer HRMS: High resolution mass spectrometry HATU: Azabenzotriazole tetramethyluronium hexafluorophosphate MW: molecular weight SP: sterically pure UPLC: Ultra-high performance liquid chromatography MS: Mass spectrometry MTBE: Methyl tert-butyl ether DCM: dichloromethane DMB: 2,4-dimethoxybenzyl DIPEA: N,N-diisopropylethylamine TFA: Trifluoroacetic acid THF: tetrahydrofuran RT: room temperature H: Time Min: minutes EA or EtOAc: ethyl acetate HPRT1: Hypoxanthine phosphoribosyltransferase 1 GAPDH1: glyceraldehyde-3-phosphate dehydrogenase 1 NTC: non-targeting control WP: Well plate Bz: benzoyl Cbz: benzyloxycarbonyl CE: 2-cyanoethyl Trt: Trityl IPr: Isopropyl Sar: Sarcosine ESI-TOF-MS: Electrospray ionization-time of flight mass spectrometry

[0194] Example 1: Reduction or interference with full-length CD33 SNP rs3865444 has been reported to be associated with increased skipping of CD33 exon-2 and reduced levels of full-length CD33 on the surface of monocytes. This allele was found to be associated with reduced full-length CD33 levels in human cerebrospinal fluid (CSF) and plasma as measured using Somascan technology (Figure 1). A study by the Alzheimer's Disease Neuroimaging Initiative (ADNI) found that this allele was associated with reduced ventricular volume and increased midtemporal volume, both of which are consistent with protection from Alzheimer's disease (Figure 2). Furthermore, in longitudinal analyses, this allele was associated with trends of improved scores on the Alzheimer's Disease Assessment Scale (ADAS), Mini-Mental State Examination (MMSE), Reay Auditory Verbal Learning Test (RAVLT)-immediate, Trail Making Test-B (TRABSCOR), Functional Activities Questionnaire (FAQ), 18F-fluorodeoxyglucose-positron emission tomography (FDG PET), ventricular volumes, fusiform gyrus, and midtemporal volumes (Figure 3), pointing to protection from the disease.

[0195] On the other hand, rs201074739 is a four-base pair deletion in exon 3 of the CD33 gene. This causes a frameshift in the open reading frame and premature translation termination. This indel was associated with decreased full-length CD33 levels in human CSF and plasma as measured using SomaScan technology (Figure 4). However, this indel has not been associated with reduced disease risk to date. Moreover, it was associated with increased ventricular volume and worsened Functional Activities Questionnaire (FAQ) scores, suggesting a deleterious effect (Figure 2).

[0196] Thus, successful induction of CD33 exon-2 skipping may be associated with therapeutic benefit.

[0197] Example 2: General Formula of ASO PMO oligonucleotides for screening were designed. The designed oligonucleotides listed in Tables 11 and 12 below were prepared by GeneTools LLC (www.gene-tools.com). Table 11 lists the top PMO oligonucleotides along with their deconvoluted MS data. Table 1 includes the top PMO oligonucleotides in Table 11 as well as other PMO oligonucleotides. All of the PMO oligonucleotides listed in Tables 11 and 1 have a sarcosine linker (Sar) attached to a phosphorodiamidate at the 5' end. All of the PMO oligonucleotides in Tables 11 and 1 were synthesized with an unmodified cytosine PMO nucleotide. All of the PMO oligonucleotides listed in Tables 11 and 1 have sterically random internucleotide linkages and are therefore referred to as sterically random PMO oligonucleotides. The general formula of the PMO oligonucleotides listed in Tables 11 and 1 below is: [ka]

[0198] [Table 19]

[0199] MOE oligonucleotides for screening were designed. The designed oligonucleotides listed in Tables 12 and 2 were generated by either Integrated DNA Technologies (www.idtdna.com) or GeneDesign (Ajinomoto Bio Pharma, https: / / ajibio-pharma.com / ). Table 12 lists the top MOE sequences along with their deconvoluted MS data. All MOE oligonucleotides listed in Tables 12 and 2 have a hydroxyl at the 5' end. All MOE oligonucleotides listed in Tables 12 and 2 have a 2'-O-MOE modified ribonucleotide containing a phosphorothioate backbone, except where noted. All MOE oligonucleotides listed in Tables 12 and 2 were synthesized with 5-methylcytosine 2'-O-MOE ribonucleotides. All MOE oligonucleotides listed in Tables 12 and 2 have sterically random internucleotide linkages and are therefore referred to as sterically random MOE oligonucleotides. The general formula for the MOE oligonucleotides listed in Table 12 and Table 2, depicted in free form, is: [ka]

[0200] [Table 20]

[0201] Example 3: Synthesis of PMO-302 (sterically pure internucleotide bond (Sp)) Synthesis of stereopure PMO-302 oligonucleotide (CCTCACCTGTCACATGCACAGAGAG (SEQ ID NO: 2)) with an unfunctionalized 5'-OH. The monomers used in the synthesis of PMO-302 are as follows (reported in WO2017024264A2): [ka]

[0202] Synthesis of PMO-302 with a 5'-OH and a stereopure internucleotide linkage: 2mer synthesis: [ka] Unless otherwise noted, all liquid components were added via appropriately sized syringes. All reactions were carried out under a N2 atmosphere. Filtrations and workups were performed open to the atmosphere. Filtrations were performed with sintered glass funnels.

[0203] A flask containing the amine ((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)morpholin-2-yl)methyl benzoate (130 mg) was equipped with a stir bar and a rubber septum. The atmosphere was replaced with nitrogen and sparged. After 5 minutes, 1,3-dimethyl-2-imidazolidinone (2.2 mL) was added via syringe at room temperature, followed by 1,2,2,6,6-pentamethylpiperidine (164 μL), and the mixture was allowed to form a solution. Solid ((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidochloridate (219 mg) was added all at once, and the flask was sealed with a rubber septum. Stirring was continued for 3 hours, and the reaction was monitored by UPLC MS. Upon completion, MTBE (11.7 mL) was added over 1 minute with stirring. A precipitate formed upon completion of the addition. n-Heptane (10 mL) was added. The oily mixture was allowed to stand for 10 minutes. The heavy oil was allowed to settle, while the cloudy supernatant was decanted into a 30 mL vial and centrifuged. This resulted in the formation of an additional oily residue at the bottom. The solvent was removed by decantation, and the two oily residues were combined by dissolving in 1 mL of DCM and purified by flash chromatography using 0-5% MeOH in DCM. Fractions containing the desired product were dried under vacuum to give ((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-((S)-(((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methoxy)(dimethylamino)phosphoryl)morpholin-2-yl)methyl benzoate (270 mg) as a white foam. MS (ESI) m / z: [M+H] + C 60 H 58 N9O 10 Calculated value for P: 1096.40; Found: 1096.53.

[0204] Deprotection of the 2mer: [ka] To a flask containing ((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-((S)-(((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2yl)methoxy)(dimethylamino)phosphoryl)morpholin-2-yl)methyl benzoate (350 mg) was added DCM (3.5 mL). Ethanol (186 μL) was added via syringe at room temperature. TFA (160 μL) was added dropwise over 30 seconds at room temperature. The reaction mixture was stirred for 30 minutes and monitored by UPLC-MS. Upon completion, MTBE (14 mL) was added via syringe over 1 minute. The suspension was stirred for 10 minutes and then sonicated. The suspension was filtered through a sintered filter funnel and rinsed with 10 mL of MTBE (2 x 5 mL). The solid was dried and transferred to a new flask, then dissolved by adding DCM (3.5 mL). 1,2,2,6,6-Pentamethylpiperidine (292 μL) was added via syringe. After 10 min at room temperature, MTBE (15.8 mL) was added over 1 min. A white solid formed. After 10 min, the slurry was sonicated, filtered, and rinsed with MTBE (2 x 10 mL). Drying under airflow for 20 minutes and under vacuum for 1 hour gave ((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-((S)-(((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)morpholin-2-yl)methyl benzoate (243 mg). MS (ESI) m / z: [M+H] + C 41 H 44 N9O 10 Calculated value for P: 854.29; Found value: 854.65.

[0205] 3mer synthesis: [ka] To a flask containing ((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-((S)-(((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)morpholin-2-yl)methyl benzoate (215 mg) was added 1,3-dimethyl-2-imidazolidinone (2 mL) and 1,2,2,6,6-pentamethylpiperidine (138 μL) under nitrogen. After 2 minutes, ((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl-(R)-dimethylphosphoramidochloridate (169 mg) was added and the reaction was stirred at room temperature for 3 hours. Upon completion, ethyl acetate (2.6 mL) was added followed by MTBE (14 mL). The resulting white precipitate was filtered, rinsed with MTBE (2 x 5 mL), and dried under vacuum to give ((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-((S)-(((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-((S)-(dimethylamino)(((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methoxy)phosphoryl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)morpholin-2-yl)methyl benzoate (350 mg). MS (ESI) m / z: [M+H] + C 72 H 77 N 13 O 15 Calculated value for P2: 1426.51; Measured value: 1427.74.

[0206] Deprotection of the 3mer: [ka] A flask was charged with ((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-((S)-(((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-((S)-(dimethylamino)(((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methoxy)phosphoryl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)morpholin-2-yl)methyl benzoate (350 mg) and DCM (4.2 mL). Ethanol (143 μL) was added slowly at room temperature, followed by TFA (95 μL). The reaction mixture was stirred at room temperature for 2 hours. Upon completion, MTBE (15 mL) was added. The solid was filtered and rinsed with MTBE (10 mL). The solid was dried, then transferred to a flask and dissolved by adding DCM (2.7 mL). 1,2,2,6,6-Pentamethylpiperidine (224 μL) was added at room temperature, and the reaction mixture was stirred at room temperature for 10 minutes. MTBE (15 mL) was added, and the resulting slurry was stirred for 10 minutes, sonicated, filtered, and rinsed with MTBE (10 mL). The trimer was obtained as the free base (297 mg). MS (ESI) m / z: [M+H] + C 53 H 63 N 13 O 15 Calculated value for P2: 1184.40; Measured value: 1185.

[0207] 4mer synthesis: [ka] A flask was charged with ((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-((S)-(((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-((S)-(dimethylamino)(((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholin-2-yl)methoxy)phosphoryl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)morpholin-2-yl)methyl benzoate (292 mg) and the flask was purged with nitrogen. 1,3-Dimethyl-2-imidazolidinone (2.9 mL) was added, followed by 1,2,2,6,6-pentamethylpiperidine (135 μL). ((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidochloridate (207 mg) was added all at once and the reaction was stirred at room temperature for at least 1 hour while being monitored for completion by HPLC-MS. Ethyl acetate (2.9 mL) was charged followed by MTBE (14 mL). The slurry was stirred for 15 minutes, filtered, and washed with 2 x 5 mL of MTBE. The resulting solid was dried under vacuum for 10 minutes, then collected in a new flask and dried under vacuum to give 430 mg of the 4mer. MS (ESI) m / z: [M+H] + C 90 H 99 N 18 O 20 Calculated value for P3: 1846.65; Measured value: 1847.

[0208] Deprotection of the 4mer: [ka] A flask was charged with ((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-((S)-(((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-((S)-(((2S,6R)-4-((S)-(((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-trimethylsilyl) (dimethylamino)phosphoryl)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)morpholin-2-yl)methyl benzoate (430 mg) was added. DCM (4.3 mL) and ethanol (272 μL) were added. After a solution formed, TFA (135 μL) was added. The reaction mixture was stirred for 2.5 hours and deemed complete by HPLC analysis. Ethyl acetate (3.0 mL) and MTBE (10.8 mL) were added over 1 minute. A solid precipitate formed during the MTBE addition. Upon completion of the MTBE addition, the solid was stirred for 10 minutes and sonicated three times. It was filtered and rinsed with 2×5 mL of MTBE. The solid was then dried and then dissolved in DCM (4.3 mL) and treated with 1,2,2,6,6-pentamethylpiperidine (319 μL). After 5 min, the desired product was precipitated by the addition of ethyl acetate (3.0 mL) and MTBE (10.8 mL) over 1 min.The solid was filtered, rinsed with MTBE, and dried under vacuum overnight to give ((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-((S)-(((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-((S)-(((2S,6R)-4-((S)-(((2S,6R)-6-(4-benzamido-2-oxopyrimidin (Dimethylamino)phosphoryl)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)morpholin-2-yl)methyl benzoate (330 mg) was obtained. MS (ESI) m / z: [M+H]. + C 71 H 85 N 18 O 20 Calculated value for P3: 1603.54; Measured value: 1605.

[0209] 5mer synthesis: [ka] ((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-((S)-(((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-((S)-(((2S,6R)-4-((S)-(((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)-6-(5- To a flask containing methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)morpholin-2-yl)methyl benzoate (330 mg), 1,3-dimethyl-2-imidazolidinone (3.3 mL) and 1,2,2,6,6-pentamethylpiperidine (113 μL) were added. After the residue was completely dissolved, ((2S,6R)-6-(6-benzamido-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidochloridate (178 mg) was added at room temperature. The reaction mixture was stirred at room temperature for 5 hours, and then ethyl acetate (6.6 mL) and MTBE (13.2 mL) were added. The white precipitate was filtered and dried. The solid was dissolved in 2 mL of DCM and purified by automated silica gel chromatography on a 25 g cartridge using 0-20% MeOH in DCM. 345 mg of the desired product 5mer was obtained. MS (ESI) m / z: C 109 H 121 N 25 O 24 Calculated value for P4: [(M+2H) / 2] + 1145.4; Actual value: 1145.6.

[0210] Deprotection of the 5mer: [ka] In a flask, ((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-((S)-(((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-((S)-(((2S,6R)-4-((S)-(((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-((S)-(((2S,6R)-6-(6-benzamido-9H-purin-9-yl) To the reaction mixture was added 328 mg of 4-tritylmorpholin-2-yl)methoxy)(dimethylamino)phosphoryl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)morpholin-2-yl)methyl benzoate. 3.1 mL of DCM was added, followed by 167 μL of ethanol. 66.2 μL of TFA was added at room temperature and stirred for 3 hours. 3 additional drops (approximately 15 μL) of TFA were added. The reaction was monitored by HPLC-MS, and upon completion (disappearance of the starting material peak), 11.8 mL of ethyl acetate was added, followed by stirring for 5 minutes. The mixture was filtered and rinsed with 2 mL of EtOAc and 5 mL of MTBE. Additional solid formed in the mother liquor and was also collected by a second filtration. The combined solids were placed in a reaction flask. DCM (2.3 mL) and 1,2,2,6,6-pentamethylpiperidine (209 μL) were added. Stirred for 15 minutes, then EtOAc (2.6 mL) and MTBE (10.5 mL) were added. The resulting solid was filtered, rinsed with 2×3 mL of MTBE, and then dried under vacuum and collected to give 280 mg of the deprotected 5-mer. MS (ESI) m / z: C 90 H 107 N 25 O 24 Calculated value for P4: [M+2H / 2] + 1023.8; Actual value: 1024.12.

[0211] 6mer synthesis: [ka] ((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-((S)-(((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-((S)-(((2S,6R)-4-((S)-(((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-((S)-(((2S,6R)-6-(6-benzamido-9H-purin-9-yl)morpholin-2-yl)methoxy)( To a flask containing 265 mg of (dimethylamino)phosphoryl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)morpholin-2-yl)methyl benzoate, 2.7 mL of 1,3-dimethyl-2-imidazolidinone was added. 71.0 μL of 1,2,2,6,6-pentamethylpiperidine was added. 108 mg of ((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidochloridate was added as a solid at room temperature. After 3 h at room temperature and completion as judged by HPLC analysis, EtOAc (5.3 mL) and MTBE (10.6 mL) were added over 2-3 min each. Filtered and rinsed with 2 x 3 mL of MTBE. After drying with a stream of air for 2-3 min, the solid turned into a sticky mass. This solid was transferred to the same flask containing 10 mL of DCM and concentrated under vacuum.((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-((S)-(((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-((S)-(((2S,6R)-4-((S)-(((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-((S)-(((2S,6R)-4-((S)-(((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl) Methoxy)(dimethylamino)phosphoryl)-6-(6-benzamido-9H-purin-9-yl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)morpholin-2-yl)methyl benzoate (354 mg) was isolated. MS (ESI) m / z: C. 127 H 143 N 30 O 29 Calculated value for P5: [M+2H / 2] + 1354.97; Actual value: 1354.73.

[0212] Deprotection of the 6mer: [ka] To a flask containing the dried, evaporated solid (6mer) from the previous step, DCM (3.2 mL) and ethanol (155 μL) were added. After the solid was completely dissolved, TFA (71.7 μL) was added. The mixture was stirred for 2 h, and HPLC analysis showed that the reaction was not complete. An additional 50 μL of TFA was added, and stirring was continued for an additional 6 h. EtOAc (2.9 mL) was added, followed by MTBE (11 mL). The resulting solid was filtered and rinsed with 4:1 MTBE / EtOAc (12 mL). ((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-((S)-(((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-((S)-(((2S,6R)-4-((S)-(((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-((S)-(((2S,6R)-4-((S)-(((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)morpholin-2-yl)methoxy )(dimethylamino)phosphoryl)-6-(6-benzamido-9H-purin-9-yl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)morpholin-2-yl)methyl benzoate (295 mg) was isolated. MS (ESI) m / z: C 108 H 129 N 30 O 29 Calculated value for P5: [(M+2H) / 2] + 1233.92; Actual value: 1233.68.

[0213] Synthesis of 7mer: [ka] ((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-((S)-(((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-((S)-(((2S,6R)-4-((S)-(((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl))-4-((S)-(((2S,6R)-4-((S)-(((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)-6-(6-benzamido-9H-purine To a flask containing 6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)morpholin-2-yl)methoxy)(dimethylamino)phosphoryl)morpholin-2-yl)methyl benzoate (295 mg), 1,3-dimethyl-2-imidazolidinone (2.9 mL) and then 1,2,2,6,6-pentamethylpiperidine (65.6 μL) were added at room temperature. ((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidochloridate was added as a solid (100 mg) at room temperature. The reaction was stirred at room temperature for 3 hours. After completion by HPLC analysis, EtOAc (5.9 mL) and MTBE (11.8 mL) were added over 2-3 minutes each. The solid was filtered and rinsed with 2 x 5 mL of MTBE. After drying with a stream of air for 2-3 minutes, the solid was transferred to a flask and dried under vacuum for 1 hour to give the 7mer (430 mg). MS (ESI) m / z: C 145 H 165 N 35 O 34 Calculated value for P6: [(M+2H) / 2] + 1564.85; Actual value: 1564.77.

[0214] Deprotection of the 7mer:

change

[0215] From the 8mer to the 25mer, a general procedure was used for coupling, deprotection and freebasing:

[0216] General Coupling Procedure A: To a flask containing dried PMO oligonucleotide (free-base PMO oligonucleotide) (1 wt, 1 eq) was added 1,3-dimethyl-2-imidazolidinone (6–10 volumes relative to the free-base PMO oligonucleotide), followed by 1,2,2,6,6-pentamethylpiperidine (3–5 eq). The mixture was stirred and sonicated until all solids dissolved. Activated monomer (R)-dimethylphosphoramidochloridate (1.3–2.5 eq) was added all at once as a solid under a N2 atmosphere. The reaction mixture was stirred for a minimum of 3 h (18–24 h for stages 15–25 mer) and monitored for completion (>99.5% target or starting material mass undetectable by UV) by UPLC MS. Optionally, additional (R)-dimethylphosphoramidochloridate was added if the target conversion criterion was not reached. Upon completion, the reaction mixture was charged with 10-40 volumes of EtOAc and MTBE (10-40 volumes relative to the free-base PMO oligonucleotide), resulting in the formation of a white precipitate. This solid was filtered through a sintered funnel, purified with 1:1 EtOAc / MTBE, dried under vacuum, and collected to yield the "trityl-protected PMO oligonucleotide" for the next step. Overall yields were 90-100%.

[0217] General Procedure B for Trityl Deprotection and Freebasing: A trityl deblocking solution was prepared as follows: a flask was charged with DCM (8 mL), 2,2,2-trifluoroethanol (2 mL), 4-cyanopyridine (100 mg), ethanol (100 μL), and trifluoroacetic acid (105 mg) in that order. The solution was mixed until all components were dissolved and then used directly for deprotection.

[0218] Step 1—Trityl Deprotection: To a flask containing the "trityl-protected PMO oligonucleotide" (1 wt, 1 eq.) was added the trityl deblocking solution (8 volumes relative to the trityl-protected PMO oligonucleotide mass). The reaction mixture was stirred for 5–30 min and monitored by UPLC MS. Upon completion (>99.5% of target), EtOAc (10–40 volumes) and MTBE (10–40 volumes) were added, resulting in the formation of a white precipitate. The solid was filtered through a sintered funnel, rinsed with 1:1 EtOAc / MTBE, dried under vacuum, and collected to yield the "TFA salt PMO oligonucleotide" for the next step.

[0219] Step 2—Freebasing: To a flask containing the "TFA salt PMO oligonucleotide" (1 wt, 1 eq) was added DCM (7–10 volumes relative to the TFA salt PMO oligonucleotide mass) and EtOH (0.3–0.5 volumes). This solution was treated with 1,2,2,6,6-pentamethylpiperidine (5 eq). The reaction mixture was stirred for 5–10 min and then treated with EtOAc (10–40 volumes) and MTBE (10–40 volumes), resulting in the formation of a white precipitate. The solid was rinsed with 1:1 EtOAc / MTBE, dried under vacuum, and collected for the next step.

[0220] Coupling to 8mer: [ka] Using general procedure A: 7mer (340 mg) was reacted with ((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidochloridate (86 mg) to give 8mer (403 mg). MS (ESI) m / z: C 157 H 184 N 39 O 39 Calculated value for P7: [M+2H / 2] + 1730.09; Actual value: 1730.

[0221] Deprotection of the 8mer: [ka] Using general procedure B: Trityl protected 8mer (380 mg) was reacted to give the free base 8mer (353 mg). MS (ESI) m / z: C 138 H 170 N 39 O 39 Calculated value for P7: [M+2H / 2] + 1608.53; Actual value: 1609.

[0222] Coupling to 9mer: [ka] Using general procedure A: 8mer (370 mg) was reacted with ((2S,6R)-6-(6-(2-cyanoethoxy)-2-isobutyramido-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidochloridate (105 mg) to give 9mer (453 mg).

[0223] Deprotection of the 9mer: [ka] Using general procedure B: The trityl protected 9mer (453 mg) was reacted to give the free base 9mer (411 mg).

[0224] Coupling to 10mer: [ka] Using general procedure A: the 9-mer (405 mg) was reacted with ((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidochloridate (80 mg) to give the 10-mer (469 mg). MS (ESI) m / z: C 188 H 230 N 51 O 49 Calculated value for P9: [M+3H / 3] + 1422.8; Actual value: 1423.3.

[0225] Deprotection of the 10mer: [ka] Using general procedure B: The trityl protected 10mer (450 mg) was reacted to give the free base 10mer (435 mg).

[0226] Coupling to 11mer: [ka] Using general procedure A: the 10-mer (435 mg) was reacted with ((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidochloridate (98 mg) to give the 11-mer (512 mg).

[0227] Deprotection of the 11mer: [ka] Using general procedure B: The trityl protected 11mer (500 mg) was reacted to give the free base 11mer (481 mg).

[0228] Coupling to 12mer: [ka] Using general procedure A: The 11mer (481 mg) was reacted with ((2S,6R)-6-(6-benzamido-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidochloridate (102 mg) to give the 12mer (525 mg).

[0229] Deprotection of the 12mer: [ka] Using general procedure B: The trityl protected 12mer (525 mg) was reacted to give the free base 12mer (490 mg).

[0230] Coupling to 13mer: [ka] Using general procedure A: the 12mer (484 mg) was reacted with ((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidochloridate (86 mg) to give the 13mer (550 mg).

[0231] Deprotection of the 13mer: [ka] Using general procedure B: The trityl protected 13mer (550 mg) was reacted to give the free base 13mer (550 mg).

[0232] Coupling to 14mer: [ka] Using general procedure A: the 13mer (550 mg) was reacted with ((2S,6R)-6-(6-benzamido-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidochloridate (94 mg) to give the 14mer (621 mg).

[0233] Deprotection of the 14mer: [ka] Using general procedure B: The trityl protected 14mer (621 mg) was reacted to give the free base 14mer (596 mg).

[0234] Coupling to 15mer: [ka] Using general procedure A: 14mer (596 mg) was reacted with ((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidochloridate (84 mg) to give 15mer (655 mg). MS (ESI) m / z: C 274 H 337 N 79 O 72 P 14 Calculated value as: [M+4H / 4] + 1581.28; Actual value: 1582.

[0235] Deprotection of the 15mer: [ka] Using general procedure B: Trityl protected 15mer (650 mg) was reacted to give the free base 15mer (613 mg). MS (ESI) m / z: C 255 H 323 N 79 O 72 P 14Calculated value as: [M+4H / 4] + 1520.76; Actual value: 1521.

[0236] Coupling to 16mer: [ka] Using general procedure A: The 15mer (613 mg) was reacted with ((2S,6R)-6-(6-(2-cyanoethoxy)-2-isobutyramido-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidochloridate (103 mg) to give the 16mer (680 mg).

[0237] Deprotection of the 16mer: [ka] Using general procedure B: The trityl protected 16mer (680 mg) was reacted to give the free base 16mer (623 mg).

[0238] Coupling to 17mer: [ka] Using general procedure A: the 16mer (623 mg) was reacted with ((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidochloridate (93 mg) to give the 17mer (690 mg).

[0239] Deprotection of the 17mer: [ka] Using general procedure B: The trityl protected 17mer (690 mg) was reacted to give the free base 17mer (670 mg).

[0240] Coupling to 18mer: [ka] Using general procedure A: The 17mer (673 mg) was reacted with ((2S,6R)-6-(6-benzamido-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidochloridate (98 mg) to give the 18mer (740 mg).

[0241] Deprotection of the 18mer: [ka] Using general procedure B: The trityl protected 18mer (739 mg) was reacted to give the free base 18mer (675 mg).

[0242] Coupling to 19mer: [ka] Using general procedure A: the 18mer (675 mg) was reacted with ((2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidochloridate (127 mg) to give the 19mer (735 mg).

[0243] Deprotection of the 19mer: [ka] Using general procedure B: The trityl protected 19mer (735 mg) was reacted to give the free base 19mer (732 mg).

[0244] Coupling to 20mer: [ka] Using general procedure A: 19mer (732 mg) was reacted with ((2S,6R)-6-(6-benzamido-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidochloridate (135 mg) to give 20mer (790 mg). MS (ESI) m / z: C 367 H 452 N 111 O 95 P 19 Calculated value as: [M+5H / 5] + 1706; Actual value: 1707.

[0245] Deprotection of the 20mer: [ka] Using general procedure B: Trityl protected 20mer (790 mg) was reacted to give the free base 20mer (743 mg). MS (ESI) m / z: C 348 H 438 N 111 O 95 P 19 Calculated value as: [M+5H / 5] + 1657.6; Actual value: 1658.

[0246] Coupling to 21mer: [ka] Using general procedure A: The 20mer (743 mg) was reacted with ((2S,6R)-6-(6-(2-cyanoethoxy)-2-isobutyramido-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidochloridate (129 mg) to give the 21mer (795 mg).

[0247] Deprotection of the 21mer: [ka] Using general procedure B: The trityl protected 21mer (800 mg) was reacted to give the free base 21mer (756 mg).

[0248] Coupling to 22mer: [ka] Using general procedure A: The 21mer (753 mg) was reacted with ((2S,6R)-6-(6-benzamido-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidochloridate (137 mg) to give the 22mer (806 mg).

[0249] Deprotection of the 22mer: [ka] Using general procedure B: The trityl protected 22mer (806 mg) was reacted to give the free base 22mer (785 mg).

[0250] Coupling to 23mer: [ka] Using general procedure A: The 22mer (780 mg) was reacted with ((2S,6R)-6-(2-isobutylamido-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidochloridate (161 mg) to give the 23mer (837 mg).

[0251] Deprotection of the 23mer: [ka] Using general procedure B: The trityl protected 23mer (837 mg) was reacted to give the free base 23mer (830 mg).

[0252] Coupling to 24mer: [ka] Using general procedure A: The 23mer (830 mg) was reacted with ((2S,6R)-6-(6-benzamido-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidochloridate (177 mg) to give the 24mer (800 mg).

[0253] Deprotection of the 24mer: [ka] Using general procedure B: The trityl protected 24mer (800 mg) was reacted to give the free base 24mer (793 mg).

[0254] Coupling to 25mer: [ka] Using general procedure A: 24mer (793 mg) was reacted with ((2S,6R)-6-(2-isobutylamido-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl (R)-dimethylphosphoramidochloridate (150 mg) to give 25mer (818 mg). MS (ESI) m / z: C 456 H 571 N 147 O 118 P 24 Calculated value as: [M+7H / 7] + 1535.53; Actual value: 1535.56.

[0255] 25mer base deprotection: [ka] To a 100 mL flask containing the 25mer (710 mg) was added methanol (19.5 mL) and 28% aqueous ammonium hydroxide (19.5 mL) under a nitrogen atmosphere. The reaction mixture was stirred at 50°C for 2 days to give a clear solution. This solution was then evaporated under vacuum at 35°C to a volume of approximately 20 mL. The slightly cloudy mixture was filtered through a plastic fritted funnel and rinsed with approximately 5-10 mL of water to a total volume of 25 mL. The resulting solution was used for purification by reverse-phase HPLC using the following conditions: Evaporation of the desired peak fractions gave a total of 148 mg of the deprotected trityl-tagged 25mer as a white solid. MS (ESI) m / z: C 308 H 464 N 144 O 96 P 24 Calculated as: [M+1H] + 8462.97; Found: 8463.00 (HRMS spectrum after deconvolution).

[0256] [Table 21]

[0257] Trityl deprotection of the 25mer: [ka] To a vial containing the trityl-protected 25mer (3.7 mg), 0.1 M phosphoric acid (250 μL) was added. The vial was stirred at room temperature for 4 hours, after which the reaction was deemed complete (two consecutive UPLC MS runs showed the starting material peak had converted to an early-eluting peak). 0.1 M ammonium hydroxide (250 μL) was added and filtered through a 0.2 μM syringe filter. The filter was rinsed with 0.4 mL of water and collected in a vial. The sample was purified by reverse-phase HPLC using the method in the table below. The desired fractions were combined, evaporated under vacuum, and then lyophilized to yield 1.2 mg of the desired product, the 25mer PMO (PMO-302). MS (ESI) m / z: C 289 H450 N 144 O 96 P 24 Calculated as: [M+1H] + 8220.86; Found: 8220.87 (HRMS spectrum after deconvolution).

[0258] [Table 22]

[0259] Example 4: Additional exemplary PMO-ASOs PMO oligonucleotides for screening were designed. The designed oligonucleotides were produced by solid-phase methods by GeneTools LLC (website: www.gene-tools.com). Table 13 below lists the synthesized PMO oligonucleotides along with their deconvoluted MS data. These PMO oligonucleotides are complementary to sections of SEQ ID NO: 1 that exhibit increased exon-2 skipping activity. Specifically, PMO-221 to PMO-240, PMO-324, PMO-424, PMO-402, and PMO-502 are complementary to region 1; and PMO-241 to PMO-244 are complementary to region 2. All PMO oligonucleotides listed in Table 13 below contain a sarcosine (Sar) linker with a phosphorodiamidate attached at the 5' end. All PMO oligonucleotides listed in Table 13 below were synthesized with unmodified cytosine PMO nucleotides. All PMO oligonucleotides listed below in Table 13 have sterically random internucleotide linkages and are therefore referred to as sterically random PMO oligonucleotides. The structure of PMO-224 is: [ka]

[0260] [Table 23]

[0261] Example 5: Synthesis of PMO oligonucleotides with stereopure internucleotide linkages and 5'-sarcosine linkers

[0262] [Table 24]

[0263] Solution-phase synthesis of stereopure PMO oligonucleotides: Solution phase synthesis of the 5'-sarcosine capped stereopure oligonucleotides in Table 14 was carried out using a method similar to that described in Example 3 (using general procedures A and B), except that step 1 begins with coupling sarcosine benzyl ester to stereopure cytosine dimethylphosphoramidochloridate. Briefly, the synthesis involves repeated deprotection / freebasing / coupling steps as depicted here for all Sp internucleotide linkages:

[0264] [ka] [ka] General scheme for the synthesis of PMO-424 and PMO-502 by liquid phase. Briefly, the synthesis involves repeated deprotection / freebasing / coupling steps as depicted here for all Rp internucleotide linkages): [ka] [ka] General scheme for the synthesis of PMO-324 and PMO-402 by liquid phase. At the end of each individual step, precipitation of the oligonucleotides was achieved by adding a non-polar solvent such as MTBE and / or EtOAc. For the elongation steps up to the 6-mer, purification of the 3'-N-trityl-protected oligonucleotides was performed by silica gel chromatography using DCM / MeOH as the eluent.

[0265] Once the desired oligonucleotide length was reached (21 mer for PMO-324, PMO-424, and 25 mer for PMO-402 and PMO-502), the 3'-N-trityl protected sequences were subjected to base deprotection as follows.

[0266] Base deprotection for solution phase synthesis: The 3'-N-trityl-protected PMO oligonucleotide residue (1 wt.) from the final coupling step was dissolved in MeOH (8 volumes), followed by the addition of 7N NH3 in MeOH (20 volumes). The reaction mixture was heated to 50-55°C for at least 48 hours. The solution was filtered to remove any solids and rinsed with 1:1 MeOH / 7N NH3 in MeOH. Preparative-scale chromatographic purification using a reverse-phase gradient as outlined in Table 15 afforded the 3'-N-trityl-protected PMO after solvent evaporation.

[0267] [Table 25]

[0268] Final trityl deprotection: To the base-protected PMO oligonucleotide from the HPLC purification, 0.1 N phosphoric acid (at least 20 equivalents) was added, and the reaction was monitored by HPLC. After the completion of trityl deprotection was assessed by two consecutive HPLC runs, the reaction mixture was basified by adding ammonium hydroxide (at least 40 equivalents). The solution was filtered, and the final PMO oligonucleotide was purified by HPLC under the conditions in Table 16.

[0269] [Table 26]

[0270] Example 6: Analytical Data of Stereopure PMO Oligonucleotides Melting temperature (Tm) of PMO oligonucleotide: Tm measurement device: Shimadzu UV-2700 UV-visible spectrophotometer ASO samples were prepared by dissolving approximately 0.6–0.8 mg of solid in nuclease-free water to a concentration of approximately 3.2 μg / mL. Reverse-complementary RNA (obtained from IDT Technologies Inc.) was dissolved in nuclease-free water to a concentration of 400 μM. A 10 μL aliquot of each stock solution was diluted to 1 mL with nuclease-free water, and the concentration was determined using a UV-visible spectrophotometer. Test samples (500 μL) containing 4.0 μM PMO with 4.0 μM reverse-complementary RNA in a buffer solution containing 100 mM NaCl, 10 mM Na phosphate pH 7.0, and 0.1 mM EDTA were prepared. Test samples were incubated in 1 mL cuvettes and heated from 15°C to 105°C at 0.5°C / min. The increase in UV absorbance due to strand melting was monitored at 260 nm. Prior to this experiment, samples were melted and reannealed by heating from 25°C to 95°C at 5°C / min and cooling to the starting temperature to ensure complete annealing. Shimadzu Tm analysis software was used to calculate the Tm using the derivative (curve inflection point: 50% melting).

[0271] Analytical data for stereo-pure PMO oligonucleotides. PMO-424: [ka] P 31 NMR(D2O,162MHz)δ 21.5,18.7,18.6,18.5,18.4,18.3,18.3,18.1,18.0,17.9. ESI-TOF-MS calculated value: C246 H 390 N 119 O 84 P 21 as 7009.02; Actual value: 7008.51. Tm = 80.1°C (sterically random Tm = 75.0°C). See Figure 7. See Figure 5 for HPLC and HRMS data.

[0272] PMO-324: [ka] ESI-TOF-MS calculated value: C 246 H 390 N 119 O 84 P 21 as 7009.02; Actual value: 7008.50. Tm = 66.5°C (sterically random Tm = 75.0°C). See Figure 7. See Figure 6 for HPLC and HRMS data.

[0273] PMO-502: [ka] ESI-TOF-MS calculated value: C 294 H 462 N 147 O 98 P 25 as 8398.20 Actual value: 8397.98. Tm=87.8°C (sterically random Tm=79.3°C). See Figure 10. See Figure 8 for HPLC and HRMS data.

[0274] PMO-402: [ka] ESI-TOF-MS calculated value: C 294 H 462 N 147 O 98 P 25 as 8398.20 Actual value: 8397.99. Tm = 69.0°C (sterically random Tm = 79.3°C). See Figure 10. See Figure 9 for HPLC and HRMS data.

[0275] Example 7: Solid-phase synthesis of stereopure PMOs using a peptide synthesizer Deprotection of Fmoc on Sar-Wang resin: [ka] Fmoc-SAR-Wang resin (purchased from Aapptec, RWG103, lot number 9953380, 0.65 mmol / g, 110-200 mesh) (1 g, 650 mmol) was treated with DMF (8 mL), the resin was allowed to swell for 2 h, and the DMF was drained. The resin was treated with 20% piperidine in DMF (6 mL), shaken for 3 min, the solvent removed, and dried under N gas for 1 min (the same procedure was repeated four times). Finally, the resin was washed with DMF (5 mL x 5), CHCl (5 mL x 5), and dried under vacuum using N gas overnight to give 0.8 g of resin.

[0276] Resin loading calculation: The collected piperidine solution was adjusted to a final volume of 40 mL with 20% piperidine in DMF. A 0.1 mL aliquot was diluted 100-fold with DMF, and the UV absorbance at 301 nm of the Fmoc groups per gram was measured. The resin loading was >700 μmol / g. UV measurement conditions Solvent: 20% piperidine in DMF Wavelength: 301nm ε=7800

[0277] General procedure for solid phase synthesis of PMOs: [ka] [ka] The Fmoc-deprotected resin (1.10 g, loading: 0.650 mmol / g) was transferred to a peptide synthesizer reaction vessel, washed with CHCl (20 mL x 5), acetonitrile (20 mL x 5), and dried. Stereopure cytosine dimethylphosphoramidochloridate (1 equiv.) was added as a solid to the flask. Next, 1,2,2,6,6-pentamethylpiperidine (PMP, 10.0 equiv.) and anhydrous 1,3-dimethyl-2-imidazolidinone (DMI, 5.0 mL) were added to the vessel, and the mixture was shaken at room temperature for 20 h. LCMS of a reaction aliquot showed no monomer in solution (indicating that all monomer had been loaded onto the resin). Steps 5–9 in Table 17 were then carried out.

[0278] [Table 27]

[0279] Preparation of detritylation solution: To a solution of 4-cyanopyridine (10.1 g; 1.055 eq) in dichloromethane (790 mL) is added trifluoroacetic acid (10.5 g; 1.0 eq), followed by 2,2,2-trifluoroethanol (198 mL) and ethanol (10 mL), and the solution is stirred for 3 hours.

[0280] After the first loading of the monomer onto the resin, a synthesis cycle (as shown in Table 17) was initiated. The synthesis involved a series of repeated steps including deprotection / neutralization / coupling / capping. The required monomers (the purity of the monomers was characterized by HPLC-Mass before use) were added in each cycle to obtain the title nucleotide sequence.

[0281] In each synthesis cycle, after the coupling reaction (Step 4, Table 17), a small amount of resin was subjected to cleavage conditions (0.1 mL of 7N NH3 / MeOH, 55°C, 4 hours), and the RP HPLC-Mass was recorded for coupling efficiency (RP HPLC-Mass showed two peaks, methyl ester and amide, in a ratio of approximately 2:1. To ensure complete conversion of the methyl ester to the amide, the cleavage reaction was left stirring at 55°C overnight). The cleavage protocol was repeated from the 2-mer to the 21-mer for PMO-324 and PMO-424, and down to the 25-mer for PMO-402 and PMO-502. The RP HPLC-Mass was recorded using the conditions in Table 18.

[0282] [Table 28]

[0283] For example, Figure 11 shows the UV chromatogram of the trityl-protected 21-mer (all Sp-Sar-CCTCACCTGTCACATGCACAG-Tr) after cleavage from the resin.

[0284] Cleavage from resin and base deprotection: After achieving the desired oligonucleotide length, the synthesized PMO-loaded resin was dried, transferred to a centrifuge bottle, and charged with 7N NH3 / MeOH (approximately 0.5 mL / 1 μmol). The mixture was stirred at 50-55°C for 60 hours. The reaction was cooled to room temperature, and the solids were filtered and washed with methanol. The resulting filtrate was concentrated under reduced pressure to an approximate final volume of approximately 20 mL, and any solids were then filtered through a 0.4 micron membrane filter. The filtrate was concentrated to dryness and weighed. The resulting crude residue was dissolved in 60 mL of a solvent mixture of 50 mM Et3NHOAc (using cell culture water) / MeCN (1 / 1) containing 0.1% Et3N. The filtrate was purified by reverse-phase HPLC according to the conditions listed in Table 19.

[0285] [Table 29]

[0286] Final detritylation: Freshly prepared 0.1 M aqueous phosphoric acid (20 equiv.) was added to the flask containing the recovered 3'-N-Tr-PMO (1 equiv.), and the mixture was stirred at room temperature for 2 h (a cloudy white solution formed within 10 min). Two consecutive LCMS runs confirmed the reaction was complete (showing that the starting material peak had converted to an early-eluting peak; the HPLC sample was prepared in water only). The reaction was basified by adding 28% ammonium hydroxide (40 equiv.), stirred for 30 min, and the solid was filtered through a membrane filter (0.45 μm) and washed with water. The resulting filtrate was purified by reverse-phase HPLC (Table 20).

[0287] [Table 30]

[0288] Each fraction was analyzed (by HPLC), and the fractions containing the product were dried using a Genevac. The final product was dissolved in endotoxin-free water, and the solution was filtered through an Amicon 3K filter to remove any inorganic salt impurities. The resulting aqueous solution was freeze-dried to give the title compound as a white, cotton-like solid.

[0289] Analytical data for stereopure PMOs prepared by solid phase synthesis: PMO-424: P 31 NMR(D2O,162MHz)δ 21.5,18.7,18.6,18.5,18.4,18.3,18.3,18.1,18.0,17.9. LRMS:C 246 H 390 N 119 O 84 P 21 (m / z=7009.02) [M+5H] 5+ Calculated m / z for ion: 1402.80; Found: 1402.62 PMO-324: LRMS:C246 H 390 N 119 O 84 P 21 (m / z=7009.02) [M+5H] 5+ Calculated m / z for ion: 1402.80; Found: 1403.4 PMO-402: LRMS:C 294 H 462 N 147 O 98 P 25 (m / z=8396.92) [M+6H] 6+ Calculated m / z for ion: 1400.66; Found: 1401.2

[0290] Example 8: Additional exemplary MOE-ASOs Phosphorothioate oligonucleotides were designed for screening. All oligonucleotides listed in Table 21 below contain ribonucleotides with a phosphorothioate backbone, except where noted (e.g., solid line (-) = phosphodiester (PO) linkage). All oligonucleotides listed in Table 21 below were synthesized with 5-methylcytosine ribonucleotides. All oligonucleotides listed in Table 21 below have sterically random phosphorothioate internucleotide linkages and are therefore referred to as sterically random oligonucleotides. All oligonucleotides listed in Table 21 below are complementary to region 6: (SEQ ID NO: 218).

[0291] [Table 31]

[0292] [Table 32]

[0293] All oligonucleotides listed in Table 22 below contain 2'-O-MOE modified ribonucleotides and a 5'-terminal hydroxyl group. The oligonucleotides in Table 22 contain stereochemically pure phosphorothioate internucleotide linkages and are therefore referred to as stereochemically pure MOE oligonucleotides. All oligonucleotides listed in Table 22 are complementary to region 6: (SEQ ID NO: 218).

[0294] [Table 33]

[0295] [Table 34]

[0296] [Table 35]

[0297] Example 9: Preparation of stereopure 2'-MOE phosphorothioate oligonucleotides Protected 2'-O-MOE-3'-OH monomer [ka] (Compound 1) 2,2-Diethoxy-1-methylpyrrolidine: A mixture of NMP (100 mL, 1039.008 mmol) and dimethyl sulfate (99 mL, 1039.008 mmol) was stirred and heated to 80 °C (sand bath) overnight, then allowed to cool to room temperature. After cooling, the homogeneous liquid was washed with ether (2 × 100 mL), and residual solvent was removed in vacuo. The resulting residue was dissolved in CHCl (400 mL), dried over anhydrous MgSO, filtered, washed with CHCl (100 mL), and concentrated under reduced pressure to give 5-methoxy-1-methyl-3,4-dihydro-2H-pyrrol-1-ium as a brown viscous liquid (solidified upon storage at -20 °C); 1H NMR (400MHz, CDCl3)δ 4.35-4.40(m,3H),3.98-4.05(m,2H),3.69-3.73(m,3H),3.31-3.38(m,2H),3.19-3.22(m,3H),2.37-2.48(m,2H).

[0298] The crude product (obtained above) was added via cannula or dropping funnel to a solution of sodium ethanolate (370 g, 1142.909 mmol, 21% sodium ethoxide in ethanol) over 1 h at 50-55 °C under a N2 atmosphere. After stirring at the same temperature for 3 h, the reaction was cooled to room temperature. The precipitated white solid was filtered and washed with ethanol (50 mL), and the filtrate was concentrated (water bath temperature maintained at approximately 30 °C). The crude residue was fractionally distilled under house vacuum at 55-65 °C to give 2,2-diethoxy-1-methylpyrrolidine (115 g, 66% yield) as a pale yellow or colorless liquid. The pure product was stored at -20 °C. 1 H NMR (400MHz, CDCl3)δ 3.44-3.60(m,4H),2.83-2.91(m,3H),2.33-2.40(m,4H),1.90-1.98(m,2H),1.72-1.87(m,2H),1.15-1.22(m,6H).

[0299] General Procedure 1: Pya (N-methylpyrrolidine) Protection of 2'-O-MOE G, A, and mC [ka] (Compound 2-1) 9-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxy-3-(2-methoxyethoxy)tetrahydrofuran-2-yl)-2-(1-methylpyrrolidin-2-ylidene)amino)-1,9-dihydro-6H-purin-6-one: 2-Amino-9-((2R,3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3-(2-methoxyethoxy)tetrahydrofuran-2-yl)-1,9-dihydro-6H-purin-6-one (13.8 g, 40.431 mmol) was chased twice with anhydrous pyridine (100 mL) under vacuum. To the concentrated residue was added anhydrous pyridine (114 mL, 1418.073 mmol) followed by 2,2-diethoxy-1-methylpyrrolidine (14.01 g, 80.862 mmol) slowly at room temperature. The reaction was stirred overnight at room temperature, changing from a white, cloudy solution to a brown, clear solution. Water (0.1 mL / 6 mmol) was added, and the mixture was concentrated under vacuum and then chased three times with pyridine and MeCN. To the resulting residue, pyridine (105 mL, 1298.197 mmol) and 1-[chloro-(4-methoxyphenyl)-phenylmethyl]-4-methoxybenzene (15.62 g, 46.108 mmol) were added at room temperature. After stirring overnight at room temperature, the reaction mixture was worked up with saturated NaHCO (150 mL) and EtOAc (300 mL × 2). The residue was purified by silica gel column chromatography (100 g Star silica, EtOAc / Hept 30 to 100%, then EtOAc / MeOH 0 to 30%) to give compound 2-1 as a foamy solid in 77% yield. 1 H NMR(400MHz,CDCl3)δ 9.28-9.36(m,1H),7.67-7.72(m,1H),7.32-7.39(m,2H),7.16-7.28(m,6H),7.08-7.16(m ,1H),6.69-6.78(m,4H),5.92-5.96(m,1H),4.29-4.37(m,2H),4.11-4.17(m,1H),3.74-3 .82(m,1H),3.68-3.73(m,7H),3.55-3.63(m,1H),3.45-3.52(m,1H),3.34-3.41(m,3H),3 .25-3.33(m,5H),3.01-3.09(m,2H),2.92-2.96(m,3H),1.90-2.00(m,2H);MS(ESI,m / z)[C 39 H 44 N5O8+H + ] Calculated value: 725.33 Measured value: 725.4

[0300] [ka] (Compound 2-2) (2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(2-methoxyethoxy)-5-(6-1-methylpyrrolidin-2-ylidene)amino)-9H-purin-9-yl)tetrahydrofuran-3-ol: Prepared according to general procedure 1, foamy solid, 89% yield; 1 H NMR(400MHz,DMSO-d6)δ 8.36(d,J=8.0Hz,2H),7.40-7.31(m,2H),7.29-7.16(m,7H),6.87-6.77(m,4H),6.07(d,J=4.8Hz ,1H),5.18(d,J=6.0Hz,1H),4.69(t,J=5.2Hz,1H),4.44(q,J=5.2Hz,1H),4.11-4.05(m,1H),3.76 -3.71(m,7H),3.62(dt,J=11.2,4.8Hz,1H),3.49(t,J=7.2Hz,2H),3.42(t,J=4.8Hz,2H),3.23(d, J=4.8Hz,2H),3.14(s,3H),3.04(s,3H),2.85(t,J=8.0Hz,2H),2.02-1.93(m,2H);MS(ESI,m / z)[C 39 H 44 N6O7+H + ] Calculated value: 709.33 Measured value: 709.20

[0301] [ka] (Compound 2-3) 1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxy-3-(2-methoxyethoxy)tetrahydrofuran-2-yl)-5-methyl-4-(1-methylpyrrolidin-2-ylidene)amino)pyrimidin-2(1H)-one: Prepared according to general procedure 1, 87% yield; foamy solid;1 H NMR(400MHz,CDCl3)δ 7.77-7.81(m,1H),7.46-7.51(m,2H),7.34-7.41(m,4H),7.27-7.33(m,2H),7.20-7.27(m,1H),6.82-6 .88(m,4H),5.99-6.04(m,1H),4.34-4.43(m,1H),4.25-4.33(m,1H),4.08-4.15(m,1H),3.99-4.05(m, 1H),3.90-3.99(m,1H),3.74-3.83(m,6H),3.54-3.64(m,3H),3.42-3.50(m,3H),3.42(s,3H),3.29-3. 34(m,1H),3.07-3.29(m,2H),3.03-3.07(m,3H),2.00-2.11(m,2H),1.53-1.58(m,3H);MS(ESI,m / z)[C 39 H 44 N6O8+H + ] Calculated value: 699.33 Measured value: 699.25

[0302] Pivaloylmethyl (POM) protection of T: [ka] (Compound 2-4) (3-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxy-3-(2-methoxyethoxy)tetrahydrofuran-2-yl)-5-methyl-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl pivalate: Step 1: To 1-((2R,3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3-(2-methoxyethoxy)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (14.2 g, 44.893 mmol) in pyridine (99 mL, 1228.96 mmol) was added 1-[chloro-(4-methoxyphenyl)-phenylmethyl]-4-methoxybenzene (18.25 g, 53.871 mmol) at room temperature. Upon completion as monitored by UPLC-MS, the mixture was added saturated NaHCO3 (80 mL), extracted with EtOAc (200 mL × 2), and purified by silica gel column chromatography (100 g, Star silica, EtOAc / Hept 10 to 100%) to give 1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxy-3-(2-methoxyethoxy)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (25 g, 40.408 mmol) in 90% yield. 1 H NMR(400MHz,DMSO-d6)δ 11.37(s,1H),7.49(s,1H),7.39(d,J=7.6Hz,2H),7.35-7.21(m,8H),6.90(d,J=8.8Hz,4H),5.85(d,J=4.8Hz,1H),5.12(d,J=6.0Hz,1H), 4.23(q,J=5.2Hz,1H),4.09(t,J=4.8Hz,1H),4.02-3.95(m,1H),3.79-3.67(m,8H),3.48(t,J=4.7Hz,2H),3.26-3.20(m,5H),1.40(s,3H).

[0303] Step 2: To an aqueous solution of NaCO (242 mL, 121.225 mmol), 1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxy-3-(2-methoxyethoxy)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (25 g, 40.408 mmol), tetrabutylammonium hydrogen sulfate (5.49 g, 16.163 mmol), and chloromethyl pivalate (7.30 g, 48.49 mmol) in DCM (250 mL, 3885.69 mmol) were added at room temperature. The reaction mixture was stirred at room temperature for 16 hours. UPLC-Mass analysis showed that some starting material remained unreacted, so 700 mg of chloromethyl pivalate was added at room temperature. After stirring at room temperature for an additional 2 days, the mixture was worked up with saturated NaHCO3 (50 mL) and extracted with EtOAc (100 mL × 3) and purified by column chromatography (100 g snap, EtOAc / Hept 10 to 60%) to give (3-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxy-3-(2-methoxyethoxy)tetrahydrofuran-2-yl)-5-methyl-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl pivalate (23 g, 31.4 mmol, 78% yield) along with recovered starting material (3.25 g). 1 H NMR (400 MHz, DMSO-d6) δ 7.62(s,1H),7.40(d,J=7.6Hz,2H),7.36-7.20(m,7H),6.90(d,J=8.8Hz,4H) ,5.89(d,J=4.8Hz,1H),5.84-5.73(m,2H),5.17(d,J=6.0Hz,1H),4.26(q,J= 5.6Hz,1H),4.12(t,J=4.8Hz,1H),4.02-3.98(m,1H),3.78-3.70(m,8H),3.5 1-3.40(m,2H),3.28-3.20(m,5H),1.44(s,3H),1.10(s,9H);MS(ESI,m / z)[C 40 H 48 N2O 11 +Na+ ] Calculated value: 755.32 Measured value: 755.1

[0304] 2'-O-MOE-3'-PSI activated monomer General Procedure 2 1 :PSI activation [ka] (Compound 3-1) (3-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-(2-methoxyethoxy)-4-(((2R,3aS,6R,7aS)-3a-methyl-6-(prop-1-en-2-yl)-2-sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-2-yl)oxy)tetrahydrofuran-2-yl)-5-methyl-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl pivalate: (2S,3aS,6R,7aS)-3a-methyl-2-((perfluorophenyl)thio)-6-(prop-1-en-2-yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide (3.70 g, 8.29 mmol) ((-)-PSI reagent) and (3-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl (4.50 g, 6.141 mmol) was dissolved in THF (20.47 mL, 6.141 mmol) and acetonitrile (20.47 mL, 6.141 mmol), and the solution was cooled in an ice bath. DBU (1.203 mL, 7.983 mmol) was added to the mixture, which was stirred at 0°C until the reaction was complete (0.5-2 h), as monitored by UPLC-MS. The reaction mixture was diluted with EtOAc, washed with saturated NaHPO (aq) solution, then saturated NaHCO (aq), dried over NaSO, and purified by silica gel chromatography (50 g Star, Hept: EtOAc gradient up to 70%) to give 3-1 as a white solid (5.3 g, 88% yield). 1H NMR(400MHz,CD3CN)δ ppm 7.46-7.54(3H,m),7.33-7.39(6H,m),7.26-7.32(1H,m),6.91(4H,d,J=8.75Hz), 5.97(1H,d,J=6.38Hz),5.86-5.93(2H,m),5.45-5.52(1H,m),5.02(1H,s),4.93(1 H,s),4.45-4.54(2H,m),4.26(1H,d,J=2.88Hz),3.77-3.84(8H,m),3.47-3.62(2 H,m),3.42(1H,dd,J=11.01,2.88Hz),3.29-3.33(1H,m),3.28(3H,s),2.64(1H,br s),2.25-2.32(1H,m),2.12-2.14(3H,m),2.07(1H,br dd,J=13.70,4.44Hz),1.99-1.99(1H,m),1.81-1.95(2H,m),1.80(3H,s),1.69(3H,s),1.44(3H,s),1.18(9H,s); 31 P NMR(162MHz,CD3CN)δ ppm 101.69;MS(ESI,m / z)[C 50 H 63 N2O 12 PS2+Na + ] Calculated value: 1001.35 Actual value: 1001.4

[0305] [ka] (Compound 3-2) (2R,3aS,6R,7aS)-2-(((2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(2-methoxyethoxy)-5-(6-(((E)-1-methylpyrrolidin-2-ylidene)amino)-9H-purin-9-yl)tetrahydrofuran-3-yl)oxy)-3a-methyl-6-(prop-1-en-2-yl)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide: Prepared according to general procedure 2 with (-)-PSI reagent, 78% yield; foamy solid; 1H NMR(400MHz, CDCl3)δ 8.40-8.46(m,1H),8.00-8.03(m,1H),7.36-7.41(m,2H),7.24-7.30(m,4H),7.17-7.22(m,2H),7.08-7.16(m,1H),6.69-6.78(m, 4H),6.05(d,J=7.5Hz,1H),5.45-5.59(m,1H),5.04(dd,J=7.5,4.7Hz,1H),4.95(s,1H),4.78-4.93(m,1H),4.50(dt,J=12.6,3.3H z,1H),4.27-4.33(m,1H),3.59-3.79(m,10H),3.31-3.46(m,5H),3.10-3.15(m,3H),3.06-3.10(m,3H),2.83-2.97(m,2H),2.47-2 13C NMR(101MHz,CDCl3)δ 166.9,160.9,158.6,158.5,152.8,151.6,144.8,144.5,140.1,135.6,135.6,130.2,130.1,128.2,128.0,126.9,126.6,113.3,112.2,86.8,85.4,85.4, 83.6,83.5,80.1,80.0,77.3,76.9,72.3,70.6,68.0,65.7,63.0,58.9,55.2,51.6,38.9,33.7,33.7,32.0,30.1,27.8,27.6,25.6,23.5,22.7,21.8,19.7; 31 P NMR (162MHz, CDCl3) δ 101.34; MS (ESI, m / z) [C 49 H 59 N6O8PS2+H + ]としてのcalculated value is 955.36 and measured value is 956.3.

[0306]

change

[0307] [ka] (Compound 3-4) 9-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-(2-methoxyethoxy)-4-(((2R,3aS,6R,7aS)-3a-methyl-6-(prop-1-en-2-yl)-2-sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-2-yl)oxy)tetrahydrofuran-2-yl)-2-((-1-methylpyrrolidin-2-ylidene)amino)-1,9-dihydro-6H-purin-6-one: Prepared according to general procedure 2 with (-)-PSI reagent, foamy solid, 80% yield; 1 H NMR(400MHz,CD3CN)δ ppm 9.18(1H,br s),7.74(1H,s),7.43(2H,d,J=7.38Hz),7.22-7.33(7H,m),6.85(4H,dd,J=9.01,2.63Hz),5.89(1 H,d,J=5.50Hz),5.48(1H,dt,J=13.54,4.80Hz),4.98(1H,s),4.92(1H,s),4.85(1H,t,J=5.38Hz), 4.50(1H,dt,J=12.69,3.22Hz),4.23(1H,q,J=4.09Hz),3.79(6H,s),3.67-3.77(2H,m),3.43-3.5 1(4H,m),3.32(2H,qd,J=10.94,4.06Hz),3.21(3H,s),3.03(3H,s),2.99-3.02(1H,m),2.63(1H,br s),2.27(1H,br d,J=13.13Hz),2.12-2.15(1H,m),2.01-2.06(1H,m),1.99-1.99(4H,m),1.79-1.93(2H,m),1.77(3H,s),1.68(3H,s); 31 P NMR(162MHz,CD3CN)δ ppm 101.36;MS(ESI,m / z)[C 49 H 59 N6O9PS2+H + ] Calculated value: 971.35 Measured value: 971.4

[0308] [ka] (Compound 3-5) 9-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-(2-methoxyethoxy)-4-(((2S,3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2-yl)-2-sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-2-yl)oxy)tetrahydrofuran-2-yl)-2-((1-methylpyrrolidin-2-ylidene)amino)-1,9-dihydro-6H-purin-6-one: A white foamy solid prepared according to general procedure 2 with (+)-PSI reagent; 1 H NMR(400MHz,CD3CN,296K)δ(ppm)=9.56(br s,1H),7.74(s,1H),7.44(d,J=7.5Hz,2H),7.34-7.28(m,6H),7.28-7.21(m,1H),6.86(dd,J=2. 4,8.9Hz,4H),5.89(d,J=6.4Hz,1H),5.44-5.36(m,1H),4.99(s,1H),4.89(s,1H),4.78(t,J=5. 8Hz,1H),4.45(td,J=3.0,12.7Hz,1H),4.27(q,J=3.9Hz,1H),3.78(s,6H),3.75-3.70(m,1H),3 .67-3.57(m,1H),3.47-3.40(m,2H),3.39-3.32(m,4H),3.12(s,3H),3.09-2.92(m,5H),2.63(br s,1H),2.30-2.15(m,2H),2.04(br dd,J=4.0,12.9Hz,1H),2.00-1.90(m,4H),1.82(br s,1H),1.79-1.75(m,3H),1.68(s,3H); 13C NMR (101 MHz, CDCN, 298 K)δ(ppm)=170.8,160.1,159.3,158.3,152.1,147.2,146.2,137.9,136 .9,136.9,131.5,131.4,129.4,129.3,128.3,114.5,112.4,87.9,87.6 ,87.3,83.5,83.4,81.8,78.2,78.1,73.1,72.3,66.4,64.4,59.4,56.3 ,52.4,40.2,34.9,34.8,32.5,32.4,28.7,28.6,24.2,23.2,22.3,20.8; 31 P NMR(162MHz,CD3CN)δ 101.9;MS(ESI,m / z)[C 49 H 59 N6O9PS2+H + ] Calculated value: 971.35 Measured value: 971.1

[0309] [ka] (Compound 3-6) 1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-(2-methoxyethoxy)-4-(((2S,3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2-yl)-2-sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-2-yl)oxy)tetrahydrofuran-2-yl)-5-methyl-4-(((E)-1-methylpyrrolidin-2-ylidene)amino)pyrimidin-2(1H)-one: a white foamy solid prepared according to general procedure 2 with (+)-PSI reagent; 1H NMR(400MHz,CD3CN,296 K)δ(ppm)=7.57(s,1H),7.50(d,J=7.5Hz,2H),7.40-7.31(m,6H),7.31-7.23(m,1H),6.90(d,J=8.9Hz,4H),6.05(d,J= 5.8Hz,1H),5.49-5.40(m,1H),4.99(s,1H),4.88(s,1H),4.43(td,J=3.1,12.6Hz,1H),4.34(t,J=5.4Hz,1H),4.26(br d,J=3.4Hz,1H),3.85-3.72(m,8H),3.54-3.45(m,4H),3.38(d,J=2.8Hz,2H),3.26(s,3H),3.11-3.05(m,2H),3.03(s,4H),2.62(br s,1H),2.22(br d,J=12.3Hz,1H),2.13-2.01(m,3H),2.01-1.92(m,2H),1.92-1.79(m,2H),1.76(s,3H),1.68(s,3H),1.56(s,3H); 13 C NMR (101MHz, CD3CN, 298) K)δ(ppm)=172.6,170.0,160.2,147.2,146.1,138.4,137.0,136.8,131.5,131.5,129.5,129.4,128.4,114.6,112.5,88.7,88.2,87.8,82. 9,82.9,82.3,82.2,77.7,77.6,73.3,71.8,66.7,63.9,59.5,56.3,5 2.5,40.2,34.9,34.8,32.4,31.8,28.7,28.6,24.2,23.2,22.3,20.8; 31 P NMR(162MHz,CD3CN)δ 101.8;MS(ESI,m / z)[C 49 H 61 N4O9PS2+H + ]としてのcalculated value is 945.36 and measured value is 946.5.

[0310]

change

[0311] [ka] (Compound 3-8) (3-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-(2-methoxyethoxy)-4-(((2S,3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2-yl)-2-sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-2-yl)oxy)tetrahydrofuran-2-yl)-5-methyl-2,6-dioxo-3,6-dihydropyrimidin-1(2H)-yl)methyl pivalate: A foamy solid prepared according to general procedure 2 with (+)-PSI reagent; 1H NMR(400MHz,CD3CN)δ ppm 7.53(1H,s),7.48(2H,d,J=7.63Hz),7.33-7.39(6H,m),7.26-7.32(1H,m),6.92(4H,d,J=8.76Hz),6.01(1H,d,J=6.88H z),5.86-5.92(2H,m),5.45(1H,ddd,J=11.60,4.78,2.75Hz),5.01(1H,s),4.90(1H,s),4.44-4.49(2H,m),4.29(1H,br d,J=2.63Hz),3.80(6H,s),3.76-3.79(1H,m),3.32-3.54(4H,m),3.23(3H,s),2.60-2.66(1H,m),2.24(2H,br d,J=12.76Hz),2.07(1H,br dd,J=13.01,3.75Hz),1.99-2.01(2H,m),1.80-1.92(2H,m),1.78(3H,s),1.68(3H,s),1.46(3H,s),1.18(9H,s); 31 P NMR(162MHz,CD3CN)δ ppm 102.18;MS(ESI,m / z)[C 50 H 63 N2O 12 PS2+Na + ] Calculated value: 1001.35 Actual value: 1001.1

[0312] General Procedure 3: PO-PSI Monomer to PS-PSI Monomer [ka] (Compound 4-1) N-(9-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-(2-methoxyethoxy)-4-(((2S,3aR,6S,7aR)-3a-methyl-2-oxido-6-(prop-1-en-2-yl)hexahydrobenzo[d][1,3,2]oxathiaphosphol-2-yl)oxy)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide: To N-(9-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-(2-methoxyethoxy)-4-(((2S,3aR,6S,7aR)-3a-methyl-6-(prop-1-en-2-yl)-2-sulfidohexahydrobenzo[d][1,3,2]oxathiaphosphol-2-yl)oxy)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide (1 g, 1.042 mmol) in MeCN (15.00 mL, 15 vol) was added SeO (1.0 equiv, 0.116 g, 1.042 mmol) in an ice bath. Additional SeO2 (0.116 g, 1.042 mmol) was added at 0 °C until the reaction was complete. A total of 3 equivalents of SeO2 were used. After completion, as monitored by UPLC-MS, the mixture was filtered through Celite and dry SiO2 (EtOAc / THF). The filtrate was washed with saturated NaHCO3 (10 mL), dried over Na2SO4, filtered (dry SiO2), and concentrated. The residue was purified by silica gel column chromatography (50 g, Hept / EtOAc, 20 to 100%, then EtOAc / THF 0 to 100%) to give 4-1 (0.55 g, 56% yield). MS (ESI, m / z) [C 48 H 58 N5O 11 PS-H + ] Calculated value: 942.36 Measured value: 942.53

[0313] [ka] (Compound 4-2) N-(1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-(2-methoxyethoxy)-4-(((2R,3aS,6R,7aS)-3a-methyl-2-oxido-6-(prop-1-en-2-yl)hexahydrobenzo[d][1,3,2]oxathiaphosphol-2-yl)oxy)tetrahydrofuran-2-yl)-5-methyl-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide: Prepared according to general procedure 3, white foamy solid; 59% yield; 31 P NMR (162 MHz, acetonitrile-d3) δ 40.36; MS (ESI, m / z) [C 51 H 58 N3O 11 PS+H + ] Calculated value: 952.35 Measured value: 952.35.

[0314] [ka] (Compound 4-3) 1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-(2-methoxyethoxy)-4-(((2R,3aS,6R,7aS)-3a-methyl-2-oxido-6-(prop-1-en-2-yl)hexahydrobenzo[d][1,3,2]oxathiaphosphol-2-yl)oxy)tetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione: Prepared according to general procedure 3, white foamy solid; 46% yield; 31 P NMR (162 MHz, acetonitrile-d) δ 40.42; MS (ESI, m / z) [C 44 H 53 N2O 11 PS+Na + ] Calculated value: 871.30 Measured value: 871.28

[0315] PO-PSI Reagent from Cyclohexyl Epoxide: [ka] (Compound 5) rac-2-((4-bromophenyl)thio)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide: A solution of triethylamine bis(4-bromophenyl)phosphorotetrathioate (50.0 g, 87.2 mmol) and cyclohexene oxide (13.2 mL, 131 mmol) in chloroform (175 mL) was treated with dibutyl phosphate (16.2 mL, 87.2 mmol) and dichloroacetic acid (10.8 mL, 131 mmol). After stirring at room temperature for 15 hours, the mixture was concentrated in vacuo. The residue was diluted with water (125 mL) and n-heptane (125 mL), cooled in an ice bath, and stirred at 0 °C for 2 hours. The resulting precipitate was filtered and subsequently washed with water (100 mL) and n-heptane (125 mL). The filter cake was dissolved in CHCl (200 mL), and the aqueous layer was removed. The organic layer was concentrated in vacuo to approximately 50 mL and treated with n-heptane (75 mL). The mixture was stirred at room temperature for 20 minutes and concentrated in vacuo to approximately 50 mL. The resulting precipitate was filtered, washed with n-heptane (20 mL), and dried under N2 purging for 2 hours to give the title compound (30.1 g, 91%). 1 H NMR(400MHz,CDCl3,296K)(a 1:2 mixture of diastereomers) δ(ppm)=7.58-7.51(m,8H),7.47-7.41(m,4H),4.04(dt,J=3.9,10.7Hz,1H),3.65-3.56(m,4H),2.27 -2.12(m,6H),1.89(m,3H),1.81(m,3H),1.75-1.58(m,3H),1.49-1.25(m,8H),1.23-1.16(m,1H),1.07-0.86(m,1H); 31 P NMR(162MHz,CDCl3,296 K)δ(ppm)=107.01(s,1P),103.23(s,2P);MS(ESI)m / z:[M+H] + C 12 H 15 Calculated for BrOPS3: 380.91; found: 380.84.

[0316] [ka] (Compound 6) rac-2-((4-bromophenyl)thio)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-oxide: A solution of (3aR,7aR)-2-((4-bromophenyl)thio)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-sulfide (10.0 g, 26.2 mmol) in CHCl (170 mL) was treated with SeO (2.91 g, 26.2 mmol) and stirred at room temperature for 2 h. Additional SeO (2.91 g, 26.2 mmol) was added, and stirring was continued at room temperature for an additional 19 h. The reaction mixture was filtered through a pad of dry silica gel and rinsed with CHCl. ​​The filtrate was washed with 10% NaHPO (70.0 mL), dried over MgSO, and concentrated in vacuo. The residue was treated with n-heptane (46 mL), and the resulting slurry was stirred at room temperature for 20 min. The precipitate was filtered, washed with n-heptane (20 mL), and dried with a N2 purge to give the title compound (6.18 g, 64.5%). 1 H NMR (400 MHz, CDCl3, 296 K) (ca. 1:2 mixture of two diastereomers) δ (ppm) = 7.58-7.48 (m, 12H), 4.10 (dt, J = 4.1, 10.8 Hz, 1H), 3.60 (dt, J = 3.6, 10.8 Hz, 2H), 3.37 (dt, J = 3.9, 10.8 Hz, 2H), 2.43-2.36 (m, 1H), 2.25-2.07 (m, 5H), 1.98-1.83 (m, 4H), 1.83-1.73 (m, 3H), 1.63-1.48 (m, 3H), 1.46-1.23 (m, 8H), 1.11-0.99 (m, 1H); 31 P NMR(162MHz,CDCl3,297 K)δ(ppm)=62.54(s,1P),56.98(s,2P);MS(ESI)m / z:[M+H] + C 12 H 15 Calculated for BrO2PS2: 364.94; Found: 364.97.

[0317] General Procedure 4: PO-PSI Monomer [ka] (Compound 7-1) 1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-(2-methoxyethoxy)-4-(((3aR,7aR)-2-oxidohexahydrobenzo[d][1,3,2]oxathiaphosphol-2-yl)oxy)tetrahydrofuran-2-yl)-5-methyl-4-(((E)-1-methylpyrrolidin-2-ylidene)amino)pyrimidin-2(1H)-one: 1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxy-3-(2-methoxyethoxy)tetrahydrofuran-2-yl)-5-methyl-4-(((E)-1-methylpyrrolidin-2-ylidene)amino)pyrimidin-2(1H)-one (4.30 g, 6.15 mmol) and (3aR,7aR)-2-((4-bromophenyl)thio)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-oxide (3.15 g, 8.62 mmol) were azeotroped three times with acetonitrile (43 mL). The residue was dissolved in acetonitrile (43 mL), cooled to 0 °C, and treated with DBU (1.6 mL, 8.3 mmol). The mixture was stirred at 0° C. for 2 hours, quenched with saturated NaHPO (40 mL), and diluted with ethyl acetate (50 mL). The organic layer was separated, and the aqueous layer was extracted twice with ethyl acetate (50 mL). The organic layers were combined, washed with saturated NaHCO (20 mL), dried over MgSO, and concentrated in vacuo. The residue was purified by silica gel column chromatography (ethyl acetate in n-heptane = 17% to 100% and then THF in ethyl acetate = 0% to 100%) to give the title compound (3.07 g, 57.1%) as a foamy solid. MS(ESI) m / z:[M+H] + C 45 H 56 N4O 10 Calculated PS value 875.3; measured value 875.1.

[0318] [ka] (Compound 7-2) (3aR,7aR)-2-(((2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(2-methoxyethoxy)-5-(6-(((E)-1-methylpyrrolidin-2-ylidene)amino)-9H-purin-9-yl)tetrahydrofuran-3-yl)oxy)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-oxide: (2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(2-methoxyethoxy)-5-(6-(((E)-1-methylpyrrolidin-2-ylidene)amino)-9H-purin-9-yl)tetrahydrofuran-3-ol (2.70 g, 3.81 mmol) and (3aR,7aR)-2-((4-bromophenyl)thio)hexahydrobenzo[d][1,3,2]oxathiaphosphole 2-oxide (1.95 g, 5.33 mmol) were azeotroped three times with acetonitrile (25.4 mL) on a rotary evaporator. The residue was dissolved in acetonitrile (25.4 mL), cooled to 0 °C, and treated with DBU (0.78 mL, 5.1 mmol). The mixture was stirred at 0° C. for 2 hours, quenched with saturated NaHPO (30 mL), and diluted with ethyl acetate (30 mL). The organic layer was separated, and the aqueous layer was extracted twice with ethyl acetate (30 mL). The organic layers were combined, washed with saturated NaHCO (20 mL), dried over MgSO, and concentrated in vacuo. The residue was purified by column chromatography (ethyl acetate in n-heptane = 17% to 100%, then THF in ethyl acetate = 0% to 100%) to give the title compound (2.10 g, 62.3%) as a foamy solid. MS(ESI) m / z:[M+H] + C 45 H 54 Calculated value for N6O9PS: 884.33. Measured value: 884.45.

[0319] [ka] (Compound 7-3) 9-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-(2-methoxyethoxy)-4-(((3aR,7aR)-2-oxidohexahydrobenzo[d][1,3,2]oxathiaphosphol-2-yl)oxy)tetrahydrofuran-2-yl)-2-((1-methylpyrrolidin-2-ylidene)amino)-1,9-dihydro-6H-purin-6-one: Prepared according to general procedure 4, white foamy solid; 80% yield; MS (ESI, m / z) [C 45 H 53 NO 10 PS+H + ] Calculated value: 901.33 Measured value: 901.1

[0320] General Procedure 5: Synthesis of succinic acid monomers To the protected nucleoside (1.0 equiv.) and succinic anhydride (1.5 equiv.) were added DCM (8 vol.) and EtN (3.0 equiv.) at room temperature. The mixture was stirred overnight at room temperature. Phosphate buffer (pH 7, 6 vol.) was added to the mixture, and the mixture was extracted three times with DCM (8 vol.). The organic layer was then concentrated and purified by column chromatography (heptane / EtOAc, 10 to 100%).

[0321] (5-Methyl succinate-C-MOE): [ka] (Compound 8) 4-(((2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(2-methoxyethoxy)-5-(5-methyl-4-(((E)-1-methylpyrrolidin-2-ylidene)amino)-2-oxopyrimidin-1(2H)-yl)tetrahydrofuran-3-yl)oxy)-4-oxobutanoic acid: To 1-((2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-hydroxy-3-(2-methoxyethoxy)tetrahydrofuran-2-yl)-5-methyl-4-(((E)-1-methylpyrrolidin-2-ylidene)amino)pyrimidin-2(1H)-one (5 g, 7.155 mmol) and succinic anhydride (1.074 g, 10.732 mmol) in DCM (40.0 mL, 621.71 mmol) was added EtN (2.99 mL, 21.465 mmol) at room temperature. The mixture was stirred at room temperature overnight. To the mixture was added phosphate buffer (pH 7, 30 mL) and extracted with DCM (50 mL × 3). The organic layer was then concentrated and purified by column chromatography (Hept / EtOAc, 10 to 100%) to give 4-(((2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(2-methoxyethoxy)-5-(5-methyl-4-(((E)-1-methylpyrrolidin-2-ylidene)amino)-2-oxopyrimidin-1(2H)-yl)tetrahydrofuran-3-yl)oxy)-4-oxobutanoic acid (4.92 g, 6.16 mmol, 86% yield). 1 H NMR(400MHz,CD3CN)δ ppm 7.59(1H,s),7.47(2H,d,J=7.50Hz),7.31-7.39(6H,m),7.24-7.31(1H,m),6.91(4H,d,J=8.63Hz),6.04 (1H,d,J=5.25Hz),5.35(1H,t,J=5.13Hz),4.35(1H,t,J=5.32Hz),4.14-4.25(1H,m),3.79(6H,s),3.74- 3.78(1H,m),3.66(1H,dt,J=11.44,4.28Hz),3.44-3.52(4H,m),3.33-3.40(2H,m),3.26(3H,s),3.05-3 .11(2H,m),3.04(3H,s),2.50-2.65(4H,m),2.00-2.08(2H,m),1.99(1H,s),1.61(3H,s);MS(ESI,m / z)[C 43 H 50 N4O 11 +H +] Calculated value 799.35; measured value 799.9.

[0322] General Procedure 6: Solid-phase synthesis of stereocontrolled PS MOE ASOs A general procedure for the automated solid-phase synthesis of stereocontrolled PS-oligonucleotides was modified from previously reported procedures in Knouse et al., “Unlocking P(V):Reagents for chiral phosphorothioate synthesis,” Science 2018,361(6408),1234-1238; and Huang et al., “AP(V) platform for oligonucleotide synthesis,” Science 2021,373(6560),1265-1270.

[0323] Automated solid-phase oligonucleotide synthesis: Part 1. Loading onto the resin: Preparation of 1mer [ka] TentaGel S-NH2 (AC354610050, ACROS Organics, loading 0.2-0.3 mmol / g) (4 g, approximately 1 mmol) was placed in a 50 mL solid-phase reaction flask and washed with DMF (10 mL × 3), DCM (10 mL × 3), and DMF (10 mL × 3). To this resin, N-(((9H-fluoren-9-yl)methoxy)carbonyl)-N-methylglycine (3.11 g, 10.00 mmol) in DMF (5.00 mL) and ((3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)oxy)tri(pyrrolidin-1-yl)phosphonium hexafluorophosphate (V) (5.21 g, 10.00 mmol) in DMF (5 mL) were added at room temperature, followed by N-4-methylmorpholine (2199 mL, 20.00 mmol). The mixture was shaken at 400 rpm. After 24 h, the liquid was drained, and the resin was rinsed with DMF (10 mL × 3), DCM (10 mL × 3), and DMF (10 mL × 3). To this resin was added premixed pyridine (4.85 mL, 60.00 mmol) and AcO (0.944 mL, 10.00 mmol) at room temperature. After 3 minutes, the solution was drained, and premixed pyridine (4.85 mL, 60.00 mmol) and AcO (0.944 mL, 10.00 mmol) were added at room temperature. After 3 minutes, the liquid was drained, and the resin was washed with DMF (10 mL x 3), DCM (10 mL x 3), and DMF (10 mL x 3).

[0324] The resin was then treated with 30 mL of 20% piperidine in DMF, and the solution was collected after 3 minutes. This procedure was repeated five times, and the resin was washed with DMF (10 mL x 3), DCM (10 mL x 3), and DMF (10 mL x 3). In a volumetric flask, the collected solution was made up to 300 mL with a solution of 20% piperidine in DMF. An aliquot of this solution was diluted 10-fold with 20% piperidine in DMF, and the UV absorbance of the piperidine-fulvene adduct was measured (λ = 301 nm, ε = 7800 nm). -1 cm -1 , A = 2.41), and an estimated loading of 230 μmol / g was obtained.

[0325] The resin was washed with DMF (10 mL × 3), DCM (10 mL × 3), and DMF (10 mL × 3). The resin was then added with 4-(((2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(2-methoxyethoxy)-5-(5-methyl-4-(((E)-1-methylpyrrolidin-2-ylidene)amino)-2-oxopyrimidin-1(2H)-yl)tetrahydrofuran-3-yl)oxy)-4-oxobutanoate in DMF (5 mL). To the resulting solution was added (3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)oxy)tri(pyrrolidin-1-yl)phosphonium hexafluorophosphate (V) (1.7 equiv., 0.886 g, 1.7 mmol) and N-4-methylmorpholine (2 equiv., 0.258 g, 2 mmol) in DMF (5 mL). The mixture was shaken at room temperature for 3 days and washed with DMF (10 mL × 3), DCM (10 mL × 3), and DMF (10 mL × 3).

[0326] The resin was washed with DMF (10 mL x 3), DCM (10 mL x 3). The DMTr group was removed by treating it with 3% dichloroacetic acid (DCA) in DCM (20 mL) for 2 minutes, followed by a DCM (20 mL) wash. This procedure was repeated (more than 5 times) until no color was observed. The resin was then washed with DCM (10 mL x 3), DMF (10 mL x 3), and MeCN (10 mL x 3).

[0327] The combined deprotection solution was diluted with 3% DCA in DCM. The UV absorbance of the DMTr cation was measured (λ = 410 nm, ε = 30,400 M). -1 cm -1 ), and the loading was quantified (0.2 mmol / g).

[0328] Part 2. Automated synthesis on the K&A H-8-SE oligosynthesizer The prepared 5'-O-DMTr-nucleotide-loaded TentaGel-SAR (20 μmol, 200 μmol / g) was loaded into an empty 6 mL syringe column (Biocomma Limited, catalog number RSSC-6) and washed with MeCN. Stereopure oligonucleotides were synthesized on a K&A H-8-SE oligosynthesizer using stereopure PSI and PO-PSI monomers after the cycles shown in Table 23. As shown in the following scheme: Sp phosphorothioate linkages were obtained using Rp-PSI-monomers prepared from (-)-PSI reagent; Rp phosphorothioate linkages were obtained using Sp-PSI-monomers synthesized from (+)-PSI, and PO internucleotide linkages were obtained using PO-PSI-monomers. 1 . [ka]

[0329] Monomers in the synthesis of Sp, Rp phosphorothioate and PO (phosphodiester) internucleotide linkages.

[0330] [Table 36]

[0331] Analytical HPLC Method 1 - RP HPLC - Mass: Column: Acquity UPLC BEH C18 1.7 μm 2.1 × 50 mm (Part Number: 186002350); Solvent: Buffer A (10 mM ammonium bicarbonate in water), Buffer B (100 mM ammonium bicarbonate / MeOH / MeCN = 10 / 10 / 80); Temperature: 60 °C; Flow Rate: 0.8 mL / min; Gradient: 5 to 99% B gradient (6 min).

[0332] Part 3. Cleavage from the resin and deprotection: After the final cycle (DMTr-on), the resin in cleavage solution (28% NH4OH / NH4OAc / EtOH (10 / 1 / 1, approximately 1 mL / 1 μmol) was heated at 65 °C for 2 days in a sealed bottle. It was cooled to room temperature, filtered, and then concentrated. The failed sequences were removed, and the DMTr group was deprotected using the following C18 cartridge protocol. The collected fractions were concentrated and purified by ion-pairing reversed-phase (IR-RP) HPLC.

[0333] C18 Column Protocol: A Sep-Pak cartridge [Waters, Sep-Pak Vac 35 cc (10 g) C18 cartridge] was equilibrated with MeOH (2 CV), MeCN (2 CV), followed by 2N Et3NHOAc (2 column volumes (CV)). The crude sample in 0.1N Et3NHOAc was loaded onto the cartridge. The truncated sequence was eluted by washing the cartridge with 2N NaCl / MeCN (5 / 1, v / v), 3% TFA in water (150 mL), and then water (50 mL). The crude DMTr-off PS-oligonucleotide was eluted with 50 mL of acetonitrile-water (1:1, v / v) containing 0.5% of 28% NH4OH. The solution containing the DMTr-off oligonucleotide was dried under vacuum. The weight was measured using a Nanodrop (RNA-40). 31 P NMR was taken and analyzed by RP-HPLC, IEX-HPLC and UPLC / MS.

[0334] Analytical HPLC Method 2 - Ion Pairing RP HPLC - Mass: Column: XBridge Premier BEH C18 (2.5 μm, 150 × 2.1 mm); Temperature: 60 °C; Flow rate: 1 mL / min; Detection wavelength: 260 nm; Solvents: Buffer A: 100 mM HFIP / 8.6 mM EtN(HO), Buffer B: 100% MeOH; Gradient: 5% to 30% B gradient (15 min).

[0335] Analytical HPLC Method 3 - Ion Pairing RP HPLC - Mass: Column: XBridge Premier BEH C18 (300 Å, 2.5 μm, 150 × 2.1 mm); Temperature: 60 °C; Flow Rate: 0.5 mL / min; Detection Wavelength: 260 nm; Solvent: Buffer A: 100 mM n-C6H 13 NH3OAc(H2O / MeCN 9 / 1) Buffer B: 100mM C6H 13 NH3OAc (HO / MeCN 1 / 1); Gradient: 80% to 100% B gradient (15 min).

[0336] Analytical HPLC Method 4 - Ion Pairing RP HPLC - Mass: Column: XBridge Premier BEH C18 (300 Å, 2.5 μm, 150 × 2.1 mm); Temperature: 60 °C, Flow Rate: 0.5 mL / min. Detection Wavelength: 260 nm; Solvents: Buffer A: 10 mM n-hexylamine / 50 mM HFIP in water, Buffer B: MeCN; Gradient: 23 to 28% Buffer B gradient (15 min).

[0337] Part 4. HPLC purification and desalting: The crude material after SepPak treatment was purified by ion-pairing RP HPLC using sterile water (WFI from Baxter, VWR, catalogue 68000-955) according to the following method.

[0338] Preparative HPLC method 1: Column: XBridge Prep C18 OBD Prep (10 μm, 19 x 250 mm); flow rate: 30 mL / min. Detection wavelength: 260nm; Solvent: Buffer A: 8.6mM TEA / 100mM HFIP in water, Buffer B: MeOH; Gradient: 10-37% Buffer B gradient (30 min).

[0339] Preparative HPLC Method 2: Column: Xbridge BEH C18 (10 μm, 10 × 250 mm); Flow rate: 14 mL / min. Detection wavelength: 260 nm; Solvent: Buffer A: 100 mM C6H 13 NH3OAc(H2O / MeCN 9 / 1), Buffer B: 100mM C6H 13NH3OAc (H2O / MeCN 1 / 1); Gradient: 50% to 75% gradient (26 min)

[0340] Preparative HPLC method 3: Column: XBridge C18 OBD Prep (300 Å, 5 μm, 19 × 250 mm); flow rate: 30 mL / min; detection wavelength: 260 nm; solvent: buffer A: 10 mM HA / 50 mM HFIP in water, buffer B: MeCN; gradient: 23–28% buffer B gradient (30 min).

[0341] Fractions containing the desired compound were concentrated and dissolved in 0.2 N NaCl in EtOH / water (1 / 4). The resulting solution was desalted by membrane filtration using a 3000 MW cutoff (3K centrifuge membrane tube, Amicon Ultra-15, Ultracel-3K (3400 rpm, 45 min) (Sigma-Aldrich catalog UFC900396) or Macrosep Devices (PALL catalog MAP003C38), 3400 rpm, 40 min, 15 mL WFI x 3). The final desalted solution was filtered (0.2 micron sterile syringe filter). The absorbance of the diluted solution was measured at 260 nm on a Nanodrop UV-Vis spectrophotometer to determine the yield (7-15% yield), and the endotoxin level was confirmed to be less than 0.06 EU / mg by the kinetic chromogenic LAL method (Charles River, Endosafe® nexgen-PTS).

[0342] Part 5. Reverse complementary RNA and Tm measurement by NMR Tm measurement device: Shimadzu UV-2700 UV-visible spectrophotometer Protocol 1: ASO samples were prepared at a concentration of 400 μM using deionized water. IDT reverse complementary RNA (rcRNA) was dissolved in ultrapure distilled water to a concentration of 400 μM. A 10 μL aliquot of each stock solution was diluted to 1 mL using ultrapure distilled water, and its actual concentration was measured using a UV-visible spectrophotometer. Test samples (500 μL) containing 4.0 μM ASO with 4.0 μM rcRNA in a buffer solution (100 mM NaCl, 10 mM Na phosphate pH 7.0, 0.1 mM EDTA) were prepared. Test samples were incubated in 1 mL cuvettes and heated from 15°C to 105°C at 0.5°C / min. The increase in UV absorbance due to strand melting was monitored at 260 nm. Prior to this experiment, samples were melted and reannealed by heating from 25°C to 95°C at 5°C / min and cooling to the starting temperature to ensure complete annealing. Shimadzu Tm analysis software was used to calculate the Tm using the derivative (curve inflection point: 50% melting).

[0343] Protocol 2: Prepare ASO samples at a concentration of 200 μM using PBS, then follow the same procedure as in Protocol 1 with adjusted volumes.

[0344] In stock phosphate buffer (100 mM, pD = 7.4) prepared with 135.5 mg KDPO and 31.2 mg KDPO in 10 mL D0 after C18 purification and DMTr deprotection. 31 P NMR (162MHz) 3 See Evstigneev et al., “Hexamer oligonucleotide topology and assembly under solution phase NMR and theoretical modeling scrutiny,” Biopolymers 2010, 93(12), 1023-1038.

[0345] Exemplary Compounds All nucleotides are 2'-MOE unless specified, and "C" represents 5'-methylcytosine.

[0346] A. Compound MOE-277:20mer, all Sp [ka] Purified by preparative HPLC method 1:C 260 H 372 N 83 O 133 P 19 S 19 Mw=7985.47 was used as the most abundant natural isotope [M-4H] 4- The theoretical ion value was m / z 1995.36, but it was detected by low-resolution mass spectrometry at m / z 1995.05; Tm = 57.8°C by Protocol 1 (Tm = 66.5°C with sterically random). The Tm of MOE-277 is shown in Figure 12.

[0347] B. Compound MOE-278:20mer, all Rp [ka] Purified by preparative HPLC method 2: C 260 H 372 N 83 O 133 P 19 S 19 Mw=7985.47 was used as the most abundant natural isotope [M-4H] 4- The theoretical ion value was m / z 1995.36, but it was detected by low-resolution mass spectrometry at m / z 1995.492; Tm = 71.5°C by Protocol 1 (Tm = 66.5°C for sterically random). The Tm of MOE-278 is shown in Figure 12.

[0348] C. Compound MOE-279:20mer, 4Rp [ka] Purified by preparative HPLC method 1:C 260 H 372 N 83 O 133 P 19 S 19 Mw=7985.47 was used as the most abundant natural isotope [M-4H] 4- The theoretical ion mass was m / z 1995.36, but was detected by low-resolution mass spectrometry at m / z 1995.25. Tm = 61.4°C by Protocol 1 (Tm = 66.5°C for sterically random).

[0349] D. Compound MOE-280:20mer, 5Rp [ka] Purified by preparative HPLC method 1:C 260 H 372 N 83 O 133 P 19 S 19 Mw=7985.47 was used as the most abundant natural isotope [M-4H] 4- The theoretical ion mass was m / z 1995.36, but was detected by low-resolution mass spectrometry at m / z 1994.88. Tm = 62.7°C by Protocol 1 (Tm = 66.5°C for sterically random).

[0350] E. Compound MOE-281:20mer, 7Rp [ka] Purified by preparative HPLC method 1:C 260 H 372 N 83 O 133 P 19 S 19 Mw=7985.47 was used as the most abundant natural isotope [M-4H] 4-The theoretical ion mass was m / z 1995.36, but it was detected by low-resolution mass spectrometry at m / z 1995.06. Tm = 62.3°C by protocol 1 (Tm = 66.5°C by sterically random).

[0351] F. Compound MOE-282:20mer, 7Rp [ka] Purified by preparative HPLC method 1:C 260 H 372 N 83 O 133 P 19 S 19 Mw=7985.47 was used as the most abundant natural isotope [M-4H] 4- The theoretical ion mass was m / z 1995.36, but was detected by low-resolution mass spectrometry at m / z 1994.81. Tm = 63.5°C by Protocol 1 (Tm = 66.5°C for sterically random).

[0352] G. Compound MOE-283:20mer, 9Rp [ka] Purified by preparative HPLC method 1:C 260 H 372 N 83 O 133 P 19 S 19 Mw=7985.47 was used as the most abundant natural isotope [M-4H] 4- The theoretical ion mass was m / z 1995.36, but was detected by low-resolution mass spectrometry at m / z 1995.43. Tm = 64.8°C by Protocol 1 (Tm = 66.5°C by sterically random).

[0353] H. Compound MOE-284:20mer, 10Rp [ka] Purified by preparative HPLC method 1:C 260 H 372 N 83 O 133 P 19 S 19 Mw=7985.47 was used as the most abundant natural isotope [M-4H] 4- The theoretical ion value was m / z 1995.36, but was detected by low-resolution mass spectrometry at m / z 1994.96; Tm = 66.2°C by Protocol 1 (Tm = 66.5°C with sterically random).

[0354] I. Compound MOE-285:20mer, 9Rp [ka] Purified by preparative HPLC method 1:C 260 H 372 N 83 O 133 P 19 S 19 Mw=7985.47 was used as the most abundant natural isotope [M-4H] 4- The theoretical ion value was m / z 1995.36, but was detected by low-resolution mass spectrometry at m / z 1995.50; Tm = 63.4°C by Protocol 1 (Tm = 66.5°C with sterically random).

[0355] J. Compound MOE-286:20mer, 13Rp [ka] Purified by preparative HPLC method 2: C 260 H 372 N 83 O 133 P 19 S 19 Mw=7985.47 was used as the most abundant natural isotope [M-4H] 4- The theoretical ion mass was m / z 1995.36, but was detected at m / z 1994.55 by low-resolution mass spectrometry.

[0356] K. Compound MOE-287:20mer, 3Rp [ka] Purified by preparative HPLC method 2: C 260 H 372 N 83 O 133 P 19 S 19 Mw=7985.47 was used as the most abundant natural isotope [M-4H] 4- The theoretical ion value was m / z 1995.36, but was detected by low-resolution mass spectrometry at m / z 1995.08; Tm = 59.7°C by Protocol 1 (Tm = 66.5°C with sterically random).

[0357] FIG. 12 shows the Tm of MOE-012, MOE-277, and MOE-278.

[0358] L. Compound MOE-288:18mer, all Sp [ka] Purified by preparative HPLC method 3:C 234 H 335 N 76 O 119 P 17 S 17 Mw=7186.34 was used as the most abundant natural isotope [M-4H] 4- The theoretical ion value was m / z 1795.59, but was detected by low-resolution mass spectrometry at m / z 1795.45; Tm = 58.4°C by protocol 2 (Tm = 65.9°C with sterically random). 31 P NMR(162MHz)δ ppm 56.09,55.82,55.78,55.56,55.52,55.32,55.21,55.15,55.06

[0359] M. Compound MOE-289:18mer, all Rp [ka] Purified by preparative HPLC method 3:C 234 H 335 N 76 O 119 P 17 S 17 Mw=7186.34 was used as the most abundant natural isotope [M-4H] 4- The theoretical ion was m / z 1795.59, but was detected by low-resolution mass spectrometry at m / z 1795.38; Tm = 70.4°C by protocol 2 (Tm = 65.9°C for sterically random). 31 P NMR(162MHz)δ ppm 56.09,55.82,55.78,55.56,55.52,55.32,55.21,55.15,55.06

[0360] N. Compound MOE-290:18mer, 11Rp [ka] Purified by preparative HPLC method 3:C 234 H 335 N 76 O 119 P 17 S 17 Mw=7186.34 was used as the most abundant natural isotope [M-4H] 4- The theoretical ion value was m / z 1795.59, but was detected by low-resolution mass spectrometry at m / z 1795.57; Tm = 66.6°C by protocol 2 (Tm = 65.9°C for sterically random). 31 P NMR(162MHz)δ ppm 58.53,58.22,58.08,57.82,57.60,57.40,57.12,55.67,55.54,55.30,55.12

[0361] O. Compound MOE-291:18mer, 8Rp [ka] Purified by preparative HPLC method 3:C 234 H 335 N 76 O 119 P 17 S 17 Mw=7186.34 was used as the most abundant natural isotope [M-4H] 4- The theoretical ion was m / z 1795.59, but was detected by low-resolution mass spectrometry at m / z 1795.31; Tm = 62.5°C by protocol 2 (sterically random Tm = 65.9°C). 31 P NMR(162MHz)δ ppm 57.07,56.83,56.71,56.54,56.31,55.16,54.81,54.33,54.24,54.12,54.23

[0362] P. Compound MOE-292:18mer, 8Rp [ka] Purified by preparative HPLC method 3:C 234 H 335 N 76 O 119 P 17 S 17 Mw=7186.34 was used as the most abundant natural isotope [M-4H] 4- The theoretical ion was m / z 1795.59, but was detected by low-resolution mass spectrometry at m / z 1794.95; Tm = 62.6°C by protocol 2 (Tm = 65.9°C with sterically random). 31 P NMR(162MHz)δ ppm 60.01,59.40,59.36,58.87,58.50,58.14,57.67,57.37,57.15,56.66,56.48,55.83,55.55,55.26

[0363] Figure 13 shows an example of an overlay HPLC chromatogram (MOE-252 and MOE-288 to MOE-292 by analytical HPLC method 4.

[0364] Q. Compound MOE-293: 18mer, 4Rp [ka] Purified by preparative HPLC method 3:C 234 H 335 N 76 O 119 P 17 S 17 Mw=7186.34 was used as the most abundant natural isotope [M-4H] 4- The theoretical ion value was m / z 1795.59, but it was detected by low-resolution mass spectrometry at m / z 1795.95; Tm = 59.5°C by Protocol 2 (Tm = 65.9°C with sterically random). 31 P NMR(162MHz)δ ppm 56.79,56.19,55.09,54.93,54.85,54.67,54.53

[0365] R. Compound MOE-294:18mer, 6Rp [ka] Purified by preparative HPLC method 3:C 234 H 335 N 76 O 119 P 17 S 17 Mw=7186.34 was used as the most abundant natural isotope [M-4H] 4- The theoretical ion was m / z 1795.59, but was detected by low-resolution mass spectrometry at m / z 1795.54; Tm = 59.7°C by Protocol 2 (Tm = 65.9°C with sterically random). 31P NMR(162MHz)δ ppm 57.84,57.43,57.17,56.92,56.80,55.98,55.86,55.62,55.58,55.46,55.27,55.11,55.06,55.00

[0366] S. Compound MOE-295:18mer, 4Rp [ka] Purified by preparative HPLC method 3:C 234 H 335 N 76 O 119 P 17 S 17 Mw=7186.34 was used as the most abundant natural isotope [M-4H] 4- The theoretical ion was m / z 1795.59, but was detected by low-resolution mass spectrometry at m / z 1795.82; Tm = 60.6°C by protocol 2 (Tm = 65.9°C with sterically random). 31 P NMR(162MHz)δ ppm 57.71,57.25,57.06,56.11,55.79,55.68,55.48,55.35,55.21,55.11

[0367] T. Compound MOE-296:18mer, 2Rp / 2PO [ka] Purified by preparative HPLC method 3:C 234 H 335 N 76 O 121 P 17 S 15 Mw=7154.38 was used as the most abundant natural isotope [M-4H] 4- The theoretical ion value was m / z 1787.59, but was detected by low-resolution mass spectrometry at m / z 1787.56; Tm = 61.3°C by protocol 2 (Tm = 65.9°C for sterically random). 31 P NMR(162MHz)δ ppm 58.26,58.20,57.84,57.65,57.49,57.40,57.16,56.97,0.33

[0368] U. Compound MOE-297:18mer, 4Rp / 2PO [ka] Purified by preparative HPLC method 3:C 234 H 335 N 76 O 121 P 17 S 15 Mw=7154.38 was used as the most abundant natural isotope [M-4H] 4- The theoretical ion value was m / z 1787.59, but was detected by low-resolution mass spectrometry at m / z 1787.35; Tm = 62.7°C by protocol 2 (Tm = 65.9°C with sterically random). 31 P NMR(162MHz)δ ppm 57.24,57.04,56.42,56.36,55.83,55.69,55.56,55.36,55.17,55.07,54.64,-1.03,-1.13

[0369] V. Compound MOE-298:18mer, 2Rp / 2PO [ka] Purified by preparative HPLC method 3:C 234 H 335 N 76 O 121 P 17 S 15 Mw=7154.38 was used as the most abundant natural isotope [M-4H] 4- The theoretical ion was m / z 1787.59, but was detected by low-resolution mass spectrometry at m / z 1787.40; Tm = 61.6°C by protocol 2 (Tm = 65.9°C with sterically random). 31 P NMR(162MHz)δ ppm 57.20,57.08,56.77,56.55,56.17,56.10,-0.72

[0370] W. Compound MOE-299:20mer, 2Rp / 2PO [ka] Purified by preparative HPLC method 3:C 260 H 372 N 83 O 135 P 19 S 17 Mw=7950.51 was used as the most abundant natural isotope [M-4H] 4- The theoretical ion was m / z 1986.62, but was detected by low-resolution mass spectrometry at m / z 1987.01; Tm = 61.5°C by Protocol 1 (Tm = 69.6°C with sterically random). 31 P NMR(162MHz)δ ppm 57.48,57.22,56.14,55.90,55.65,55.77,55.38,55.30,55.25,55.21,55.06,54.94,-0.95,-0.99

[0371] X. Compound MOE-300:18mer, 6Rp / 2PO [ka] Purified by preparative HPLC method 3:C 234 H 335 N 76 O 121 P 17 S 15 Mw=7154.38 was used as the most abundant natural isotope [M-4H] 4- The theoretical ion value was m / z 1787.59, but was detected by low-resolution mass spectrometry at m / z 1787.49; Tm = 63.2°C by Protocol 2 (Tm = 65.9°C for sterically random). 31 P NMR(162MHz)δ ppm 58.39,58.07,57.85,57.69,56.36,56.06,55.78,55.70,55.57,55.38,55.33,55.29,-0.97

[0372] Y. Compound MOE-301:18mer, 5Rp / 2PO [ka] Purified by preparative HPLC method 3:C 234 H 335 N 76 O 121 P 17 S 15 Mw=7154.38 was used as the most abundant natural isotope [M-4H] 4- The theoretical ion value was m / z 1787.59, but was detected by low-resolution mass spectrometry at m / z 1787.66; Tm = 62.2°C by Protocol 2 (Tm = 65.9°C with sterically random). 31 P NMR(162MHz)δ ppm 58.57,58.02,57.81,57.65,56.31,56.02,55.73,55.64,55.52,55.33,55.27,55.25,55.11,-1.00

[0373] Z. Compound MOE-303:18mer, 3PO [ka] Purified by preparative HPLC method 3:C 234 H 335 N 76 O 122 P 17 S 14 Mw=7137.40 was used as the most abundant natural isotope [M-4H] 4-The theoretical ion was m / z 1783.25, but was detected by low-resolution mass spectrometry at m / z 1782.76; Tm = 59.5°C by protocol 2 (Tm = 65.9°C for sterically random). 31 P NMR(162MHz)δ ppm 56.36,56.11,55.93,55.84,55.69,55.64,55.53,55.38,-0.88,-0.97

[0374] AA. Compound MOE-304: 18mer, 5PO [ka] Purified by preparative HPLC method 3:C 234 H 335 N 76 O 124 P 17 S 12 Mw=7106.45 was used as the most abundant natural isotope [M-4H] 4- The theoretical ion was m / z 1775.61, but was detected by low-resolution mass spectrometry at m / z 1775.83; Tm = 61.6°C by protocol 2 (Tm = 65.9°C with sterically random). 31 P NMR (162MHz, solvent) δ ppm 56.29, 55.75, 55.71, 55.65, 55.53, 55.42, -0.62, -0.79, -0.89, -0.98

[0375] BB. Compound MOE-305: 18mer, 4PO [ka] Purified by preparative HPLC method 3:C 234 H 335 N 76 O 123 P 17 S 13 Mw=7122.43 was used as the most abundant natural isotope [M-4H] 4-The theoretical ion value was m / z 1779.50, but was detected by low-resolution mass spectrometry at m / z 1779.42; Tm = 61.4°C by protocol 2 (Tm = 65.9°C with sterically random). 31 P NMR(162MHz)δ ppm 55.54,55.49,55.72,55.26,55.14,55.11,54.98,-1.02,-1.06,-1.14,-1.47

[0376] CC. Compound MOE-306:18mer, 3PO [ka] Purified by preparative HPLC method 3:C 234 H 335 N 76 O 122 P 17 S 14 Mw=7137.40 was used as the most abundant natural isotope [M-4H] 4- The theoretical ion value was m / z 1783.25, but was detected by low-resolution mass spectrometry at m / z 1783.54; Tm = 60.3°C by protocol 2 (Tm = 65.9°C with sterically random). 31 P NMR(162MHz)δ ppm 56.18,55.88,55.53,55.37,55.30,55.64,55.16,55.07,-0.84,-0.90,-0.95

[0377] DD. Compound MOE-307:18mer, 2PO [ka] Purified by preparative HPLC method 3:C 234 H 335 N 76 O 121 P 17 S 15 Mw=7154.38 was used as the most abundant natural isotope [M-4H] 4-The theoretical ion value was m / z 1787.59, but it was detected by low-resolution mass spectrometry at m / z 1788.19; Tm = 59.0°C by Protocol 2 (Tm = 65.9°C for sterically random). 31 P NMR(162MHz)δ ppm 56.03,55.84,55.70,55.56,55.45,55.27,55.24,55.11,54.95,-1.10,-1.20

[0378] EE. Compound MOE-308: 20mer, 2PO [ka] Purified by preparative HPLC method 3:C 260 H 372 N 83 O 135 P 19 S 17 Mw=7950.51 was used as the most abundant natural isotope [M-4H] 4- The theoretical ion value was m / z 1986.62, but was detected by low-resolution mass spectrometry at m / z 1986.92; Tm = 58.5°C by Protocol 1 (Tm = 69.6°C with sterically random). 31 P NMR(162MHz)δ ppm 55.91,55.76,55.52,55.20,55.10,54.99,54.89,-0.99,-1.05,-1.09

[0379] FF. Compound MOE-309:20mer, 4PO [ka] Purified by preparative HPLC method 3:C 260 H 372 N 83 O 137 P 19 S 15 Mw=7919.50 was used as the most abundant natural isotope [M-4H] 4-The theoretical ion value was m / z 1978.87, but was detected by low-resolution mass spectrometry at m / z 1978.66; Tm = 62.8°C by Protocol 1 (Tm = 69.6°C with sterically random). 31 P NMR(162MHz)δ ppm 55.83,55.65,55.50,55.41,55.19,55.02,55.11,54.77,-1.04,-1.11,-1.15,-1.55

[0380] GG. Compound MOE-310:18mer, 5R / 2PO [ka] Purified by preparative HPLC method 3:C 234 H 335 N 76 O 121 P 17 S 15 Mw=7154.38 was used as the most abundant natural isotope [M-4H] 4- The theoretical ion value was m / z 1787.59, but was detected by low-resolution mass spectrometry at m / z 1787.64; Tm = 63.4°C by protocol 2 (Tm = 65.9°C with sterically random). 31 P NMR(162MHz)δ ppm 58.22,57.54,56.08,55.88,55.41,55.26,55.17,55.10,55.04,54.95,-1.02

[0381] HH. Compound MOE-311:18mer, 4R / 2PO [ka] Purified by preparative HPLC method 3:C 234 H 335 N 76 O 121 P 17 S 15 Mw=7154.38 was used as the most abundant natural isotope [M-4H]4- The theoretical ion value was m / z 1787.59, but was detected by low-resolution mass spectrometry at m / z 1787.34; Tm = 61.0°C by Protocol 2 (Tm = 65.9°C with sterically random). 31 P NMR(162MHz)δ ppm 58.21,57.66,56.05,55.89,55.57,55.48,55.38,55.28,55.26,55.05,54.96,-1.03,-1.25

[0382] Example 10: Structures of PMO-002 and PMO-424 PMO-002 The stereorandom PMO-002 oligonucleotide sequence was synthesized according to the method disclosed in Example 2 above. [ka]

[0383] PMO-424 Stereopure PMO-424 was synthesized according to the method disclosed above in Example 5. The structure of the all-Sp phosphorodiamidate PMO-424 is shown here: [ka]

[0384] Example 11: DBCO-functionalized PMO-002 (Compound 9) [ka] PMO-002 (40 mg) and DMSO (500 μL) were added to a vial, and the mixture was warmed to 39 °C until a clear solution was obtained, then cooled to room temperature. To this PMO solution was added DBCO-C6 acid (CAS 1425485-72-8) (4.8 mg, 3.0 equiv.) and Hunig's base (4.2 μL, 5 equiv.) in NMP (500 μL). HOBt (2.2 mg, 3 equiv.) was added to the mixture, followed by HBTU (5.4 mg, 3 equiv.) in NMP (50 μL) via syringe. The reaction mixture was immediately heated to 40 °C for 3 h. After complete conversion by HPLC, the reaction mixture was cooled to room temperature, and then aqueous NH4OH (28%, 200 μL) was added and stirred for 2 h. The solvent was partially evaporated under a stream of nitrogen, followed by the addition of MeCN (3 mL) and EtOAc (3 mL). The white precipitate was solidified by centrifugation and the supernatant was decanted. The residue was dissolved in water (3 mL) and filtered through a syringe filter to remove particulate matter. Purification by reverse-phase chromatography using the method in Table 24 below gave compound 9 DBCO-PMO-002 (13 mg). LRMS:C 315 H 479 N 148 O 100 P 25 (8709.04) [M+6H] 6+ Calculated m / z for ion: 1452.51; Found: 1452.81

[0385] [Table 37]

[0386] Example 12: BCN-functionalized-PMO-002 (Compound 10) [ka] To a flask were added PMO-002 (25 mg, 2.98 μmol), aqueous sodium tetraborate (0.1 M, 300 μL), and 30 μL aqueous NaHCO3 (saturated solution). After dissolution of PMO-002, the flask was charged with ((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)methyl (2,5-dioxopyrrolidin-1-yl)carbonate (2.60 mg, 8.93 μmol) dissolved in DMSO (250 μL). The reaction mixture was stirred overnight, and then MeCN (10 mL) was added. The precipitate was solidified by centrifugation, and the solvent was then decanted. The solid compound 10 was washed with MeCN, dried, and then used directly (without further purification) in the click reaction described in Example 15 below. LRMS:C 305 H 474 N 147 O 100 P 25 (8570.0) [M+6H] 6+ Calculated m / z for ion: 1429.33; Found: 1429.58

[0387] Example 13: Peptides for conjugation with ASO Peptides were synthesized using standard Fmoc solid-phase synthesis protocols. R. Behrendt et al., 22 J. PEPT. SCI. 4-27 (2016). Structures and low-resolution (LR) MS data for peptides are included where applicable. B is β-alanine; X is 6-aminohexanoic acid; other standard single-letter amino acid abbreviations are used.

[0388] compound 11 [ka] Compound 11: RXRRBRRXRRBRXB-azidolidine; MW=1974.42, LRMS: C 81 H 156 N 42 O 16 (1974.27) [M+5H] 5+ Calculated m / z for ion: 395.85; Found: 395.8

[0389] Compounds 12 and 13 [ka] For click reactions, see R 1 and R 2 are the choices shown for (compound 12) in the box above: RXRRBRRXRYQFLIRXRBRXRB-azidolidine; (MW=3064.7, LRMS: calculated m / z for [M+7H]7+ ion of C134H239N57O26 (3063.92): 438.7; found: 438.8) was used. For amide coupling, R 1 and R 2 is the choice shown for (compound 13) in the box above: Ac-RXRRBRRXRYQFLIRXRBRXRB-OH.

[0390] compound 14 [ka] Compound 14: Ac-LRKLRKRLLRXB-azidolidine: MW=1731.23, LRMS: C 77 H 147 N 31 O 14 (1731.17) [M+2H] 2+ Calculated m / z for ion: 866.58; found: 866.4.

[0391] compound 15 [ka] Compound 15: K(azidoacetyl)-LYENKPRRPYIL: MW=2520.89, LRMS: C 114 H 178 N 34 O 31 (2519.34) [M+4H] 4+ Calculated m / z for ion: 630.8; found: 631.2.

[0392] compound 16 [ka] Compound 16: K(azidoacetyl)-PPPAGSSPGLYENKPRRPYIL: MW=1773.08, LRMS: C 134 H 239 N 57 O 26 (1771.98) [M+2H] 2+ Calculated m / z for ion: 886.9; observed: 885.7.

[0393] compound 17 [ka] Compound 17-cyclo[f-[2-Nal]RrRrQ(-[Ava]-[PEG2]-acid)]: MW=1342.58, LRMS: C 62 H 95 N 21 O 13 (1341.74) [M+2H] 2+ Calculated m / z for ion: 671.9; found: 672.2. f = D-phenylalanine; Nal = L-2-naphthylalanine; R = L-arginine, r = D-arginine, Ava = 5-aminovaleric acid (5-aminopentanoic acid).

[0394] compound 18 [ka] Compound 18:MW=1370.11, LRMS:C 63 H 98 N 22 O 13 (1371.62) [M+2H] 2+ Calculated m / z for ion: 686.8; found: 686.7.

[0395] compound 19 [ka] Compound 19: MW=1322.77, LRMS:C 59 H 98 N 22 O 13 (1323.57) [M+2H] 2+ Calculated m / z for ion: 662.8; found: 662.6.

[0396] compound 20 [ka] Compound 20: MW=1313.74, LRMS:C 62 H 95 N 19 O 13 (1314.56) [M+2H] 2+ Calculated m / z for ion: 657.3; found: 658.1.

[0397] Example 14: General procedure C for attaching (R)-lipoic acid (LA) to compounds 18, 19 and 20. A solution of (R)-lipoic acid-NHS ester (3 equivalents, 10 μL NMP / mg peptide) and DIPEA (7 equivalents; 10 μL NMP / mg peptide) was added to either compound 18, 19, or 20 (1 equivalent) in NMP (10 μL / mg). The reaction was stirred overnight until complete by UPLC and quenched with water (10 equivalents) in NMP. The solution was diluted with MTBE, the solid was centrifuged, and the pellet was washed with MTBE. The product was monitored by UPLC and the desired mass was identified. This product was used without further purification in Example 16.

[0398] compound 21 [ka] Compound 21: (5.4 mg, 47%) Prepared according to general procedure A for 18 (10 mg). MW=1558.80, LRMS: C 71 H110 N 22 O 14 S2(1559.92) [M+2H] 2+ Calculated m / z for ion: 780.0; Found: 780.5

[0399] compound 22 [ka] Compound 22: (10.2 mg, quantitative) Prepared according to general procedure A for 19 (10 mg). MW=1510.8, LRMS: C 67 H 110 N 22 O 14 S2(1511.9) [M+2H] 2+ Calculated m / z for ion: 755.9; found: 756.6.

[0400] compound 23 [ka] Compound 23 (5.9 mg, 51%) was prepared according to general procedure A for 20 (10 mg). MW=1501.77, LRMS: C 70 H 107 N 19 O 14 S2(1502.87) [M+2H] 2+ Calculated m / z for ion: 751.4; found: 752.1.

[0401] Example 15: General Procedure D for the Synthesis of Peptide-PMO Conjugates via Click Reaction To a vial was added compound 9 (1.0 equiv.) or compound 10 (1.0 equiv.), water (30 vol.), and Et3NHOAc (3 vol., 1.0 M solution in water). The peptide-containing azide (amounts listed follow specific examples) was dissolved in DMSO (10 vol.) and added to the reaction mixture. After completion of the reaction as monitored by HPLC-MS (high-performance liquid chromatography-mass spectrometry), MeCN (40–100 vol.) was added, resulting in the formation of a white precipitate. The mixture was centrifuged to compress the solid, and the supernatant was decanted. The solid was purified by size-exclusion chromatography using the general purification conditions listed in Table 25. The resulting fractions were desalted, filtered through a sterile Paal Mustang® 0.2 μm syringe filter, and lyophilized to yield the peptide-PMO conjugate as a white solid. The product of the click reaction contains a mixture of triazole regioisomers.

[0402] [Table 38]

[0403] compound 24 [ka] Compound 24: (5.4 mg, 53% yield) Prepared by general procedure B using compound 9 (7.5 mg, 0.861 μmol) and compound 12 (2.64 mg, 0.861 μmol). MW=11778.336, LRMS: C 450 H 720 N 205 O 126 P 25 (11785.97) [M+16H] 16+ Calculated m / z for ion: 737.6; found: 737.0.

[0404] compound 25 [ka] Compound 25: Prepared by general procedure B using compound 9 (13 mg) and compound 11 (5.89 mg). MW=10687.99, LRMS: C 396 H 635 N 190 O 116 P 25 (10682.31) [M+12H] 12+ Calculated m / z for ion: 891.2; found: 891.4.

[0405] compound 26 [ka] Compound 26: Prepared from compound 10 (12 mg) and compound 14 (7.3 mg) by general procedure B. MW=10305.65, LRMS: C 382 H 621 N 178 O 114 P 25 (10300.17) [M+11H] 11+ Calculated m / z for ion: 937.4; found: 938.0.

[0406] compound 27 [ka] Compound 27: 4.4 mg (46% yield) Prepared from compound 10 (8 mg) and compound 15 (4.9 mg) by general procedure B. MW=10347.98, LRMS: C 386 H 603 N 172 O 120 P 25 (10341.98) [M+8H] 8+ Calculated m / z for ion: 1293.7; found: 1294.3.

[0407] compound 28 [ka] Compound 28: 2.0 mg (31% yield) Prepared from compound 9 (5 mg) and compound 16 (4.7 mg) by general procedure B. MW=11234.46, LRMS: C 429 H 657 N 182 O 131 P 25 (11228.38) [M+9H] 9+ Calculated m / z for ion: 1248.6; found: 1249.1.

[0408] Example 16: General Procedure E for Peptide-PMO Conjugates via Amide Bonds 29: To one vial was added PMO-002 (12 mg, 1.43 μmol) and DMSO (100 μL), and the suspension was warmed to 37 °C until a clear solution was formed. To a second vial was added 13 (7.5 mg, 2.57 μmol, 1.8 equiv) and N-methylpyrroldinone (50 μL). To the second vial was added HOBT (0.5 mg, 2.8 μmol, 2.0 equiv), Hunig's base (1 μL, 5 μmol, 3.5 equiv), and N-methylpyrroldinone (50 μL) and mixed to ensure homogeneity. To this solution was added HBTU (1.4 mg, 3.57 μmol, 2.5 equiv.) and vortexed to achieve dissolution. After 1 min, the solution was transferred via syringe to the first vial containing PMO-002 in DMSO. The vial was sealed and microwaved at 65° C. for 15 min. After cooling to room temperature, the product was precipitated by adding MeCN (9 mL). The solid was collected by decanting, dissolved in water, and purified by ion exchange chromatography, General Procedure F, using a Resource S column as shown in Table 26. The collected fractions were desalted and lyophilized to give the product (3 mg).

[0409] [Table 39]

[0410] compound 29 [ka] Compound 29:MW=11332.81, LRMS:C 424 H 691 N 200 O 123 P 25 (11326.74) [M+9H] 9+ Calculated m / z for ion: 872.3; found: 872.8.

[0411] compound 30 [ka] Compound 30: (5.3 mg, 46%) Prepared according to general procedure E using PMO-002 (10 mg) and compound 20 (6.4 mg). MW=9722.77, LRMS: C 356 H 555 N 168 O 110 P 25 (9717.64) [M+10H] 10+ Calculated m / z for ion: 972.8; found: 973.3.

[0412] compound 31 [ka] Compound 31: (7.3 mg, 61%) Prepared according to general procedure E using (Sp)PMO-424 (10 mg) and compound 17 (7.6 mg). MW=8333.59, LRMS: C 308 H 483 N 140 O 96 P 21 (8329.17) [M+10H] 10+ Calculated m / z for ion: 833.9; found: 834.3.

[0413] [Table 40]

[0414] compound 32 [ka] Compound 32: (0.180 mg, 1.5%) Prepared according to general procedure E using PMO-002 (10 mg) and compound 21 (5.4 mg). Purified by general procedure G. MW=9940.1, LRMS: C 365 H 570 N 169 O 111 P 25 S2 (9934.70) [M+10H] 10+ Calculated m / z for ion: 994.5; found: 994.0.

[0415] compound 33 [ka] Compound 33: (4.3 mg, 36%) Prepared according to general procedure E using PMO-002 (10 mg) and compound 22 (10.0 mg). Purified by general procedure G. MW=9892.1, LRMS: C 361 H 570 N 169 O 111 P 25 S2 (9886.70) [M+10H] 10+ Calculated m / z for ion: 989.7; found: 989.9.

[0416] compound 34 [ka] Compound 34: (0.340 mg, 2.9%) Prepared according to general procedure E using PMO-002 (10 mg) and compound 23 (5.9 mg). Purified by general procedure G. MW=9883.1, LRMS: C 363 H 565 N 166 O 111 P 25S2(9877.67) [M+10H] 10+ Calculated m / z for ion: 988.8; found: 989.1.

[0417] Example 17: Exon skipping efficiency assay in mouse bone marrow-derived macrophage (mBMDM) cells in vitro Freshly isolated mBMDM cells from humanized CD33 mice were cultured and maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum with recombinant mouse CSF. Approximately 50,000 cells were seeded per well and treated with each peptide-PMO ASO at concentrations of 0.078 μM, 0.156 μM, 0.31 μM, 0.63 μM, 1.25 μM, 2.5 μM, and 10 μM without transfection reagent. Exon skipping efficiency assays were performed in a 96-well plate format. Cells were incubated at 37°C in a cell culture incubator for 48 hours before total RNA isolation. Total RNA was isolated and converted to cDNA according to the supplier's protocol, and then Taqman gene expression assays were used to quantify exon-2-skipped CD33 (forward primer: CGCTGCTGCTACTGCTG (SEQ ID NO: 207); reverse primer: TTCTAGAGTGCCAGGGATGA (SEQ ID NO: 208); and probe: TGTGGGCAGACTTGACCCACAG (SEQ ID NO: 209)) and non-skipped CD33 (forward primer: GGATGGAGAGAGGAAGTA (SEQ ID NO: 210); reverse primer: GTGCCAGGGATGAGGATTT (SEQ ID NO: 211); and probe: TGCATGTGACAGACTTGACCCACA (SEQ ID NO: 212)) mRNA transcripts. Target transcript expression was normalized using the mouse housekeeping gene HPRT1.

[0418] Selected conjugates were tested for CD33 D2 skipping efficiency in mBMDMs under gymnastic (non-transfected) conditions. As shown in Figure 14, all conjugates tested showed improved activity compared to naked PMO-002.

[0419] The activity / properties of CD33-targeted oligonucleotides can be determined using a variety of techniques, using a variety of human, mouse, and non-human primate cell lines.

[0420] Example 18: In vivo assay Exon skipping activity assay in the whole brain of the hCD33 mouse model after intracerebroventricular administration. We used a humanized CD33 mouse model to study CD33 exon-2 skipping CPP-ASO in vivo. Using CRISPR / Cas9-mediated gene editing, we replaced mouse CD33 with human genomic CD33, including the signal peptide. The mouse 3' and 5' untranslated regions were retained. For this in vivo assay, we used a mixed-sex cohort of human CD33 mice on a C57BL / 6 background. Mice were 12–24 weeks old at the time of administration. On day 1, CPP-ASO was administered intracerebroventricularly (ICV) into the right lateral ventricle in a 10 μL bolus injection in PBS. One week after injection, or longer for duration studies, mice were autopsied. At autopsy, mice were transcardially perfused with PBS under Avertin anesthesia. Brains were rapidly removed from the skull, and the cortex and hippocampus were dissected from the injected hemisphere for exon skipping assessment. For RNA isolation, frozen tissue was homogenized in 9x volume of Trizol for 3 minutes. 500 μL of Trizol lysate was transferred to a 1 mL deep-well plate. 100 μL of chloroform was added to each sample, shaken vigorously, and centrifuged at 4000 × g for 5 minutes. The supernatant (250 μL) was transferred to a binding plate from the SV96 Total RNA Extraction Kit (Promega), and RNA was extracted using the same protocol. Total RNA was isolated and converted to cDNA according to the SV96 protocol (Promega), and exon-2-skipped CD33 mRNA transcripts were then quantified using TaqMan gene expression assays. Target transcript expression was normalized using the mouse housekeeping gene HPRT1.

[0421] Determination of CPP-PMO conjugate concentrations in mouse cortex and hippocampus using HELISA Compounds 30, 31, and 33 were quantified in mouse cortex and hippocampus using a hybridization-based immunoassay method (HELISA). Tissues were lysed in TRIzol 1:10 (Thermo Fisher Scientific, Waltham, MA) and diluted in hybridization buffer (1:100, 1 M NaCl in TE buffer and 0.1% Tween® 20). Compound 30 was spiked into diluted tissue homogenates to generate standards and quality controls (QCs). 35 μL of diluted samples, standards, and QCs were transferred to a 96-well PCR plate. To the PCR plate containing the standards and samples, 35 μl of detection probe solution (5′-GTGACAGGTGAGG / 3Bio / -3′ (for compounds 30 and 33, Integrated DNA Technologies, Inc., Coralville, IA), 5′- / 5DigN / CTGTGCATGT-3′ (for compound 31, Integrated DNA Technologies, Inc., Coralville, IA), 100 nM in hybridization buffer) was added. Samples and detection probes were hybridized in a thermal cycler under the following conditions: 95°C for 10 minutes, 37°C for 60 minutes, and a final hold at 4°C.

[0422] An MSD Gold 96-well streptavidin SECTOR plate (Meso Scale Diagnostics, LLC., Rockville, MD) was blocked with 150 μL of casein in TBS blocker (Thermo Fisher Scientific, Waltham, MA) for 1.5 hours at room temperature. After washing with wash buffer (Tris-buffered saline containing Tween® 20, Sigma-Aldrich, St. Louis, MO), 25 μL of capture probe (5′-DigN / CTCTCTGTGCAT-3′ (for compounds 30 and 33, Integrated DNA Technologies, Inc., Coralville, IA), 5′-GACAGGTGAGG / 3Bio / -3′ (for compound 31, Integrated DNA Technologies, Inc., Coralville, IA), 200 nM in hybridization buffer) was added to the MSD plate and incubated at 37°C and 300 rpm for 1 hour. After a washing step, 25 μL of samples, standards, and QCs were transferred in duplicate to an MSD plate and incubated for 1 h at 37° C. on a shaking platform (300 rpm). The plate was then washed three times and incubated for 1 h with 25 μL of 1 μg / mL MSD Gold SULFO-TAG NHS-labeled: anti-digoxigenin, Fab fragment (generated in-house from the conjugation of MSD Gold SULFO-TAG NHS-ester (Meso Scale Diagnostics, LLC., Rockville, MD) and anti-digoxigenin, Fab fragment (Sigma-Aldrich, St. Louis)) in casein-TBS blocking buffer and 0.05% Tween 20. After a final wash step, 150 μL of 2× MSD Read Buffer T (Meso Scale Diagnostics, LLC., Rockville, MD) was added, and the plate was read on an MSD Sector S 600 instrument (Meso Scale Diagnostics, LLC., Rockville, MD).Nonlinear regression analysis was performed, and the concentrations of reference compounds were calculated from the signal intensities by interpolation from the standard curve using a four-parameter logistic (4PL) model (weighting factor = 1 / Y2) in Discovery Workbench 4.012.1 (Meso Scale Diagnostics, LLC., Rockville, MD). The lower limit of quantitation (LLOQ) was 9.8 nM in the cortex and hippocampus.

[0423] In vivo results Compounds 25, 29, 30, 31, 32, 33, and 34 were administered in vivo via ICV administration as outlined above. When administered at 30 μg according to the in vivo assay protocol, compounds 25 and 29 caused acute toxicity in mice. Therefore, their in vivo exon-2 skipping activity could not be assessed. Compounds 30, 31, 32, 33, and 34 were well tolerated. The in vivo skipping efficiency of compound 30 is shown in Figure 15. When administered at 30 μg, compound 30 demonstrated a 10-fold improvement in skipping efficiency compared to PMO-002 (e.g., 30 μg of compound 30 resulted in a similar level of skipping as 300 μg of PMO-002).

[0424] The duration of compound 30 was also evaluated. A single 30 μg dose of compound 30 maintained exon skipping in mouse brain for up to 60 days (cortex and hippocampus, FIG. 16). In comparison, PMO-002 showed peak activity at 7 days and decreased activity after 14 days. Analysis of the brain concentrations of compound 30 compared to PMO-002 showed a dramatically improved PK profile with higher exposure. Compound 30 exhibited a 10-fold improvement in brain exposure compared to PMO-002 (FIG. 17).

[0425] Sp-PMO-424 was conjugated with compound 17 to form compound 31, which showed a 10-fold improvement in skipping efficacy compared to Sp-PMO-424 (FIG. 18).

[0426] Both Compound 30 and Compound 31 were tolerated in vivo up to the 60 μg dose tested when administered ICV.

[0427] Lipoic acid contains a five-membered disulfide ring, which may increase peptide interaction with proteins and improve cellular uptake. Lipoic acid was incorporated into conjugate compounds 32, 33, and 34 by attachment to a lysine residue. Compounds 32, 33, and 34 were tested in vivo at a 10 μg dose ( FIG. 19 ). Compound 33 contains a lysine-N-lipoic acid conjugate in the macrocyclic ring instead of the phenylalanine in compound 30. Compound 33 exhibited improved skipping efficacy compared to compounds 32 and 34 and compared to compound 30.

[0428] Those skilled in the art will appreciate that the present disclosure may be modified in ways not specifically described herein. The present disclosure is not limited in scope by the specific embodiments described herein, which are for illustrative purposes only. The present disclosure encompasses any modifications and variations, including all functionally equivalent products, compositions, and methods.

[0429] The entire disclosures of all publications cited herein are hereby incorporated by reference, without any admission that any such publication constitutes prior art or is part of the general knowledge of those skilled in the art.

[0430] Example 19: Further examples of cell-penetrating peptides Further examples of cell-penetrating peptides include peptides that contain cyclic lactams. In some embodiments, the cyclic lactams can contain 8-, 9-, or 10-membered rings. In some embodiments, the cyclic lactams can be peptides that contain aromatic linear or branched alkyl groups and functionalized alkyl groups in side chains (R 1 , R 2) side chain. Additionally, in some embodiments, this side chain may contain a guanidine group, such as that found in arginine, which promotes cell-penetrating activity. In some embodiments, optimal cell-penetrating efficacy may be achieved by varying the stereochemistry of each center accordingly. Examples of lactam amino acids are listed in FIG. 20. Examples of cell-penetrating peptides with lactam amino acids AA1-AA10 are listed in FIG. 21. In some embodiments, the synthesis of lactam amino acids AA1-AA10 used in peptide synthesis follows the synthetic pathways shown in FIGS. 22-27. Further examples of cell-penetrating peptides include peptides containing chemically modified proline residues (FIG. 28). Proline residues provide conformational bias that may not be achieved with acyclic amino acids. In some embodiments, the modified proline is constructed so that it contains a side chain functional group with an aromatic, linear, and / or branched alkyl group. Additionally, in some embodiments, the side chain contains one or more guanidine groups, such as those found in arginine. In some embodiments, the stereochemistry at each center may be varied accordingly to achieve optimal cell penetration efficacy. In Examples 19-47, when a specific stereochemical configuration is not depicted for a compound, all possible stereochemical configurations are included.

[0431] In some embodiments, the synthesis of prolines modified with guanidine side chains is carried out according to a previously reported method (Ishiguro et al. J. Med. Chem. 2004, 47, 489-492).

[0432] Another example includes novel peptides containing peptide bond isosteres, such as 1,3,4-oxadiazoles, as shown in Figure 29. In some embodiments, the peptides are constructed using previously reported methodologies (Yudin AK et al. Nature Chem. 8, 2016, 1104).

[0433] In some embodiments, synthesis of the cyclic peptide portion follows a general solid-phase synthesis protocol. In some embodiments, the linker between the peptide and the PMO is selected from the linkers exemplified in the compounds of Examples 15 and 16. In some embodiments, attachment of the peptide to the PMO is achieved as described for the construction of peptide-PMO conjugates in Examples 15 and 16 (e.g., amide bond formation, azide-alkyne click chemistry, etc.).

[0434] Example 20: Preparation of Compound 41 [ka] Compound 41: Compound 41 was prepared by the dropwise addition of DMSO (22.4 mL, 315 mmol) to a solution of oxalyl chloride (13.8 mL, 158 mmol) in DCM (400 mL) at −78°C under nitrogen. This solution was stirred at −78°C for 10 minutes. A solution of 41-I (28.1 mL, 131 mmol) in DCM (7.50 mL) was added slowly, and the reaction was stirred at −78°C for 1 hour. Triethylamine (92 mL, 656 mmol) was then added, and the reaction mixture was maintained at −78°C for 10 minutes before warming to room temperature and stirring for 2 hours. The reaction was then quenched with water, and the phases were separated. The organic layer was washed successively with 1N HCl, NaHCO3 (sat.), and brine, and dried over MgSO4. The solvent was removed by rotary evaporation to give compound 41-II (24.9 g, 131 mmol, quantitative) as a clear oil.

[0435] Ethyl 2-(diethoxyphosphoryl)acetate (78.0 ml, 393 mmol) was added to compound 41-II (24.7 g, 131 mmol) and lithium chloride (16.7 g, 393 mmol) in acetonitrile (525 ml). The solution was then cooled in an ice bath, and N,N-diisopropylethylamine (137 ml, 786 mmol) was added. The reaction was stirred until it was deemed complete by LCMS or TLC (monitoring for the disappearance of starting material) and then worked up with ethyl acetate and saturated aqueous NaHCO3. The aqueous layer was extracted once more with ethyl acetate, and the organic fractions were combined, washed with half-saturated brine, and dried over Na2SO4. The resulting organic layer was concentrated and purified by Biotage™ purification to give compound 41-III (28.0 g, 108 mmol, 83% yield).

[0436] To a stirred solution of compound 41-III (28 g, 108 mmol) in dry DCM (310 ml) at −78° C., DIBAL-H (1 M in hexane, 238 ml, 238 mmol) was added dropwise. After the addition was complete, the solution was allowed to warm to room temperature. Upon complete consumption of the starting material (as indicated by TLC), the reaction mixture was cooled again to −78° C. and quenched with saturated aqueous Rochelle's salt solution. After stirring at room temperature for 30 minutes, the biphasic mixture was poured into a separatory funnel, and the aqueous layer was extracted three times with ethyl acetate. The organic layer was washed with brine, dried over MgSO4, and concentrated to give crude compound 41 (19.3 g, 89 mmol, 82% yield), which was used directly in the next step without further purification. 1 H NMR(400MHz,CDCl3)δ ppm 5.60-5.71(2H,m),4.05(2H,br s),3.60(2H,t,J=6.75Hz),2.18-2.31(2H,m),0.84(9H,s),0.00(6H,s);MS(ESI,m / z)C 11 H 24 O2Si+H + Calculated value: 217.17; measured value: 217.1.

[0437] Example 21: Preparation of Compound 42 [ka] Compound 42: Compound 41 (4.2 g, 19.4 mmol) in DCM (40 mL) was treated successively with DIPEA (6.78 mL, 38.8 mmol) and Ms-Cl (2.269 mL, 29.113 mmol) under nitrogen at −40° C., and the resulting mixture was stirred at −40° C. for 1 h. Lithium bromide (16.9 g, 194 mmol) in THF (200 mL) was added, and the resulting mixture was stirred at room temperature for 1 h, resulting in the formation of a white precipitate. MTBE and saturated NH4Cl were added. The aqueous layer was separated and extracted with MTBE. The combined organic layers were dried over MgSO4 and concentrated to give compound 42 (5.46 g, 19.6 mmol, quantitative) as a yellowish oil, which was used in the next step without further purification. 1 H NMR(400MHz,CDCl3)δ ppm 5.69-5.83(2H,m),3.95(2H,d,J=6.25Hz),3.65(2H,t,J=6.57Hz),2.28(2H,q,J=6.17Hz),0.89(9H,s),0.05(6H,s);MS(ESI,m / z)C 11 H 23 BrOSi+H + Calculated value: 280.09; measured value: 280.1.

[0438] Example 22: Compounds made by general procedure H [ka] General Procedure H: To a suspension of indium (3.0 equiv.) and (R,E) or (S,E) ethyl-2-((tert-butylsulfinyl)imino)acetate (1.0 equiv.) in DMF (24 vol.) was added allyl bromide (2.0 equiv.) in one portion. The reaction was stirred for 5 h and quenched with NH4Cl (sat.). The mixture was filtered through Celite and washed with EtOAc. The resulting organic layer was washed three times with water and once with brine. The organic layer was dried over MgSO4 and concentrated. The residue was purified using Biotage purification to afford general compound H as a single diastereomer as a pale yellow oil.

[0439] [ka] Compound 106: Prepare 6.4 g (81% yield) of ethyl (2S,3S)-2-(((S)-tert-butylsulfinyl)amino)-3-phenylpent-4-enoate from (E)-(3-bromoprop-1-en-1-yl)benzene (7.21 ml, 48.716 mmol) and ethyl (S,E)-2-((tert-butylsulfinyl)imino)acetate (5.00 g, 24.358 mmol) using general procedure H. 1 H NMR(400MHz,CDCl3)δ ppm 7.29-7.35(2H,m),7.25(1H,m,J=6.90Hz),7.19(2H,d,J=7.75Hz),6.09(1H ,ddd,J=17.60,9.00Hz),5.13-5.22(2H,m),4.13-4.26(3H,m),3.92(1H,br d,J=9.38Hz),3.71(1H,t,J=7.94Hz),1.28(3H,t,J=7.07Hz),1.08(9H,s);MS(ESI,m / z)C 17 H 25 NO3S+Na + ] Calculated value 346.14; measured value 346.1.

[0440] [ka] Compound 107: 7.26 g, (80% yield) of ethyl (2S,3S)-2-(((S)-tert-butylsulfinyl)amino)-3-(naphthalen-2-yl)pent-4-enoate was prepared from (E)-2-(3-bromoprop-1-en-1-yl)naphthalene (12.04 g, 48.716 mmol) and ethyl (S,E)-2-((tert-butylsulfinyl)imino)acetate (5.00 g, 24.358 mmol) using general procedure H. 1H NMR(400MHz,chloroform-d)δ ppm 7.74-7.83(3H,m),7.63(1H,s),7.39-7.53(2H,m),7.32(1H,d,J=8.50H z),6.17(1H,ddd,J=18.01,9.38,9.38Hz),5.15-5.26(2H,m),4.29(1H, dd,J=9.07,7.69Hz),4.20(2H,q,J=7.09Hz),3.97(1H,d,J=9.26Hz),3.89(1H,t,J=7.82Hz),1.25(3H,t,J=6.88Hz),1.04(9H,s);MS(ESI,m / z)C 21 H 27 NO3S+H + ] Calculated value 374.18; measured value 374.2.

[0441] [ka] Compound 108: 1.81 g, (92% yield) of ethyl (2S,3R)-3-(2-((tert-butyldimethylsilyl)oxy)ethyl)-2-(((S)-tert-butylsulfinyl)amino)pent-4-enoate was prepared using general procedure H from (E)-((5-bromopent-3-en-1-yl)oxy)(tert-butyl)dimethylsilane (2.72 g, 9.74 mmol) and ethyl (S,E)-2-((tert-butylsulfinyl)imino)acetate (1.0 g, 4.872 mmol). 1 H NMR(400MHz,CDCl3)δ ppm 5.58(1H,ddd,J=16.88,9.63,7.75Hz),5.06-5.19(2H,m),4.22(2H,q,J=7.13Hz),4.12(1H,br d,J=8.51Hz),3.87(1H,dd,J=8.19,5.57Hz),3.59-3.70(1H,m),3.47-3.59(1H,m),2.61(1H,s),1.75(1H,br d,J=6.75Hz),1.39-1.55(1H,m),1.22-1.32(12H,m),0.84-0.94(9H,m),0.00-0.08(6H,m);MS(ESI,m / z)C 19 H39 NO4SSi+H + ] Calculated value 406.25; measured value 406.2. [ka]

[0442] Example 23: Compounds made by general procedure I General Procedure I: Sulfinylamino acid (1.0 equiv.) was dissolved in THF (10 vol.), followed by the addition of HCl (4N in dioxane, 5.0 equiv.). The mixture was stirred at room temperature for 40 min, neutralized with saturated NaHCO3, and diluted with DCM. The mixture was extracted three times with DCM, and the organic layer was dried over Na2SO4 and concentrated in vacuo to give general compound I as a yellow oil. The crude general compound I was used in the next step without further purification.

[0443] [ka] 4.3 g (89% yield) of compound 109 was prepared from compound 106 (7.1 g, 21.9 mmol) using general procedure I. 1 H NMR(400MHz,CDCl3)δ ppm 7.28-7.38(2H,m),7.21-7.26(3H,m),6.12(1H,ddd,J=16.88,9.13,8.50Hz),5.10-5.22( 2H,m),4.16(2H,q,J=7.13Hz),3.80(1H,d,J=7.63Hz),3.64(1H,t,J=7.94Hz),1.87(2H,br s),1.25(3H,t,J=7.13Hz);MS(ESI,m / z)[C13H17NO2+H + ] Calculated value 220.14; measured value 220.1.

[0444] [ka] 5.9 g (100% yield) of compound 110 was prepared from compound 107 (8.2 g, 21.4 mmol) using general procedure I. 1 H NMR(400MHz,CDCl3)δ ppm 7.82(3H,br t,J=8.57Hz),7.69(1H,s),7.41-7.54(2H,m),7.37(1H,d,J=8.38Hz),6.21(1H,ddd,J=17.01,9.13,8.50Hz), 5.05-5.27(2H,m),4.18(2H,q,J=7.00Hz),3.88(1H,d,J=7.63Hz),3.78(1H,dd,J=8.13,7.38Hz),1.50(2H,br s),1.25(3H,t,J=7.00Hz);MS(ESI,m / z)C 17 H 19 NO2+H + ] Calculated value 270.15; measured value 270.2.

[0445] [ka] 1.5 g (92% yield) of compound 111 was prepared from compound 108 (2.9 g, 7.1 mmol) using general procedure I. 1 H NMR(400MHz,CDCl3)δ ppm 5.73(1H,dt,J=16.95,9.72Hz),5.14-5.34(2H,m),4.28(2H,q,J=7.00Hz),3.70-3.93(1H,m),3.60-3.69(1H,m),3.5 8(1H,d,J=4.88Hz),2.58-2.71(1H,m),1.74-1.99(1H,m),1.59-1.70(1H,m),0.99(9H,s),0.13(6H,s);MS(ESI,m / z)C 15 H 31 NO3Si+H + ] Calculated value 302.22; measured value 302.3. [ka]

[0446] Example 24: Compounds made by general procedure J General Procedure J: To general compound I (1.0 eq) dissolved in MeCN (10 vol) followed by DIPEA (1.2 eq) was added di-tert-butyl dicarbonate (1.1 eq). The resulting suspension was stirred at ambient temperature for 1 hour, then diluted with CHCl, washed with H0, 10% citric acid solution, and saturated NaHCO, and then dried over anhydrous NaSO. The resulting suspension was filtered and the organics were evaporated. The resulting compound was purified by Biotage purification to give general compound JI.

[0447] General compound JI (1.0 equiv.) was dissolved in THF (8 vol.) and water (2 vol.). Lithium hydroxide monohydrate (5.0 equiv.) was added all at once at room temperature. The reaction was stirred vigorously until complete by LCMS. The solution was concentrated and partitioned between water and EtOAc. The pH of the aqueous phase was adjusted to pH 1 and extracted three times with EtOAc. The combined organics were washed with brine, dried over Na2SO4, and concentrated to give general compound J. The isolated material was sufficiently pure to proceed to the next step.

[0448] [ka] Compound 112 was synthesized using the procedure from Buschauer, A., J. Med. Chem. 2016, 59, 13, 6045. 1 H NMR(400MHz,CDCl3)δ ppm 5.67-5.82(1H,m),5.14-5.22(2H,m),5.02(1H,br d,J=7.25Hz),4.40(1H,br d,J=5.75Hz),2.44-2.68(2H,m),1.46(9H,s);MS(ESI,m / z)[C 10 H 17 NO4+Na + ] Calculated value 238.10; measured value 238.1.

[0449] [ka] 2.5 g (86% yield) of compound 113 was prepared from compound 109 (2.5 g, 17.3 mmol) using general procedure J. 1 H NMR(400MHz,CDCl3)δ ppm 7.28-7.37(3H,m),7.21-7.26(2H,m),6.11(1H,ddt,J=17.50,8.80,8.80,8.80Hz),5.23(2H,br d,J=13.51Hz),4.81(1H,br d,J=8.38Hz),4.68(1H,br t,J=6.88Hz),3.76-3.95(1H,m),1.40(9H,s);MS(ESI,m / z)[C 16 H 21 NO4+Na + ] Calculated value 314.14; measured value 314.1.

[0450] [ka] 3.1 g (77% yield) of compound 114 was prepared from compound 110 (3.0 g, 11.1 mmol) using general procedure J. 1 H NMR(400MHz,CDCl3)δ ppm 7.83(3H,br d,J=8.25Hz),7.68(1H,s),7.45-7.53(2H,m),7.37(1H,br d,J=8.38Hz),6.20(1H,ddt,J=17.50,9.00,9.00,9.00Hz),5.22-5.31(2H,m),4.86(1H,br d,J=7.75Hz),4.56-4.80(1H,m),4.06(1H,br s),1.37(9H,s);MS(ESI,m / z)[C 20 H 23 NO4+Na + ] Calculated value 364.1; measured value 364.0.

[0451] [ka] Compound 50 is prepared from compound 111 using general procedure J.

[0452] [ka] Example 25: Compounds made by general procedure K General Procedure K: The free amino ester (1.0 equiv.), equimolar amounts of 2,4-dimethoxybenzaldehyde (1.0 equiv.), and acetic acid (2.0 equiv.) were dissolved in absolute EtOH (33 vol.). Sodium cyanoborohydride (2 equiv.) was added all at once, and the reaction was allowed to proceed at ambient temperature for 12 h. Excess borohydride was quenched with 10% Na2CO3 in water, followed by the addition of DCM. The aqueous layer was extracted three times with DCM and dried over anhydrous MgSO4. The organic layer was removed under reduced pressure to give a clear yellow oil. The desired compound was purified using Biotage purification to yield the corresponding reductive amination product.

[0453] [ka] Compound 57 was synthesized using the procedure from Creighton, CJ, Bioorg. Med. Chem. 12, 2004, 4375. 1 H NMR(400MHz,chloroform-d)δ ppm 7.12(1H,d,J=7.88Hz),6.41-6.46(2H,m),5.67-5.79(1H,m),5.03-5.14(2H,m),3. 80(6H,s),3.71-3.77(1H,m),3.62-3.71(4H,m),3.35(1H,t,J=6.57Hz),2.42(2H,br t,J=6.88Hz),1.20-1.35(1H,m);MS(ESI,m / z)[C15H21NO4+Na + ] Calculated value 302.14; measured value 302.1.

[0454] [ka] Compound 115 is prepared from compound 109 using general procedure K.

[0455] [ka] 670 mg (86% yield) of compound 116 was prepared from compound 110 (500 mg, 1.86 mmol) using general procedure K. 1 H NMR (400 MHz, chloroform-d) δ ppm 7.71-7.83 (3H, m), 7.54 (1H, s), 7.42-7.50 (2H, m), 7.23 (1H, br d,J=8.38Hz),6.95-7.06(1H,m),6.34-6.40(1H,m),6.20(1H,s),5.99-6.10(1H,m),4.97-5.16(2H,m),4.12-4.24(2H,m),3.80( 3H,s),3.71-3.77(2H,m),3.58-3.71(2H,m),3.44(1H,d,J=13.63Hz),3.17(3H,s),0.80-0.95(4H,m);MS(ESI,m / z)[C25H27NO4+H + ] Calculated value 420.22; measured value 420.2.

[0456] [ka] Compound 63 is prepared from compound 111 using general procedure K.

[0457] [ka] Example 26: Compounds made by general procedure L General Procedure L: General compound J (1.0 equiv) is dissolved in THF (5 vol) and cooled to -78 °C. 9-BBN (2.5 equiv) is added dropwise to the reaction mixture. The reaction mixture is stirred at this temperature for 1 h, then allowed to warm to room temperature and stirred for an additional 2 h. The reaction is then cooled to 0 °C, and a solution of sodium acetate (5.0 equiv) in water (12 vol) mixed with hydrogen peroxide (30% wt / wt, 5.0 equiv) is added dropwise to the reaction mixture. The reaction is then allowed to warm to room temperature and stirred overnight. The aqueous layer is extracted three times with EtOAc and dried over anhydrous Na2SO4. After filtration, all solvent is removed under reduced pressure. The crude reaction mixture is purified by Biotage purification to give general compound LI.

[0458] General compound LI (1.0 equivalent) and triethylamine (4.0 equivalents) in dichloromethane (10 volumes) are reacted with sulfur trioxide pyridine complex (3.0 equivalents) in dimethyl sulfoxide (3 volumes) by dropwise addition at 0°C, followed by an additional 3 hours of reaction. The reaction is quenched with water and diluted with ethyl acetate. The organic layer is washed with water and brine, dried over MgSO4, filtered, and concentrated in vacuo to give general compound L-II.

[0459] The flask is flame-dried and then cooled to −78° C. Methyltriphenylphosphonium iodide (2.2 equiv.) and potassium tert-butoxide (2.2 equiv.) are added to the flask and dissolved in dry THF (10 vol.). This solution is then allowed to warm to 0° C. over 1.5 h. In a flask cooled to 0° C., general compound L-II (1.0 equiv.) is dissolved in THF (10 vol.). This solution is cannulated into the solution containing the phosphonium ylide and stirred at 0° C. for 30 min. The reaction mixture is quenched with saturated NH4Cl and stirred vigorously for 5 min. The reaction is then diluted with water and extracted three times with EtOAc. The pooled organic extracts are dried over Na2SO4, filtered, and concentrated in vacuo. The crude product is purified by Biotage purification to give general compound L.

[0460] [ka] Compound 117 is prepared from the methyl ester of compound 112 using general procedure L.

[0461] [ka] Compound 118 is prepared from the ethyl ester of compound 113 using general procedure L.

[0462] [ka] Compound 119 is prepared from the ethyl ester of compound 114 using general procedure L.

[0463] [ka] Compound 120 is prepared from the ethyl ester of compound 115 using general procedure L.

[0464] [ka] Example 27: Compounds made by general procedure M General Procedure M: General compound L (1.0 equiv.) is dissolved in THF (8 vol.) and water (2 vol.). Lithium hydroxide monohydrate (5.0 equiv.) is added all at once at room temperature. The reaction is stirred vigorously until completion as determined by LCMS. The solution is concentrated and partitioned between water and EtOAc. The pH of the aqueous phase is adjusted to pH 1 and extracted three times with EtOAc. The combined organics are washed with brine, dried over Na2SO4, and concentrated to give general compound M. The isolated material is sufficiently pure to proceed to the next step.

[0465] [ka] Compound 121 is prepared from compound 117 using general procedure M.

[0466] [ka] Compound 122 is prepared from compound 118 using general procedure M.

[0467] [ka] Compound 123 is prepared from compound 119 using general procedure M.

[0468] [ka] Compound 124 is prepared from compound 120 using general procedure M.

[0469] [ka] Example 28: Compounds made by general procedure N General Procedure N: General compound L (1.0 equivalent) is dissolved in 1,4-dioxane (4 volumes), and 4N hydrochloric acid / 1,4-dioxane solution (13 equivalents) is added, and the resulting mixture is stirred at room temperature for 2 hours. The solvent is evaporated under reduced pressure to give general compound NI.

[0470] General compound NI (1.0 eq), equimolar amounts of 2,4-dimethoxybenzaldehyde (1 eq) and acetic acid (2 eq) are dissolved in anhydrous EtOH (33 vol). Sodium cyanoborohydride (2 eq) is added all at once and the reaction is allowed to proceed at ambient temperature for 12 hours. Excess borohydride is quenched with 10% Na2CO3 in water, followed by the addition of DCM. The aqueous layer is extracted three times with DCM and dried over anhydrous MgSO4. The organic layer is removed under reduced pressure to give a clear yellow oil. The desired compound is purified using Biotage purification to give general compound N.

[0471] [ka] Compound 125 is prepared from compound 117 using general procedure N.

[0472] [ka] Compound 126 is prepared from compound 118 using general procedure N.

[0473] [ka] Compound 127 is prepared from compound 119 using general procedure N.

[0474] [ka] Compound 128 is prepared from compound 120 using general procedure N.

[0475] [ka] Example 29: Compounds made by general procedure O General Procedure O: General Compound J or General Compound M (1.0 equivalent) is dissolved in DMA (10 volumes), followed by the sequential addition of N-methylmorpholine (4-) (10 equivalents) and HATU (1.2 equivalents) at 0°C. The mixture is stirred at 0°C for 30 minutes, and General Compound K or General Compound N (2.0 equivalents) dissolved in DMA (10 volumes) is added dropwise. The solution is stirred overnight at room temperature. The reaction mixture is poured into water and extracted with ethyl acetate. The combined organic layers are washed with 1N HCl, aqueous NaHCO3, and brine, then dried over Na2SO4 and concentrated to give a crude oil, which is purified by biotage purification to give General Compound O as a mixture of rotamers.

[0476] [ka] Compound 60-1 is prepared from compound 112 and compound 57 using general procedure O.

[0477] [ka] Compound 129 is prepared from compound 109 and compound 57 using general procedure O.

[0478] [ka] Compound 60-2 is prepared from compound 52 and compound 57 using general procedure O.

[0479] [ka] Compound 60-3 is prepared from compound 51 and compound 57 using general procedure O.

[0480] [ka] Compound 60-4 is prepared from compound 112 and compound 51 using general procedure O.

[0481] [ka] Compound 64-1 is prepared from compound 50 and compound 57 using general procedure O.

[0482] [ka] Compound 64-2 is prepared from compound 112 and compound 63 using general procedure O.

[0483] [ka] Compound 64-3 is prepared from compound 51 and compound 63 using general procedure O.

[0484] [ka] Compound 64-4 is prepared from compound 52 and compound 63 using general procedure O.

[0485] [ka] Compound 64-5 is prepared from compound 50 and compound 63 using general procedure O.

[0486] [ka] Compound 60-5 is prepared from compound 52 and compound 58 using general procedure O.

[0487] [ka] Compound 60-6 is prepared from compound 51 and compound 59 using general procedure O.

[0488] [ka] Compound 80-1 is prepared from compound 121 and compound 57 using general procedure O.

[0489] [ka] Compound 80-2 is prepared from compound 122 and compound 57 using general procedure O.

[0490] [ka] Compound 80-3 is prepared from compound 123 and compound 57 using general procedure O.

[0491] [ka] Compound 80-4 is prepared from compound 121 and compound 58 using general procedure O.

[0492] [ka] Compound 86-1 is prepared from compound 124 and compound 57 using general procedure O.

[0493] [ka] Compound 86-2 is prepared from compound 112 and compound 128 using general procedure O.

[0494] [ka] Compound 86-3 is prepared from compound 123 and compound 63 using general procedure O.

[0495] [ka] Compound 86-4 is prepared from compound 122 and compound 63 using general procedure O.

[0496] [ka] Compound 86-5 is prepared from compound 50 and compound 128 using general procedure O.

[0497] [ka] Compound 80-5 is prepared from compound 122 and compound 51 using general procedure O.

[0498] [ka] Compound 82-1 is prepared from compound 121 and compound 125 using general procedure O.

[0499] [ka] Compound 82-2 is prepared from compound 122 and compound 125 using general procedure O.

[0500] [ka] Compound 82-3 is prepared from compound 123 and compound 125 using general procedure O.

[0501] [ka] Compound 82-4 is prepared from compound 121 and compound 127 using general procedure O.

[0502] [ka] Compound 88-1 is prepared from compound 124 and compound 125 using general procedure O.

[0503] [ka] Compound 88-2 is prepared from compound 121 and compound 128 using general procedure O.

[0504] [ka] Compound 88-3 is prepared from compound 123 and compound 128 using general procedure O.

[0505] [ka] Compound 88-4 is prepared from compound 123 and compound 128 using general procedure O.

[0506] [ka] Compound 88-5 is prepared from compound 124 and compound 128 using general procedure O.

[0507] [ka] Compound 82-5 is prepared from compound 122 and compound 127 using general procedure O.

[0508] [ka] Example 30: Compounds made by general procedure P General Procedure P: General compound O (1.0 equiv.) is dissolved in DCE (750 vol.) and deoxygenated by sparging with N for 30 minutes. To this solution is added Grubbs I catalyst (20 mol%), and the resulting solution is refluxed for 60 hours. Upon completion, the solvent is removed, and the crude product is purified by Biotage purification to give general compound P.

[0509] [ka] Compound 61-1 is prepared from compound 60-1 using general procedure P.

[0510] [ka] Compound 130 is prepared from compound 129 using general procedure P.

[0511] [ka] Compound 61-2 is prepared from compound 60-2 using general procedure P.

[0512] [ka] Compound 61-3 is prepared from compound 60-3 using general procedure P.

[0513] [ka] Compound 61-4 is prepared from compound 60-4 using general procedure P.

[0514] [ka] Compound 65-1 is prepared from compound 64-1 using general procedure P.

[0515] [ka] Compound 65-2 is prepared from compound 64-2 using general procedure P.

[0516] [ka] Compound 65-3 is prepared from compound 64-3 using general procedure P.

[0517] [ka] Compound 65-4 is prepared from compound 64-4 using general procedure P.

[0518] [ka] Compound 65-5 is prepared from compound 64-5 using general procedure P.

[0519] [ka] Compound 61-5 is prepared from compound 60-5 using general procedure P.

[0520] [ka] Compound 61-6 is prepared from compound 60-6 using general procedure P.

[0521] [ka] Compound 81-1 is prepared from compound 80-1 using general procedure P.

[0522] [ka] Compound 81-2 is prepared from compound 80-2 using general procedure P.

[0523] [ka] Compound 81-3 is prepared from compound 80-3 using general procedure P.

[0524] [ka] Compound 81-4 is prepared from compound 80-4 using general procedure P.

[0525] [ka] Compound 87-1 is prepared from compound 86-1 using general procedure P.

[0526] [ka] Compound 87-2 is prepared from compound 86-2 using general procedure P.

[0527] [ka] Compound 87-3 is prepared from compound 86-3 using general procedure P.

[0528] [ka] Compound 87-4 is prepared from compound 86-4 using general procedure P.

[0529] [ka] Compound 87-5 is prepared from compound 86-5 using general procedure P.

[0530] [ka] Compound 81-5 is prepared from compound 80-5 using general procedure P.

[0531] [ka] Compound 83-1 is prepared from compound 82-1 using general procedure P.

[0532] [ka] Compound 83-2 is prepared from compound 82-2 using general procedure P.

[0533] [ka] Compound 83-3 is prepared from compound 82-3 using general procedure P.

[0534] [ka] Compound 83-4 is prepared from compound 82-4 using general procedure P.

[0535] [ka] Compound 89-1 is prepared from compound 88-1 using general procedure P.

[0536] [ka] Compound 89-2 is prepared from compound 88-2 using general procedure P.

[0537] [ka] Compound 89-3 is prepared from compound 88-3 using general procedure P.

[0538] [ka] Compound 89-4 is prepared from compound 88-4 using general procedure P.

[0539] [ka] Compound 89-5 is prepared from compound 88-5 using general procedure P.

[0540] [ka] Compound 83-5 is prepared from compound 82-5 using general procedure P.

[0541] [ka] Example 31: Compounds made by general procedure Q General Procedure Q: General compound P (1.0 equiv.) is dissolved in THF (8 vol.) and water (2 vol.). Lithium hydroxide monohydrate (5.0 equiv.) is added all at once at room temperature and stirred overnight. The solution is concentrated and partitioned between water and EtOAc. The pH of the aqueous phase is adjusted to pH 1 and extracted three times with EtOAc. The combined organics are washed with brine, dried over Na2SO4, and concentrated to give general compound QI. The isolated material is sufficiently pure to proceed to the next step.

[0542] General compound QI (1.0 equiv.) is treated with a solution of triethylsilane (0.4 vol.) and water (0.2 vol.) in TFA (5 vol.). The reaction is allowed to proceed for 16 hours, after which the solvent is removed and the crude oil is treated with excess ethyl ether. General compound Q-II is triturated with ether for 4 hours and collected in a glass-fritted funnel, which is sufficiently pure to proceed to the next step.

[0543] General compound Q-II (1 equivalent) is dissolved in a mixture of methanol (75 volumes) and water (25 volumes). Approximately 10% Pd / C (25% weight / weight) is added to the solution, and the reaction mixture is treated with hydrogen at 1 atmosphere pressure and allowed to react at room temperature for 4 hours. After 4 hours, the reaction mixture is filtered through a pad of Celite and washed three times with methanol. The organic fractions are combined, and the solvent is removed under reduced pressure to give general compound Q-III.

[0544] To a solution of general compound Q-III (1.0 equiv.) in 1N NaOH (20 vol.) at 0 °C is added Fmoc-Cl (1.2 equiv.). After 24 h at room temperature, the reaction mixture is diluted with HO and adjusted to pH 2-3 with 2N HCl. The aqueous layer is extracted three times with EtOAc. The combined organic phases are washed with brine. After drying over NaSO and filtration, the solvent is removed by rotary evaporation to give general compound Q.

[0545] [ka] Compound 62-1 is prepared from compound 61-1 using general procedure Q.

[0546] [ka] Compound 62-2 is prepared from compound 61-2 using general procedure Q.

[0547] [ka] Compound 62-3 is prepared from compound 61-3 using general procedure Q.

[0548] [ka] Compound 62-4 is prepared from compound 61-4 using general procedure Q.

[0549] [ka] Compound 62-5 is prepared from compound 61-5 using general procedure Q.

[0550] [ka] Compound 62-6 is prepared from compound 61-6 using general procedure Q.

[0551] [ka] Compound 84-1 is prepared from compound 81-1 using general procedure Q.

[0552] [ka] Compound 84-2 is prepared from compound 91-2 using general procedure Q.

[0553] [ka] Compound 84-3 is prepared from compound 81-3 using general procedure Q.

[0554] [ka] Compound 84-4 is prepared from compound 81-4 using general procedure Q.

[0555] [ka] Compound 84-5 is prepared from compound 81-5 using general procedure Q.

[0556] [ka] Compound 85-1 is prepared from compound 83-1 using general procedure Q.

[0557] [ka] Compound 85-2 is prepared from compound 83-2 using general procedure Q.

[0558] [ka] Compound 85-3 is prepared from compound 83-3 using general procedure Q.

[0559] [ka] Compound 85-4 is prepared from compound 83-4 using general procedure Q.

[0560] [ka] Compound 85-5 is prepared from compound 83-5 using general procedure Q.

[0561] [ka] Example 32: Compounds made by general procedure R General Procedure R: General Compound P (1.0 equiv.) is treated with a solution of triethylsilane (0.4 vol.) and water (0.2 vol.) in TFA (5 vol.). The reaction is allowed to proceed for 16 hours, after which the solvent is removed and the crude oil is treated with excess ethyl ether. General Compound RI is triturated with ether for 4 hours and collected in a glass-fritted funnel, which is sufficiently pure to proceed to the next step.

[0562] General compound RI (1.0 equivalent) is dissolved in a mixture of methanol (75 volumes) and water (25 volumes). Approximately 10% Pd / C (25% weight / weight) is added to this solution, and the reaction mixture is treated with hydrogen at 1 atmosphere pressure and allowed to react at room temperature for 4 hours. After 4 hours, the reaction mixture is filtered through a pad of Celite and washed three times with methanol. The organic fractions are combined, and the solvent is removed under reduced pressure to give general compound R-II.

[0563] To a solution of general compound R-II (1.0 eq) in dichloromethane (10 vol) at 0°C, N,N-diisopropylethylamine (3.0 eq) and Fmoc-Cl (1.2 eq) are added. The reaction is stirred at room temperature for 66 h. The mixture is concentrated, taken up in diethyl ether, and washed with 10% aqueous citric acid and saturated aqueous sodium bicarbonate. The organic layer is dried over magnesium sulfate, filtered, and concentrated. General compound R is purified by Biotage purification.

[0564] [ka] Compound 66-1 is prepared from compound 65-1 using general procedure R.

[0565] [ka] Compound 66-2 is prepared from compound 65-2 using general procedure R.

[0566] [ka] Compound 66-3 is prepared from compound 65-3 using general procedure R.

[0567] [ka] Compound 66-4 is prepared from compound 65-4 using general procedure R.

[0568] [ka] Compound 66-5 is prepared from compound 65-5 using general procedure R.

[0569] [ka] Compound 90-1 is prepared from compound 87-1 using general procedure R.

[0570] [ka] Compound 90-2 is prepared from compound 90-2 using general procedure R.

[0571] [ka] Compound 90-3 is prepared from compound 90-3 using general procedure R.

[0572] [ka] Compound 90-4 is prepared from compound 87-4 using general procedure R.

[0573] [ka] Compound 90-5 is prepared from compound 87-5 using general procedure R.

[0574] [ka] Compound 91-1 is prepared from compound 89-1 using general procedure R.

[0575] [ka] Compound 91-2 is prepared from 89-2 using general procedure R.

[0576] [ka] Compound 91-3 is prepared from compound 89-3 using general procedure R.

[0577] [ka] Compound 91-4 is prepared from compound 89-4 using general procedure R.

[0578] [ka] Compound 91-5 is prepared from compound 89-5 using general procedure R.

[0579] [ka] Example 33: Compounds made by general procedure S General Procedure S: To a solution of general compound R (1.0 equivalent) in THF (20 volumes) at 0° C. is added tetrabutylammonium fluoride 1 M in THF (2.0 alcohol equivalents). The resulting mixture is stirred at 0° C. for 1 minute and then concentrated in vacuo. The resulting crude product is purified by Biotage purification to afford general compound SI.

[0580] A solution of general compound SI (1.0 equiv.) in THF (20 vol.) is cooled to 0°C. The reagents are added in the following order: solid PPh3 (1.30 alcohol equivalents), DEAD (1.33 alcohol equivalents), and diphenylphosphoryl azide (1.40 alcohol equivalents). The reaction mixture is stirred at 0°C for 2.3 hours and then concentrated under reduced pressure. Biotage purification affords general compound S.

[0581] [ka] Compound 67-1 is prepared from compound 66-1 using general procedure S.

[0582] [ka] Compound 67-2 is prepared from compound 66-2 using general procedure S.

[0583] [ka] Compound 67-3 is prepared from compound 66-3 using general procedure S.

[0584] [ka] Compound 67-4 is prepared from compound 66-4 using general procedure S.

[0585] [ka] Compound 67-5 is prepared from compound 66-5 using general procedure S.

[0586] [ka] Compound 92-1 is prepared from compound 90-1 using general procedure S.

[0587] [ka] Compound 92-2 is prepared from compound 90-2 using general procedure S.

[0588] [ka] Compound 92-3 is prepared from compound 90-3 using general procedure S.

[0589] [ka] Compound 92-4 is prepared from compound 90-4 using general procedure S.

[0590] [ka] Compound 92-5 is prepared from compound 90-5 using general procedure S.

[0591] [ka] Compound 93-1 is prepared from compound 91-1 using general procedure S.

[0592] [ka] Compound 93-2 is prepared from compound 91-2 using general procedure S.

[0593] [ka] Compound 93-3 is prepared from compound 91-3 using general procedure S.

[0594] [ka] Compound 93-4 is prepared from compound 91-4 using general procedure S.

[0595] [ka] Compound 93-5 is prepared from compound 91-5 using general procedure S.

[0596] [ka] Example 34: Compounds made by general procedure T General Procedure T: To a solution of general compound U (1.0 equiv.) in THF (20 vol.) at room temperature was added solid triphenylphosphine (3.5 azide equiv.). After stirring for 1.5 h, HO (0.5 mL) was added and the reaction vessel was heated at 50° C. for 3 days. The organic solvent was removed under reduced pressure and the residue was purified by Biotage purification to give the corresponding free amine.

[0597] To a solution of general compound T1 (1.0 equivalent) in THF (20 volumes) at room temperature, N,N'-di-tert-butoxycarbonyl-1H-pyrazole-1-carboxamidine (1.5 amine equivalents) is added. After stirring at the same temperature for 12 hours, the mixture is poured into water and the resulting solution is extracted with Et2O. The combined organic extracts are dried over MgSO4, filtered, and concentrated in vacuo. The residue is purified by Biotage purification to give general compound T.

[0598] [ka] Compound 68-1 is prepared from compound 67-1 using general procedure T.

[0599] [ka] Compound 68-2 is prepared from compound 67-2 using general procedure T.

[0600] [ka] Compound 68-3 is prepared from compound 67-3 using general procedure T.

[0601] [ka] Compound 68-4 is prepared from compound 67-4 using general procedure T.

[0602] [ka] Compound 68-5 is prepared from compound 67-5 using general procedure T.

[0603] [ka] Compound 94-1 is prepared from compound 92-1 using general procedure T.

[0604] [ka] Compound 94-2 is prepared from compound 92-2 using general procedure T.

[0605] [ka] Compound 94-3 is prepared from compound 92-3 using general procedure T.

[0606] [ka] Compound 94-4 is prepared from compound 92-4 using general procedure T.

[0607] [ka] Compound 94-5 is prepared from compound 92-5 using general procedure T.

[0608] [ka] Compound 95-1 is prepared from compound 93-1 using general procedure T.

[0609] [ka] Compound 95-2 is prepared from compound 93-2 using general procedure T.

[0610] [ka] Compound 95-3 is prepared from compound 93-3 using general procedure T.

[0611] [ka] Compound 95-4 is prepared from compound 93-4 using general procedure T.

[0612] [ka] Compound 95-5 is prepared from compound 93-5 using general procedure T.

[0613] [ka] Example 35: Compounds made by general procedure U General procedure U: To a solution of general compound T (1.0 equivalent) in CH2Cl2 (10 volumes) at 0 °C, TFA (10 volumes) is added, and the mixture is stirred at the same temperature for 10 hours. The completion of the reaction is confirmed by UPLC of the reaction mixture. After removing the solvent and excess TFA under reduced pressure, general compound UI is used in the next step without any purification.

[0614] To general compound UI (1.0 equiv.) in THF (20 vol.) at 0° C. is sequentially added 3N aqueous NaOH (5 vol.) and N-(2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfornyl chloride (1.5 guanidine equiv.). The solution is allowed to warm to room temperature and stirred for 12 h. Water is added to the solution and the mixture is acidified to pH 2 with 1N aqueous KHSO. The mixture is extracted with EtOAc. The combined organic extracts are dried over MgSO, filtered, and concentrated in vacuo. The residue is purified by column chromatography to give general compound U.

[0615] [ka] Compound 69-1 is prepared from compound 68-1 using general procedure U.

[0616] [ka] Compound 69-2 is prepared from compound 68-2 using general procedure U.

[0617] [ka] Compound 69-3 is prepared from compound 68-3 using general procedure U.

[0618] [ka] Compound 69-4 is prepared from compound 68-4 using general procedure U.

[0619] [ka] Compound 69-5 is prepared from compound 68-5 using general procedure U.

[0620] [ka] Compound 96-1 is prepared from compound 94-1 using general procedure U.

[0621] [ka] Compound 96-2 is prepared from compound 94-2 using general procedure U.

[0622] [ka] Compound 96-3 is prepared from compound 94-3 using general procedure U.

[0623] [ka] Compound 96-4 is prepared from compound 94-4 using general procedure U.

[0624] [ka] Compound 96-5 is prepared from compound 94-5 using general procedure U.

[0625] [ka] Compound 97-1 is prepared from compound 95-1 using general procedure U.

[0626] [ka] Compound 97-2 is prepared from compound 95-2 using general procedure U.

[0627] [ka] Compound 97-3 is prepared from compound 95-3 using general procedure U.

[0628] [ka] Compound 97-4 is prepared from compound 95-4 using general procedure U.

[0629] [ka] Compound 97-5 is prepared from compound 95-5 using general procedure U.

[0630] Example 36: Preparation of Compound 133 [ka] Compound 133: β-ketophosphonate is synthesized using the procedure from Ivan Kolarovic, A., Tetrahedron 2015, 71, 8876.

[0631] Example 37: Preparation of Compound 134 [ka] Compound 134: The aldehyde (1.0 equiv.) in acetonitrile (10 vol.) is added to 133 (1.4 equiv.) and lithium chloride (2.0 equiv.), which is then cooled in an ice bath, and DIPEA (4.0 equiv.) is added and stirring is continued at 0° C. The reaction is stirred until it is deemed complete by LCMS or TLC, then partitioned between EA / saturated NaHCO and then back-extracted once. The organic layer is washed with half-saturated brine, dried over NaSO, and concentrated. Biotage purification gives compound 134-I.

[0632] To a solution of compound 134-I (1.0 equiv.) and CeCl3·7H2O (1.2 equiv.) in MeOH (20 vol.) at 0 °C, sodium borohydride (1.2 equiv.) is added portionwise, and the mixture is stirred for 30 min. The mixture is quenched with saturated aqueous NH4Cl, and the solvent is removed by evaporation. The mixture is extracted with Et2O, and the organic layers are combined, dried over Na2SO4, and concentrated under reduced pressure. The residue is purified by Biotage™ purification to give compound 134-II.

[0633] A mixture of compound 134-II (1.0 equiv.), (2,2,2-trifluoroacetyl)glycine (1.1 equiv.), and DMAP (0.10 equiv.) was taken up in DCM (12 volumes) to give a slightly yellowish suspension. This was cooled in an ice bath, and DIC (1.1 equiv.) was added portionwise. The mixture was allowed to warm slowly to room temperature and stirred overnight. The mixture was taken up in EtOAc, washed with NaHCO3 (sat.) × 2, brine (150 mL), dried over sodium sulfate, and concentrated. The residue was purified by Biotage™ purification to give general compound 134.

[0634] Example 38: Preparation of Compound 135 [ka] Compound 135: Claisen Rearrangement: Zinc chloride (1.5 equiv.) was melted under house vacuum using a propane burner to form a clear liquid. This was cooled to room temperature and added to THF (5 vol.). A solution of LDA in THF (1 M, 4 equiv.) was added to the ZnCl-THF solution, followed by the dropwise addition of a solution of compound 134 (1.0 equiv.) in THF (5 vol.) at -78 °C, during which the mixture turned dark red, then brightened toward the end of the addition. The solution was allowed to slowly warm to room temperature and stirred overnight. The reaction was quenched with 1 M HCl. The mixture was extracted twice with MTBE, dried, and concentrated. Compound 135-I was used directly in the next step.

[0635] Iodolactonization: Compound 135-I (1.0 equiv.) is then added to a solution of acetonitrile (10 vol.) and water (1 vol.), followed by diiodine (3.0 equiv.). The dark red solution is stirred overnight at room temperature. The reaction is quenched with aqueous sodium thiosulfate, extracted with ethyl acetate, dried, concentrated, and purified using Biotage to give compound 135-II.

[0636] Elimination: To a sealed tube containing diazabicyclo[5.4.0]undec-7-ene (1.2 equiv.) is added a solution of compound 135-II (1.0 equiv.) in anhydrous DMF (10 vol.) under a nitrogen atmosphere. The reaction mixture is heated at 60° C. for 2 h. The mixture is quenched with water, and the organic material is extracted three times with diethyl ether. The combined organic layers are washed with brine, dried over MgSO4, and filtered. The volatile materials are removed, and the resulting residue is purified by Biotage purification to give compound 135.

[0637] Example 39: Preparation of Compound 136 [ka] Compound 136: A solution of compound 135 (1.0 equiv.) in ethanol (10 vol.) is cooled in an ice bath and treated portionwise with sodium borohydride (1.1 equiv.). The mixture is allowed to warm slowly to room temperature and quenched with NaHCO3- (sat.). The mixture is concentrated to half its original volume, and the remainder is extracted with ethyl acetate and washed with brine. The combined aqueous phases are back-extracted with ethyl acetate and washed with brine. Concentration of the combined organic phases afforded the desired amino alcohol, which was used without purification. The crude material from above is taken up in methanol (10 vol.), water (0.5 vol.) and treated with potassium carbonate (5 equiv.). This is stirred overnight at room temperature. The mixture is concentrated, combined with Celite 521 suspended in DCM, filtered, and concentrated. The residue is passed through a silica gel plug (conditioned with 1% v / v concentrated ammonia) rinsed with 20% methanol in DCM, and the filtrate is concentrated and purified by Biotage purification to give the desired amino alcohol.

[0638] Example 40: Preparation of Compound 137 [ka] Compound 137: To a solution of benzoic acid (1.0 equiv.) and compound 136 (1.0 equiv.) was added triethylamine (1.0 equiv.) in acetonitrile-pyridine (5 vol.) and 3 ml (3 vol.) of CCl4. Within 3 hours, a solution of triphenylphosphine (1.0 equiv.) in acetonitrile-pyridine (5 vol.) was added. Stirring was continued for 1 hour to complete the formation of the intermediate amide. Triethylamine (2.0 equiv.) was added, followed within 3 hours by a solution of triphenylphosphine (2.0 equiv.) in acetonitrile-pyridine (5 vol.), as the reaction darkened. After 18 hours and overnight, the reaction mixture was evaporated, and the dark, viscous residue was stirred with toluene and ice-cold 2N NaOH. The aqueous phase was extracted three times with toluene, dried over Na2SO4, and evaporated. Biotage purification gave compound 137-I.

[0639] A mixture of compound 137-I (1.0 equiv.), (2,2,2)-trifluoroacetyl)glycine (1.0 equiv.), and DMAP (0.1 equiv.) is taken up in DCM (12 volumes) and cooled in an ice bath, followed by the addition of DIC (1.1 equiv.), added portionwise. The mixture is allowed to warm slowly to room temperature and stirred overnight. The mixture is taken up in ethyl acetate / MTBE (1:1), washed twice with NaHCO3 (sat.), brine, dried over sodium sulfate, and concentrated to give a crude residue. This is taken up in isopropanol and heated to 80 °C to give a homogeneous solution, which is then allowed to warm slowly to room temperature. The solid is collected by filtration and washed with isopropanol to give compound 137.

[0640] Example 41: Preparation of Compound 138 [ka] Compound 138: Zinc chloride (1.5 equiv.) is melted under house vacuum using a propane burner to a clear liquid. This is cooled to room temperature and taken up in THF (5 vol.). A solution of LDA (1 M, 4.0 equiv.) in THF is added to the ZnCl-THF solution, followed by the dropwise addition of a solution of compound 137 (1.0 equiv.) in THF (5 vol.) at -78 °C, during which the mixture turns dark red, then lightens toward the end of the addition. The solution is allowed to warm slowly to room temperature and stirred overnight. The reaction is quenched with 1 M HCl. The mixture is extracted twice with MTBE, dried, and concentrated to give compound 138, which is sufficiently pure for the next step.

[0641] Example 42: Preparation of Compound 139 [ka] Compound 139: To a stirred solution of compound 138 (1.0 eq) in dry ether (5 vol) and dry DCM (5 vol) under nitrogen, tert-butyl-2,2,2-trichloroacetimidate (5.0 eq) is added, and the resulting reaction mixture is stirred at room temperature for 2 days. The reaction is poured into NaHCO (saturated) and water (1:1) and extracted with DCM (3 times). The combined organic extracts are dried over sodium sulfate and concentrated under reduced pressure, and the resulting residue is purified by Biotage purification to give compound 139-I.

[0642] To a solution of compound 139-I (1.0 equiv.) in THF (10 vol.) is added lithium hydroxide monohydrate (3.0 equiv.) and water (2 vol.). The temperature of the reaction mixture is warmed to 20-30°C, and the reaction mixture is stirred for 10 hours. Upon completion of the reaction, water is added to the mixture, the compound is extracted four times with methyl tert-butyl ether, and the aqueous layer is removed. The organic layers are combined and washed with water. The organic layer is evaporated under reduced pressure and purified by Biotage purification to give compound 139-II.

[0643] To a solution of phthalic anhydride (1.0 equivalent) in dioxane (10 volumes) is added compound 139-II (1.0 equivalent) in dioxane (10 volumes). The reaction is stirred until deemed complete by UPLC. The solvent is evaporated under reduced pressure. The resulting residue is purified by Biotage purification to give compound 139.

[0644] Example 43: Preparation of Compound 140 [ka] Compound 140: Compound 139 (1.0 equivalent) is dissolved in a mixture of methanol (75 volumes) and water (25 volumes). Approximately 10% Pd / C (25% weight / weight) is added to this solution, and the reaction mixture is treated with hydrogen at 1 atmosphere pressure and allowed to react at room temperature for 4 hours. After 4 hours, the reaction mixture is filtered through a pad of Celite and washed three times with methanol. The organic fractions are combined, and the solvent is removed under reduced pressure to give compound 140.

[0645] Example 44: Preparation of Compound 141 [ka] Compound 141: To a solution of compound 140 (1.0 equiv.) in CHCl (10 vol.) at 0° C., TFA (10 vol.) is added, and the mixture is stirred at room temperature for 10 h. After removing the solvent and excess TFA under reduced pressure, compound 141-I is used in the next step without any further purification.

[0646] To a solution of compound 141-I (1.0 eq) in DMA (10 vol) is added DIPEA (5 eq) and HATU (2.0 eq). The mixture is stirred at room temperature overnight. Water and NaHCO3 (saturated) are added to the reaction mixture. The aqueous layer is extracted three times with ethyl acetate. The combined organic layers are washed with NaHCO3 (saturated), water, and brine. The organic layers are dried over sodium sulfate, filtered, and evaporated to give a residue. The residue is purified by Biotage purification to give compound 141.

[0647] Example 45: Preparation of Compound 142 [ka] Compound 142: Compound 142 (1.0 equivalent) and 1,2-dimethoxyethane (20 volumes) are placed in a flask under nitrogen. 40% aqueous methylamine (5 volumes) is added and stirred at room temperature for 5 hours. Upon completion of the reaction, 10% aqueous sodium hydroxide is added and the mixture is stirred for 16 hours. The resulting solid is collected by filtration. The filter cake is washed with water and then dried to give compound 142-I.

[0648] To a solution of compound 142-I (1.0 equiv.) in 1N NaOH (20 vol.) at 0 °C is added Fmoc-Cl (1.2 equiv.). After 24 h at room temperature, the reaction mixture is diluted with HO and the pH is adjusted to 2-3 with 2N HCl. The aqueous layer is extracted three times with EtOAc. The combined organic phases are washed with brine (1 × 10 mL). After drying over NaSO and filtration, the solvent is removed by rotary evaporation to give compound 142-II.

[0649] To a stirred mixture of compound 142-II (1.0 equiv.) and TEMPO (1.50 equiv.) in MeCN (15 vol.) at 0° C. is added dropwise a solution of NaClO2 (80% w / w, 2.8 equiv.) in phosphate buffer (0.67 N, pH=7, 10 vol.). To this mixture is added NaOCl (6% bleach, 0.7 equiv.) dropwise over 30 min. The reaction mixture is allowed to stir at 0° C. for 3 h. The reaction is quenched with saturated Na2SO3 and allowed to warm to room temperature. The biphasic solution is acidified with 12 M HCl (pH ∼2) and the aqueous layer is extracted three times with DCM. The combined organic layers are dried over MgSO4, filtered, and concentrated. The material is purified by Biotage purification to give 142-enantiomer 1 and 142-enantiomer 2.

[0650] Example 46: Preparation of Compound 143 [ka] Compound 143: A guanidinylated proline substrate is synthesized using the procedure from Ishiguro, M., J. Med. Chem. 2004, 47, 489.

[0651] Example 47: Compounds made by general procedure V [ka] General Procedure V: The peptide (1.0 equiv.) is dissolved in a 1:1 mixture of dichloroethane:acetonitrile (40 vol.). To the reaction mixture is added an aldehyde (1.5 equiv.) and (N-isocyanimino)triphenylphosphorane (1 equiv.). After cyclization and reverse-phase purification, pure fractions are pooled and lyophilized to give two diastereomers.

[0652] [ka] Compound 105-1 is prepared using general procedure V from the corresponding peptide substrate.

[0653] [ka] Compound 105-2 is prepared using general procedure V from the corresponding peptide substrate.

[0654] [ka] Compound 105-3 is prepared using general procedure V from the corresponding peptide substrate.

[0655] [ka] Compound 105-4 is prepared using general procedure V from the corresponding peptide substrate.

[0656] [ka] Compound 107-1 is prepared using general procedure V from the corresponding peptide substrate.

[0657] [ka] Compound 107-2 is prepared using general procedure V from the corresponding peptide substrate.

[0658] [ka] Compound 107-3 is prepared using general procedure V from the corresponding peptide substrate.

[0659] [ka] Compound 107-4 is prepared using general procedure V from the corresponding peptide substrate.

[0660] [ka] Compound 144 is prepared by the standard Fmoc solid phase synthesis protocol described in Example 13.

[0661] [ka] Compound 145 is prepared from PMO-002 and compound 144 by using general procedure D.

[0662] [ka] Compound 146 is prepared from PMO-424 and compound 144 by using general procedure D.

[0663] Example 48: Preparation of Compounds 166 / 167 and 168 / 169 [ka] Compound 147: 15.8 g (72% yield) of compound 147 was prepared from (E)-(3-bromoprop-1-en-1-yl)benzene (14.4 ml, 97.4 mmol) and ethyl (S,E)-2-((tert-butylsulfinyl)imino)acetate (10.0 g, 48.7 mmol) using general procedure H. 1 H NMR(400MHz,CDCl3)δ ppm 7.29-7.35(2H,m),7.25(1H,m,J=6.90Hz),7.19(2H,d,J=7.75Hz),6.09(1H ,ddd,J=17.60,9.00Hz),5.13-5.22(2H,m),4.13-4.26(3H,m),3.92(1H,br d,J=9.38Hz),3.71(1H,t,J=7.94Hz),1.28(3H,t,J=7.07Hz),1.08(9H,s);LRMS(ESI,m / z)[C 17 H 25 NO3S+Na] + Calculated value 346.1; measured value 346.1.

[0664] [ka] 7.7 g (100% yield) of compound 148 was prepared from compound 147 (11.4 g, 35.2 mmol) using general procedure I. 1H NMR(400MHz,CDCl3)δ ppm 7.28-7.38(2H,m),7.21-7.26(3H,m),6.12(1H,ddd,J=16.88,9.13,8.50Hz),5.10-5.22( 2H,m),4.16(2H,q,J=7.13Hz),3.80(1H,d,J=7.63Hz),3.64(1H,t,J=7.94Hz),1.87(2H,br s),1.25(3H,t,J=7.13Hz);LRMS(ESI,m / z)[C 13 H 17 NO2 + H] + Calculated value 220.14; measured value 220.1.

[0665] [ka] 10.0 g (86% yield) of compound 149 was prepared from compound 148 (7.7 g, 34.3 mmol) using general procedure J. 1 H NMR(400MHz,CDCl3)δ ppm 7.28-7.37(3H,m),7.21-7.26(2H,m),6.11(1H,ddt,J=17.50,8.80,8.80,8.80Hz),5.23(2H,br d,J=13.51Hz),4.81(1H,br d,J=8.38Hz),4.68(1H,br t,J=6.88Hz),3.76-3.95(1H,m),1.40(9H,s);LRMS(ESI,m / z)[C 16 H 21 NO4 + Na] + Calculated value 314.14; measured value 314.1.

[0666] [ka] Compound 150 was synthesized using the procedure from Creighton, CJ, Bioorg. Med. Chem. 12, 2004, 4375. 1H NMR(400MHz,chloroform-d)δ ppm 7.12(1H,d,J=7.88Hz),6.41-6.46(2H,m),5.67-5.79(1H,m),5.03-5.14(2H,m),3. 80(6H,s),3.71-3.77(1H,m),3.62-3.71(4H,m),3.35(1H,t,J=6.57Hz),2.42(2H,br t,J=6.88Hz),1.20-1.35(1H,m);LRMS(ESI,m / z)[C 15 H 21 NO4 + Na] + Calculated value 302.14; measured value 302.1.

[0667] [ka] Compound 151: Compound 151 (940 mg, 83% yield) was prepared from compound 149 (4.90 g, 2.06 mmol) and compound 150 (1.15 g, 4.12 mmol) using general procedure O. 1 H NMR (400 MHz, chloroform-d) δ ppm 7.17-7.24 (5H, m), 7.04-7.17 (2H, m), 6.26-6.39 (2H, m), 5.96-6.10 (1H, m), 5.44-5.51 (1H, m), 4.92-5.17 (4H, m), 4.70-85 (2H, m), 4.33-4.42 (1H, m), 4.16 (1H, d, J =15.76Hz),3.82-4.03(1H,m),3.66-3.76(6H,m),3.53(3H,s),2.69(1H,dt,J=14. 07,6.85Hz),2.25(1H,dt,J=14.38,7.44Hz),1.20-1.28(9H,m);LRMS(ESI,m / z)[C 31 H 40 N2O7+H] + Calculated value 553.3; measured value 553.4.

[0668] [ka] Compound 152: Compound 151 (61.0 mg, 0.11 mmol, 1 equiv) was dissolved in toluene (53 mL, 870 vol) and the solution was sparged with N for 30 minutes. Ruthenium and [1,3-bis-(2,4,6-trimethylphenyl)-2-imidazolinylidene]dichloro[[2-(1-methylethoxy)phenyl]methylene] (41.6 mg, 0.066 mmol, 0.5 equiv) were added in two small portions over 1 hour. The resulting solution was refluxed for 6 hours. The residue was concentrated and purified by Biotage purification to give compound 152 (37 mg, 0.071 mmol, 63.9% yield). 1 H NMR(400MHz,chloroform-d)δ ppm 7.27(6H,m,J=3.63Hz),6.39-6.49(2H,m),5.80-5.89(1H,m),5.24-5.58(2H,m),5.08(1H,br t,J=10.44Hz),4.84(1H,br d,J=14.38Hz),4.34-4.45(2H,m),3.76-3.85(6H,m),3.67(3H,s),3.38-3.52(1 H,m),3.00-3.30(1H,m),2.79-2.94(1H,m),1.22-1.38(9H,m);LRMS(ESI,m / z)[C 29 H 36 N2O7+H] + Calculated value 525.3; measured value 525.5.

[0669] [ka] Compound 153: Compound 152 (1.17 g, 2.23 mmol, 1 equiv) was dissolved in MeOH (34.2 ml, 30 vol) followed by the addition of Pd—C (0.598 g, 50% wt, 10% Pd based). H was bubbled through the solution for 5 min and the reaction was placed under an atmosphere of H. The reaction was stirred overnight. The suspension was filtered through Celite and washed with water and MeOH. The residue was then concentrated and purified by Biotage purification to give compound 153 (900 mg, 1.71 mmol, 77% yield). 1H NMR(400MHz,chloroform-d)δ ppm 7.25-7.29(5H,m),7.14-7.21(1H,m),6.44-6.52(2H,m),5.25(1H,d,J=9.63Hz),5.09(1H,d,J=14.13Hz) ,4.79(1H,t,J=11.00Hz),4.41(1H,d,J=14.13Hz),4.35(1H,t,J=4.94Hz),3.80-3.84(9H,m),3.11(1H,br dd,J=11.44,2.69Hz),2.22-2.40(1H,m),1.93-2.12(1H,m),1.76-1.87(1 H,m),1.68-1.76(1H,m),1.57(9H,s),1.39-1.55(1H,m);LRMS(ESI,m / z)[C 29 H 38 N2O7+H] + Calculated value 527.3; measured value 527.5.

[0670] [ka] Compound 154: Compound 153 (527 mg, 1.00 mmol, 1 equiv.) was treated with a solution of triethylsilane (0.20 ml, 2 vol.), water (2 ml, 4 vol.), and TFA (0.10 ml, 2 vol.). The reaction was allowed to proceed for 16 hours, after which the solvent was removed and the crude oil was treated with excess heptane. The product was triturated in heptane for 4 hours and then collected. After 3 hours, the solvent was removed under reduced pressure and the compound was purified by Biotage purification to give compound 154 (391 mg, 1.04 mmol, quantitative) as the TFA salt. LRMS (ESI, m / z) [C 15 H 20 N2O3+H] + Calculated value 277.2; measured value 277.8.

[0671] [ka] Compound 155: Compound 154 (918 mg, 2.35 mmol, 1 equiv) was dissolved in THF (8 mL, 9 vol) and water (2 mL, 2 vol) at room temperature, to which lithium hydroxide hydrate (493 mg, 11.8 mmol, 5 equiv) was added. The solution was stirred overnight, and upon completion, the pH was adjusted with HCl (1 M) until the pH was approximately 1-2. The solution was extracted with 3 portions of EtOAc, washed with brine, and the organics were dried over Na2SO4. The resulting solution was concentrated to dryness and analyzed without further purification to give compound 155 (472 mg, 77% yield). This material was sufficiently pure to proceed to the next step. LRMS (ESI, m / z) [C 14 H 18 N2O3+H] + Calculated value 263.1; measured value 263.8.

[0672] [ka] Compound 156: Compound 155 (472 mg, 1.80 mmol, 1 equiv) was dissolved in 1,4-dioxane (5 mL, 10 vol) and saturated sodium bicarbonate solution (5 mL, 10 vol). Methyl (9H-fluoren-9-yl)carbonochloridate (512 mg, 1.98 mmol, 1.1 equiv) was added and stirred overnight. The pH of the solution was adjusted to approximately pH 1-2. The resulting aqueous phase was extracted with EA x 3, and the organics were washed and concentrated. The resulting residue was purified by reverse-phase Biotage purification to give compound 156 (650 mg, 1.34 mmol, 75% yield). 1H NMR(400MHz,DMSO-d6)δ ppm 12.59-13.21(1H,br,s),8.12-8.24(1H,m),7.85(2H,brd,J=7.50Hz),7.49-7.61(3 H,m),7.34-7.46(4H,m),7.19-7.34(5H,m),4.86(1H,dd,J=11.88,8.75Hz),4.64(1 H,d,J=4.25Hz),4.37-4.46(1H,m),4.02-4.12(2H,m),2.85(1H,t,J=11.13Hz),2.0 0-2.19(2H,m),1.74-1.87(2H,m),1.51-1.68(1H,m),1.42(1H,m);LRMS(ESI,m / z)[C 29 H 28 N2O5+H] + Calculated value 485.2; measured value 485.0.

[0673] [ka] Compound 157 was prepared by the standard Fmoc solid phase synthesis protocol described in Example 13. MW=1200.44, LRMS (ESI, m / z): C 54 H 81 N 21 O9S's [M+2H] 2+ Calculated m / z for ion: 600.8; found: 600.5.

[0674] [ka] Compound 158 was prepared by the standard Fmoc solid phase synthesis protocol described in Example 13. MW=1091.31, LRMS (ESI, m / z): C 49 H 74 N 18 O9S's [M+2H] 2+ Calculated m / z for ion: 546.29; found: 546.65.

[0675] [ka] Compound 159 was prepared by the standard Fmoc solid phase synthesis protocol described in Example 13. MW=1100.32, LRMS (ESI, m / z): C 46 H 77 N 21 O9S's [M+2H] 2+ Calculated m / z for ion: 550.8; found: 551.2.

[0676] General Procedure W: The peptide of choice (1 equivalent) was dissolved in DMF (15 volumes), followed by the addition of a solution of 4-methyl-2-oxo-2H-chromen-7-yl (4-(pyridin-2-yldisulfanyl)butyl)carbonate or 2-((4-azidobutyl)disulfanyl)pyridine (5 equivalents) in THF (15 volumes). The resulting mixture was stirred overnight at room temperature. This solution was subjected to preparative HPLC purification using the following purification conditions. The desired disulfide conjugate was obtained.

[0677] General peptide purification conditions by RP-HPLC:

[0678] [Table 41]

[0679] [ka] Compound 160: Compound 160 (3.95 mg, 48% yield) was prepared as the TFA salt from compound 157 (5.0 mg; 4.2 μmol) and the TFA salt of 4-methyl-2-oxo-2H-chromen-7-yl (4-(pyridin-2-yldisulfanyl)butyl) carbonate by general procedure W. MW=1200.44, LRMS (ESI, m / z): C 69 H 95 N21O 14 S2 [M+2H] 2+ Calculated m / z for ion: 753.9; Found: 753.9.

[0680] [ka] Compound 161: Compound 161 (2.1 mg, 70% yield) was prepared as the TFA salt from compound 158 (2.5 mg; 1.62 μmol) and the TFA salt of 4-methyl-2-oxo-2H-chromen-7-yl (4-(pyridin-2-yldisulfanyl)butyl) carbonate by general procedure W. MW=1397.64, LRMS (ESI, m / z): C 64 H 88 N 18 O 14 S2 [M+2H] 2+ Calculated m / z for ion: 699.3; found: 699.4.

[0681] [ka] Compound 162: Compound 162 (2.4 mg, 80% yield) was prepared as the TFA salt from compound 159 (2.5 mg; 1.61 μmol) and the TFA salt of 4-methyl-2-oxo-2H-chromen-7-yl (4-(pyridin-2-yldisulfanyl)butyl) carbonate by general procedure W. MW=1406.65, LRMS (ESI, m / z): C 61 H 91 N 21 O 14 S2 [M+2H] 2+ Calculated m / z for ion: 703.8; found: 703.9.

[0682] [ka] Compound 163: Compound 163 (1.06 mg, 36% yield) was prepared as the TFA salt from compound 158 (2.7 mg; 1.75 μmol) and the TFA salt of 2-((4-azidobutyl)disulfanyl)pyridine by general procedure W. MW=1220.49, LRMS (ESI, m / z): C 53 H 81 N 21O9S2 [M+H] + Calculated m / z for ion: 1220.6; found: 1220.4.

[0683] [ka] Compound 164: Compound 164 (1.24 mg, 44% yield) was prepared as the TFA salt from compound 159 (2.6 mg; 1.67 μmol) and the TFA salt of 2-((4-azidobutyl)disulfanyl)pyridine by general procedure W. MW=1228.63, LRMS (ESI, m / z): C 50 H 84 N 24 O9S2 [M+H] + Calculated m / z for ion: 12...

Claims

1. 1. A peptide-antisense oligonucleotide conjugate comprising a cell-penetrating peptide conjugated to an antisense oligonucleotide, wherein the antisense oligonucleotide is complementary to a portion of SEQ ID NO: 1, and the peptide-antisense oligonucleotide conjugate has a CD33 exon-2 skipping efficiency of 30% or greater according to a standard exon skipping efficiency assay for the antisense oligonucleotide.

2. A peptide-antisense oligonucleotide conjugate comprising a cell-penetrating peptide conjugated to an antisense oligonucleotide, wherein the antisense oligonucleotide comprises all or a portion of SEQ ID NO:2, SEQ ID NO:12, or SEQ ID NO:

224.

3. 3. The peptide-antisense oligonucleotide conjugate of claim 1, wherein the antisense oligonucleotide is 16 to 30 nucleotides in length, 18 to 30 nucleotides in length, 18 to 25 nucleotides in length, 18 to 21 nucleotides in length, 21 to 30 nucleotides in length, 21 to 25 nucleotides in length, or 25 to 30 nucleotides in length.

4. 3. The peptide-antisense oligonucleotide conjugate of claim 1, wherein the antisense oligonucleotide is 21 or 25 nucleotides in length.

5. The peptide-antisense oligonucleotide conjugate of any one of claims 1 to 4, wherein the antisense oligonucleotide comprises one or more non-natural sugar moieties, one or more non-natural internucleotide linkages, or one or more non-natural sugar moieties and one or more non-natural internucleotide linkages.

6. The peptide-antisense oligonucleotide conjugate of claim 5, wherein the antisense oligonucleotide comprises one or more unnatural sugar moieties.

7. The peptide-antisense oligonucleotide conjugate of claim 6, wherein the antisense oligonucleotide comprises a phosphorodiamidate morpholino oligomer (PMO).

8. 8. The peptide-antisense oligonucleotide conjugate of claim 7, wherein the antisense oligonucleotide has a CD33 exon-2 skipping efficiency of 30% or more according to a standard exon skipping efficiency assay for PMO ASOs.

9. The peptide-antisense oligonucleotide conjugate of claim 6, wherein the antisense oligonucleotide comprises a methoxyethyl ribose oligomer (MOE).

10. 10. The peptide-antisense oligonucleotide conjugate of claim 9, wherein the antisense oligonucleotide has a CD33 exon-2 skipping efficiency of 30% or greater according to a standard exon skipping efficiency assay for MOE ASO.

11. The peptide-antisense oligonucleotide conjugate of any one of claims 5 to 10, wherein the antisense oligonucleotide comprises one or more non-natural internucleotide linkages.

12. 12. The peptide-antisense oligonucleotide conjugate of claim 11, wherein the one or more non-natural internucleotide linkages comprise one or more phosphorodiamidate linkages and / or one or more phosphorothioate linkages.

13. 13. The peptide-antisense oligonucleotide conjugate of claim 11 or 12, wherein all of the one or more non-natural internucleotide linkages have an Sp configuration.

14. 13. The peptide-antisense oligonucleotide conjugate of claim 11 or 12, wherein all of the one or more non-natural internucleotide linkages have the Rp configuration.

15. The peptide-antisense oligonucleotide conjugate of claim 11 or 12, wherein the antisense oligonucleotide comprises one or more non-natural internucleotide linkages having an Sp configuration and one or more non-natural internucleotide linkages having an Rp configuration.

16. The peptide-antisense oligonucleotide conjugate of any one of claims 1 to 15, wherein the antisense oligonucleotide comprises one or more modified nucleobases.

17. A composition comprising the peptide-antisense oligonucleotide conjugate of any one of claims 1 to 16, and optionally a pharmaceutically acceptable carrier or excipient.

18. A peptide-antisense oligonucleotide conjugate comprising a cell-penetrating peptide conjugated to all or a portion of PMO-002 (SEQ ID NO: 2), MOE-012 (SEQ ID NO: 12), or PMO-424 (SEQ ID NO: 224).

19. A peptide-antisense oligonucleotide conjugate comprising a cell-penetrating peptide conjugated to an antisense oligonucleotide selected from the group consisting of PMO-002 (SEQ ID NO: 2), MOE-012 (SEQ ID NO: 12), and PMO-424 (SEQ ID NO: 224).

20. 20. The peptide-antisense oligonucleotide conjugate of any one of claims 1 to 19, wherein the peptide comprises at least one proteinogenic amino acid, at least one non-proteinogenic amino acid, or at least one proteinogenic amino acid and at least one non-proteinogenic amino acid.

21. 21. The peptide-antisense oligonucleotide conjugate of claim 20, wherein the peptide comprises 5 to 25 amino acids.

22. 22. The peptide-antisense oligonucleotide conjugate of claim 20 or 21, wherein the non-proteinogenic amino acid comprises a modified proline residue, a lipophilic group, and / or a lactam group.

23. The peptide-antisense oligonucleotide conjugate of any one of claims 1 to 22, wherein the peptide is a linear peptide.

24. 24. The peptide-antisense oligonucleotide conjugate of claim 23, wherein the linear peptide comprises at least a portion of a Pip6a, ApoE, and / or neurotensin-based peptide.

25. The peptide-antisense oligonucleotide conjugate of any one of claims 1 to 22, wherein the peptide is a cyclic peptide.

26. The cyclic peptide is CPP9 【Chemistry 1】 , peptide 1 【Chemistry 2】 , peptide 2 【Transformation 3】 , or peptide 3 【Chemistry 4】 26. The peptide-antisense oligonucleotide conjugate of claim 25,

27. Compound 31 【Transformation 5】 27. The peptide-antisense oligonucleotide conjugate of claim 26,

28. The peptide-antisense oligonucleotide conjugate of any one of claims 1 to 27, wherein the peptide comprises a lipoic acid group.

29. The peptide is compound 21 【Transformation 6】 Compound 22 【Transformation 7】 or Compound 23 【Transformation 8】 29. The peptide-antisense oligonucleotide conjugate of claim 28,

30. Compound 33 【Chemistry 9】 30. The peptide-antisense oligonucleotide conjugate of claim 29, wherein

31. 29. The peptide-antisense oligonucleotide conjugate of claim 28, wherein the lipoic acid group is an (R)-lipoic acid group.

32. 29. The peptide-antisense oligonucleotide conjugate of claim 28, wherein the lipoic acid group is an (S)-lipoic acid group.

33. The peptide-antisense oligonucleotide conjugate of any one of claims 1 to 32, wherein the peptide comprises an oxadiazole bond.

34. The peptide-antisense oligonucleotide conjugate of any one of claims 1 to 33, wherein the peptide is directly conjugated to the antisense oligonucleotide.

35. 35. The peptide-antisense oligonucleotide conjugate of claim 34, wherein the peptide is conjugated to the antisense oligonucleotide using a chemical reaction.

36. 36. The peptide-antisense oligonucleotide conjugate of claim 35, wherein the chemical reaction is a strain-promoted azide-alkyne cycloaddition reaction, a strained alkene-tetrazine cycloaddition reaction, or an amide coupling reaction.

37. The peptide-antisense oligonucleotide conjugate of any one of claims 1 to 33, wherein the peptide is indirectly conjugated to the antisense oligonucleotide, and a linker is conjugated between the peptide and the antisense oligonucleotide.

38. The peptide-antisense oligonucleotide conjugate of any one of claims 1 to 37, wherein the peptide-antisense oligonucleotide conjugate further comprises one or more nuclear localization sequences, and the one or more nuclear localization sequences are independently conjugated to the peptide and / or the antisense oligonucleotide.

39. 1. A method for inducing exon-2 skipping in the CD33 gene during pre-mRNA splicing, the method comprising introducing into a cell a peptide-antisense oligonucleotide conjugate, wherein the peptide-antisense oligonucleotide conjugate comprises a cell-penetrating peptide conjugated to an antisense oligonucleotide, the antisense oligonucleotide being complementary to a portion of SEQ ID NO: 1, and the peptide-antisense oligonucleotide conjugate has a CD33 exon-2 skipping efficiency of 30% or greater according to a standard exon skipping efficiency assay for the antisense oligonucleotide.

40. 1. A method for inducing exon-2 skipping in the CD33 gene during pre-mRNA splicing, the method comprising introducing into a cell a peptide-antisense oligonucleotide conjugate, wherein the peptide-antisense oligonucleotide conjugate comprises a cell-penetrating peptide conjugated to an antisense oligonucleotide, and the antisense oligonucleotide comprises all or a portion of SEQ ID NO:2, SEQ ID NO:12, or SEQ ID NO:

224.

41. 41. The method of claim 39 or 40, wherein the antisense oligonucleotide is 16 to 30 nucleotides in length, 18 to 30 nucleotides in length, 18 to 25 nucleotides in length, 18 to 21 nucleotides in length, 21 to 30 nucleotides in length, 21 to 25 nucleotides in length, or 25 to 30 nucleotides in length.

42. 41. The method of claim 39 or 40, wherein the antisense oligonucleotide is 21 or 25 nucleotides in length.

43. 43. The method of any one of claims 39 to 42, wherein the antisense oligonucleotide comprises one or more non-natural sugar moieties, one or more non-natural internucleotide linkages, or one or more non-natural sugar moieties and one or more non-natural internucleotide linkages.

44. 44. The method of claim 43, wherein the antisense oligonucleotide comprises one or more unnatural sugar moieties.

45. 45. The method of claim 44, wherein the antisense oligonucleotide comprises a phosphorodiamidate morpholino oligomer (PMO).

46. 46. ​​The method of claim 45, wherein the peptide-antisense oligonucleotide conjugate has a CD33 exon-2 skipping efficiency of 30% or greater according to a standard exon skipping efficiency assay for PMO ASOs.

47. 45. The method of claim 44, wherein the antisense oligonucleotide comprises a methoxyethyl ribose oligomer (MOE).

48. 48. The method of claim 47, wherein the antisense oligonucleotide has a CD33 exon-2 skipping efficiency of 30% or greater according to a standard exon skipping efficiency assay for MOE ASO.

49. 49. The method of any one of claims 43 to 48, wherein the antisense oligonucleotide comprises one or more non-natural internucleotide linkages.

50. 50. The method of claim 49, wherein all of the one or more non-natural internucleotide linkages have an Sp configuration.

51. 50. The method of claim 49, wherein all of the one or more non-natural internucleotide linkages have an Rp configuration.

52. 50. The method of claim 49, wherein the one or more non-natural internucleotide linkages comprise one or more non-natural internucleotide linkages having an Sp configuration and one or more non-natural internucleotide linkages having an Rp configuration.

53. 53. The method of any one of claims 39 to 52, wherein the antisense oligonucleotide comprises one or more modified nucleobases.

54. 54. The method of any one of claims 39 to 53, wherein the peptide-antisense oligonucleotide conjugate further comprises a pharmaceutically acceptable carrier or excipient.

55. 1. A method for inducing exon-2 skipping in the CD33 gene during pre-mRNA splicing, the method comprising introducing into a cell a peptide-antisense oligonucleotide conjugate, wherein the peptide-antisense oligonucleotide conjugate comprises an antisense oligonucleotide conjugated to a cell-penetrating peptide, and the antisense oligonucleotide comprises all or a portion of PMO-002 (SEQ ID NO: 2), MOE-012 (SEQ ID NO: 12), or PMO-424 (SEQ ID NO: 224).

56. 1. A method for inducing exon-2 skipping in the CD33 gene during pre-mRNA splicing, the method comprising introducing a peptide-antisense oligonucleotide conjugate into a cell, wherein the peptide-antisense oligonucleotide conjugate comprises an antisense oligonucleotide conjugated to a cell-penetrating peptide, and the antisense oligonucleotide is selected from the group consisting of PMO-002 (SEQ ID NO: 2), MOE-012 (SEQ ID NO: 12), and PMO-424 (SEQ ID NO: 224).

57. The method of any one of claims 39 to 56, wherein the cell is an animal cell.

58. 58. The method of claim 57, wherein the cell is a human cell.

59. 1. A method of treating a subject having a neurodegenerative disease, the method comprising administering to the subject a therapeutically effective amount of a peptide-antisense oligonucleotide conjugate, wherein the peptide-antisense oligonucleotide conjugate comprises an antisense nucleotide conjugated to a cell-penetrating peptide, the antisense oligonucleotide is complementary to a portion of SEQ ID NO: 1, and the peptide-antisense oligonucleotide conjugate has a CD33 exon-2 skipping efficiency of 30% or greater according to a standard exon skipping efficiency assay for the antisense oligonucleotide.

60. 1. A method of treating a subject having a neurodegenerative disease, the method comprising administering to the subject a therapeutically effective amount of a peptide-antisense oligonucleotide conjugate, wherein the peptide-antisense oligonucleotide conjugate comprises an antisense oligonucleotide conjugated to a cell-penetrating peptide, and the antisense oligonucleotide is complementary to all or a portion of SEQ ID NO:2, SEQ ID NO:12, or SEQ ID NO:

224.

61. 61. The method of claim 59 or 60, wherein the antisense oligonucleotide is 16 to 30 nucleotides in length, 18 to 30 nucleotides in length, 18 to 25 nucleotides in length, 18 to 21 nucleotides in length, 21 to 30 nucleotides in length, 21 to 25 nucleotides in length, or 25 to 30 nucleotides in length.

62. 62. The method of any one of claims 59 to 61, wherein the antisense oligonucleotide is 21 or 25 nucleotides in length.

63. 63. The method of any one of claims 59-62, wherein the antisense oligonucleotide comprises one or more non-natural sugar moieties, one or more non-natural internucleotide linkages, or one or more non-natural sugar moieties and one or more non-natural internucleotide linkages.

64. 64. The method of claim 63, wherein the antisense oligonucleotide comprises one or more unnatural sugar moieties.

65. 65. The method of claim 64, wherein the antisense oligonucleotide comprises a phosphorodiamidate morpholino oligomer (PMO).

66. 66. The method of claim 65, wherein the peptide-antisense oligonucleotide conjugate has a CD33 exon-2 skipping efficiency of 30% or greater according to a standard exon skipping efficiency assay for PMO ASOs.

67. 65. The method of claim 64, wherein the antisense oligonucleotide comprises a methoxyethyl ribose oligomer (MOE).

68. 68. The method of claim 67, wherein the antisense oligonucleotide has a CD33 exon-2 skipping efficiency of 30% or greater according to a standard exon skipping efficiency assay for MOE ASO.

69. 69. The method of any one of claims 63 to 68, wherein the antisense oligonucleotide comprises one or more non-natural internucleotide linkages.

70. 70. The method of claim 69, wherein all of the one or more non-natural internucleotide linkages have an Sp configuration.

71. 70. The method of claim 69, wherein all of the one or more non-natural internucleotide linkages have an Rp configuration.

72. 70. The method of claim 69, wherein the one or more non-natural internucleotide linkages comprise one or more non-natural internucleotide linkages having an Sp configuration and one or more non-natural internucleotide linkages having an Rp configuration.

73. 73. The method of any one of claims 59 to 72, wherein said antisense oligonucleotide comprises one or more modified nucleobases.

74. 74. The method of any one of claims 59 to 73, wherein the peptide-antisense oligonucleotide conjugate further comprises a pharmaceutically acceptable carrier or excipient.

75. 1. A method of treating a subject having a neurodegenerative disease, comprising administering to the subject a therapeutically effective amount of a peptide-antisense oligonucleotide conjugate, wherein the peptide-antisense oligonucleotide conjugate comprises an antisense oligonucleotide conjugated to a cell-penetrating peptide, and the antisense oligonucleotide comprises all or a portion of PMO-002 (SEQ ID NO: 2), MOE-012 (SEQ ID NO: 12), or PMO-424 (SEQ ID NO: 224).

76. 1. A method of treating a subject having a neurodegenerative disease, comprising administering to the subject a therapeutically effective amount of a peptide-antisense oligonucleotide conjugate, wherein the peptide-antisense oligonucleotide conjugate comprises an antisense oligonucleotide conjugated to a cell-penetrating peptide, and the antisense oligonucleotide is selected from the group consisting of PMO-002 (SEQ ID NO: 2), MOE-012 (SEQ ID NO: 12), or PMO-424 (SEQ ID NO: 224).

77. 77. The method of any one of claims 59 to 76, wherein the subject is a human subject.

78. 78. The method of any one of claims 59 to 77, wherein the neurodegenerative disease is Alzheimer's disease.

79. 10. The peptide-antisense oligonucleotide conjugate of claim 1 for use in a method for inducing exon-2 skipping in the CD33 gene during pre-mRNA splicing, the method comprising introducing into a cell the peptide-antisense oligonucleotide conjugate of claim 1, wherein the peptide-antisense oligonucleotide conjugate hybridizes to a target region of the CD33 gene and induces exon-2 skipping during pre-mRNA splicing of the CD33 gene.

80. 3. The peptide-antisense oligonucleotide conjugate of claim 2 for use in a method for inducing exon-2 skipping in the CD33 gene during pre-mRNA splicing, the method comprising introducing into a cell the peptide-antisense oligonucleotide conjugate of claim 2, wherein the peptide-antisense oligonucleotide conjugate hybridizes to a target region of the CD33 gene and induces exon-2 skipping during pre-mRNA splicing of the CD33 gene.

81. 81. The peptide-antisense oligonucleotide conjugate of claim 79 or 80, wherein the antisense oligonucleotide is 16 to 30 nucleotides in length, 18 to 30 nucleotides in length, 18 to 25 nucleotides in length, 18 to 21 nucleotides in length, 21 to 30 nucleotides in length, 21 to 25 nucleotides in length, or 25 to 30 nucleotides in length.

82. 81. The peptide-antisense oligonucleotide conjugate of claim 79 or 80, wherein the antisense oligonucleotide is 21 or 25 nucleotides in length.

83. 83. The peptide-antisense oligonucleotide conjugate of any one of claims 79 to 82, wherein the antisense oligonucleotide comprises one or more non-natural sugar moieties, one or more non-natural internucleotide linkages, or one or more non-natural sugar moieties and one or more non-natural internucleotide linkages.

84. 84. The peptide-antisense oligonucleotide conjugate of claim 83, wherein the antisense oligonucleotide comprises one or more modified sugar moieties.

85. 85. The peptide-antisense oligonucleotide conjugate of claim 84, wherein the antisense oligonucleotide comprises a phosphorodiamidate morpholino oligomer (PMO).

86. 86. The peptide-antisense oligonucleotide conjugate of claim 85, having a CD33 exon-2 skipping efficiency of 30% or more according to a standard exon skipping efficiency assay for PMO ASOs.

87. 85. The peptide-antisense oligonucleotide conjugate of claim 84, wherein the antisense oligonucleotide comprises a methoxyethyl ribose oligomer (MOE).

88. 88. The peptide-antisense oligonucleotide conjugate of claim 87, wherein the antisense oligonucleotide has a CD33 exon-2 skipping efficiency of 30% or greater according to a standard exon skipping efficiency assay for MOE ASO.

89. 89. The peptide-antisense oligonucleotide conjugate of any one of claims 83 to 88, wherein the antisense oligonucleotide comprises one or more non-natural internucleotide linkages.

90. 90. The peptide-antisense oligonucleotide conjugate of claim 89, wherein all of said one or more non-natural internucleotide linkages have an Sp configuration.

91. 90. The peptide-antisense oligonucleotide conjugate of claim 89, wherein all of said one or more non-natural internucleotide linkages have an Rp configuration.

92. 90. The peptide-antisense oligonucleotide conjugate of claim 89, wherein the one or more non-natural internucleotide linkages comprise one or more non-natural internucleotide linkages having an Sp configuration and one or more non-natural internucleotide linkages having an Rp configuration.

93. 93. The peptide-antisense oligonucleotide conjugate of any one of claims 79 to 92, wherein the antisense oligonucleotide comprises one or more modified nucleobases.

94. 94. The peptide-antisense oligonucleotide conjugate of any one of claims 79 to 93, further comprising a pharmaceutically acceptable carrier or excipient.

95. 20. The peptide-antisense oligonucleotide conjugate of claim 18 for use in a method for inducing exon-2 skipping in the CD33 gene during pre-mRNA splicing, the method comprising introducing into a cell the peptide-antisense oligonucleotide conjugate of claim 18, wherein the peptide-antisense oligonucleotide conjugate hybridizes to a target region of the CD33 gene and induces exon-2 skipping during pre-mRNA splicing of the CD33 gene.

96. 20. The peptide-antisense oligonucleotide conjugate of claim 19 for use in a method for inducing exon-2 skipping in the CD33 gene during pre-mRNA splicing, the method comprising introducing into a cell the peptide-antisense oligonucleotide conjugate of claim 19, wherein the peptide-antisense oligonucleotide conjugate hybridizes to a target region of the CD33 gene and induces exon-2 skipping during pre-mRNA splicing of the CD33 gene.

97. The peptide-antisense oligonucleotide conjugate of any one of claims 79 to 96, wherein the cell is an animal cell.

98. 98. The peptide-antisense oligonucleotide conjugate of claim 97, wherein the cell is a human cell.

99. 10. The peptide-antisense oligonucleotide conjugate of claim 1 for use in a method for treating a subject having a neurodegenerative disease, comprising administering to the subject a therapeutically effective amount of the peptide-antisense oligonucleotide conjugate of claim 1.

100. 3. The peptide-antisense oligonucleotide conjugate of claim 2 for use in a method for treating a subject having a neurodegenerative disease, comprising administering to the subject a therapeutically effective amount of the peptide-antisense oligonucleotide conjugate of claim 2.

101. 101. The peptide-antisense oligonucleotide conjugate of claim 99 or 100, wherein the antisense oligonucleotide is 16 to 30 nucleotides in length, 18 to 30 nucleotides in length, 18 to 25 nucleotides in length, 18 to 21 nucleotides in length, 21 to 30 nucleotides in length, 21 to 25 nucleotides in length, or 25 to 30 nucleotides in length.

102. 101. The peptide-antisense oligonucleotide conjugate of claim 99 or 100, wherein the antisense oligonucleotide is 21 or 25 nucleotides in length.

103. The peptide-antisense oligonucleotide conjugate of any one of claims 99 to 102, wherein the antisense oligonucleotide comprises one or more non-natural sugar moieties, one or more non-natural internucleotide linkages, or one or more non-natural sugar moieties and one or more non-natural internucleotide linkages.

104. 104. The peptide-antisense oligonucleotide conjugate of claim 103, wherein the antisense oligonucleotide comprises one or more unnatural sugar moieties.

105. 105. The peptide-antisense oligonucleotide conjugate of claim 104, wherein the antisense oligonucleotide comprises a phosphorodiamidate morpholino oligomer (PMO).

106. The peptide-antisense oligonucleotide conjugate of claim 105, having a CD33 exon-2 skipping efficiency of 30% or more according to a standard exon skipping efficiency assay for PMO ASOs.

107. The peptide-antisense oligonucleotide conjugate of claim 104, wherein the antisense oligonucleotide comprises a methoxyethyl ribose oligomer (MOE).

108. The peptide-antisense oligonucleotide conjugate of claim 107, wherein the antisense oligonucleotide has a CD33 exon-2 skipping efficiency of 30% or more according to a standard exon skipping efficiency assay for MOE ASO.

109. The peptide-antisense oligonucleotide conjugate of any one of claims 103 to 108, wherein the antisense oligonucleotide comprises one or more non-natural internucleotide linkages.

110. 110. The peptide-antisense oligonucleotide conjugate of claim 109, wherein all of said one or more non-natural internucleotide linkages have an Sp configuration.

111. 110. The peptide-antisense oligonucleotide conjugate of claim 109, wherein all of said one or more non-natural internucleotide linkages have an Rp configuration.

112. The peptide-antisense oligonucleotide conjugate of claim 109, wherein the one or more non-natural internucleotide linkages comprise one or more non-natural internucleotide linkages having an Sp configuration and one or more non-natural internucleotide linkages having an Rp configuration.

113. 113. The peptide-antisense oligonucleotide conjugate of any one of claims 99 to 112, wherein the antisense oligonucleotide comprises one or more modified nucleobases.

114. The peptide-antisense oligonucleotide conjugate of any one of claims 99 to 113, further comprising a pharmaceutically acceptable carrier or excipient.

115. 20. The peptide-antisense oligonucleotide conjugate of claim 18 for use in a method for treating a subject having a neurodegenerative disease, comprising administering to the subject a therapeutically effective amount of the peptide-antisense oligonucleotide conjugate of claim 18.

116. 20. The peptide-antisense oligonucleotide conjugate of claim 19 for use in a method of treating a subject having a neurodegenerative disease, comprising administering to the subject a therapeutically effective amount of the peptide-antisense oligonucleotide conjugate of claim 19.

117. The peptide-antisense oligonucleotide conjugate of any one of claims 99 to 116, wherein the neurodegenerative disease is Alzheimer's disease.

118. Peptides including cyclic peptides containing lipoic acid groups.

119. 119. The peptide of claim 118, wherein the lipoic acid group is an (R)-lipoic acid group.

120. 119. The peptide of claim 118, wherein the lipoic acid group is an (S)-lipoic acid group.

121. 121. The peptide of any one of claims 118-120, wherein the cyclic peptide comprises 4-40 amino acids, optionally wherein the cyclic peptide comprises 6-10 amino acids.

122. 122. The peptide of any one of claims 118-121, wherein the cyclic peptide comprises 1 to 5 arginine residues, and optionally the cyclic peptide comprises 2 to 4 arginine residues.

123. 123. The peptide of any one of claims 118-122, wherein the cyclic peptide comprises 1 to 5 aromatic hydrophobic amino acids, and optionally the cyclic peptide comprises 2 to 4 aromatic hydrophobic amino acids.

124. The cyclic peptide is 【Chemistry 10】 The peptide according to any one of claims 118 to 123, selected from:

125. 125. The peptide of any one of claims 118 to 124, wherein the cyclic peptide comprises two or more lipoic acid groups.

126. 126. The peptide of claim 125, wherein the two or more lipoic acid groups comprise two or more (R)-lipoic acid groups, two or more (S)-lipoic acid groups, and / or one or more (R)-lipoic acid groups and one or more (S)-lipoic acid groups.

127. 125. A method of making the peptide of claim 124, wherein the peptide is synthesized according to general procedure C.

128. Peptides containing cyclic lactam groups.

129. 129. The peptide of claim 128, which is a cell-penetrating peptide.

130. 130. The peptide of claim 128 or 129, which is a cyclic peptide.

131. 131. A peptide according to any one of claims 128 to 130, comprising 4 to 40 amino acids, optionally comprising 6 to 10 amino acids.

132. 132. The peptide of claim 131, wherein at least one amino acid of the peptide comprises the cyclic lactam group.

133. 133. The peptide of any one of claims 128 to 132, wherein the cyclic lactam group is an 8-, 9-, or 10-membered ring.

134. The cyclic lactam group is represented by Formula III 【Chemistry 11】 (In the formula, R １ and R ２ are each independently selected from the group consisting of H, aryl groups, heteroaryl groups, alkylaryl groups, arylalkyl groups, straight chain alkyl groups, branched alkyl groups, and guanidine-containing groups, wherein R １ and R ２ each of which is optionally substituted by one or more substituents; and wherein n is an integer from 1 to 3.

135. R １ and / or R ２ 135. The peptide of claim 134, wherein independently comprises a substituted or unsubstituted aryl group.

136. 136. The peptide of claim 135, wherein the aryl group is selected from the group consisting of a phenyl group, a benzyl group, and a naphthyl group.

137. R １ and / or R ２ A peptide according to any one of claims 134 to 136, wherein independently comprises a substituted or unsubstituted guanidine-containing group.

138. The guanidine-containing group is —(CH ２ ) ２ CN ３ H ４ 138. The peptide of claim 137,

139. 139. A peptide according to any one of claims 128 to 138, comprising 1 to 5 arginine residues, optionally comprising 2 to 4 arginine residues.

140. Formula IV: 【Chemistry 12】 (In the formula, R １ contains an aryl group or a guanidine-containing group, where R １ wherein the amino acid is optionally substituted by one or more substituents.

141. 141. The cyclic peptide of claim 140, wherein the aryl group is selected from the group consisting of a benzyl group, a phenyl group, and a naphthyl group.

142. The guanidine-containing group is —(CH ２ ) ２ -CN ３ H ４ 141. The cyclic peptide of claim 140, wherein:

143. Formula V: 【Chemistry 13】 A cyclic peptide comprising at least one oxadiazole bond having a structure according to the formula: wherein R comprises a substituted or unsubstituted aryl group.

144. 144. The cyclic peptide of claim 143, wherein the aryl group is selected from the group consisting of a phenyl group, a benzyl group, a naphthyl group, and a methylnaphthyl group.

145. The peptide-antisense oligonucleotide conjugate of any one of claims 1 to 16 or 18 to 38, which is a pharmaceutically acceptable salt.

146. 18. The composition of claim 17, wherein the peptide-antisense oligonucleotide conjugate is a pharmaceutically acceptable salt.

147. The method for inducing exon-2 skipping in the CD33 gene during pre-mRNA splicing according to any one of claims 39 to 58, wherein the peptide-antisense oligonucleotide conjugate is a pharmaceutically acceptable salt.

148. 79. The method of treating a subject having a neurodegenerative disease according to any one of claims 59 to 78, wherein the peptide-antisense oligonucleotide conjugate is a pharmaceutically acceptable salt.

149. 99. A peptide-antisense oligonucleotide conjugate for use in a method for inducing exon-2 skipping in the CD33 gene during pre-mRNA splicing according to any one of claims 79 to 98, wherein the peptide-antisense oligonucleotide conjugate is a pharmaceutically acceptable salt.

150. 118. A peptide-antisense oligonucleotide conjugate for use in a method of treating a subject having a neurodegenerative disease according to any one of claims 99 to 117, wherein the peptide-antisense oligonucleotide conjugate is a pharmaceutically acceptable salt.

151. 140. The peptide of any one of claims 118 to 139, wherein the peptide is a pharmaceutically acceptable salt.

152. The cyclic peptide of any one of claims 140 to 144, wherein the cyclic peptide is a pharmaceutically acceptable salt.

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