Method for preparing multimers of phosphorodiamidate morpholino oligomers

Abstract

Methods of making and using nucleotide repeat phosphorodiamidate morpholino oligonucleotides (PMO) are provided. Nucleotide repeat PMOs include dinucleotide repeat PMOs, trinucleotide repeat PMOs and tetranucleotide repeat PMOs.

Description

METHOD FOR PREPARING MULTIMERS OF PHOSPHORODIAMIDATE MORPHOLINO OLIGOMERS RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Patent Application No.63 / 619,998, filed on January 11, 2024, and U.S. Provisional Patent Application No. 63 / 620,276, filed on January 12, 2024, the disclosures of which are hereby incorporated herein by reference in their entirety. INTRODUCTION

[0002] More than 40 diseases are caused by expansions of simple sequence repeats. Although expanded trinucleotide repeat diseases were discovered first, and are the most common, tetra- penta-, hexa- and even dodeca-nucleotide repeat expansions have been identified as the cause of human disease. Paulson, H. (2019) “Repeat expansion diseases,” Handb. Clin. Neurol. 147:105- 123, doi: 10.1016 / B978-0-444-63233-3.00009-9. A common feature in nucleotide repeat disorders is the presence of a DNA tandem repeat in the disease-associated gene and the propensity of the repeats to expand in germ and somatic cells. Zain and Smith (2019) “Targeted oligonucleotides for treating neurodegenerative tandem repeat diseases,” Neurotherapeutics. 16:248-262, doi: 10.1007 / s13311-019-00712-9.

[0003] Antisense oligonucleotides (ASOs) are synthetic single-stranded oligonucleotides, typically from 8 to 50 nucleotides in length, that selectively bind via complementary Watson-Crick base-pairing to pre-mRNA or mRNA sequences to alter levels of a target protein that can be used to treat nucleotide repeat diseases. ASOs can exhibit their effects through a diverse set of mechanisms, some of which promote RNA degradation and some that do not. Wurster and Ludolph (2018) “Antisense oligonucleotides in neurological disorders,” Ther. Adv. in Neurolog. Disord. Vol. 11, doi: 10.1177 / 1756286418776932. Unmodified oligonucleotides are inherently unstable and are rapidly degraded by ubiquitously expressed endo-or exonucleases. As such, chemical oligonucleotide modifications have been developed to increase the ability of the ASO to recognize the target mRNA, increase resistance to nucleases, increase plasma half-file, and to alter tissue distribution. Id.

[0004] Phosphorodiamidate morpholino oligomers (PMOs) are a promising type of antisense oligonucleotide modification. PMOs are currently synthesized using phosphoramidate P(V) chemistry on a solid support using chlorophosphoramidate building blocks. However, PMOs are challenging and time consuming to synthesize. For example, each round of synthesis, which adds a single nucleotide to a growing PMO chain, can take from 3 to 5 hours. Accordingly, synthesizing a 20-mer PMO can take 60 to 100 hours. In addition, solid surface synthesis of PMOs having a greater number of phosphorodiamidate morpholino nucleotides may contain lower percentages of the desired PMO product due to errors. While the error rate for each round of PMO synthesis is low, the error rate compounds with each cycle. As such, compositions resulting from solid support PMO synthesis with a larger number of cycles may have a higher percentage of PMOs with a sequence other than the intended sequence.

[0005] There is a need for improved PMO synthesis methods, such as methods that reduce synthesis time and / or improve purity of the intended sequence. SUMMARY

[0006] The present disclosure describes, among other things, improved methods for synthesizing PMOs, such as therapeutic PMOs for the treatment of nucleotide repeat diseases; PMOs resulting from such synthesis methods; and methods of using such resulting PMOs.

[0007] Among other things, the disclosure describes methods of making PMOs in which PMO multimers comprising more than one PMO nucleotide are added to a growing PMO chain. By adding multimers during one or more cycles, as opposed to adding monomers, the reaction time for synthesizing the PMO may be reduced. In some instances, the reaction time may be substantially reduced when employing a synthesis method as described herein relative to a reaction scheme in which one PMO nucleotide (monomer) is added at each cycle, as may be performed when using conventional PMO reaction schemes. Reducing the number of cycles by adding multimers as opposed to multiple cycles of adding PMO monomers can also improve purity of the resulting PMO because compounded error is reduced as synthesis cycles are reduced.

[0008] The disclosure describes methods of making and using phosphorodiamidate morpholino oligonucleotides (PMO) containing nucleotide repeats. In embodiments, the disclosure provides methods for synthesizing PMOs comprising multimer repeats. In embodiments, the multimerrepeats are dinucleotide (dimer) repeats. In embodiments, the multimer repeats are trinucleotide (trimer) repeat PMOs. In embodiments, the multimer repeats are tetranucleotide (tetramer) repeats.

[0009] In embodiments, a method for synthesizing a phosphorodiamidate morpholino oligomer (PMO), is provided. The method includes (a) synthesizing a first phosphorodiamidate morpholino multimer, wherein synthesizing the first phosphorodiamidate morpholino multimer includes (i) coupling a morpholino monomer to a first morpholino nucleotide monomer including a phosphor- terminus to form a PMO dimer; and (b) coupling the first phosphorodiamidate morpholino multimer in (a) with one or more additional phosphorodiamidate morpholino multimers to form an extended PMO.

[0010] In embodiments, (a) further includes coupling the PMO dimer to a second morpholino nucleotide monomer including a phosphor-terminus to form a PMO trimer.

[0011] In embodiments, (a) further includes coupling the PMO trimer to a third nucleotide morpholino monomer including a phosphor-terminus to form a phosphorodiamidate morpholino tetramer.

[0012] In embodiments, each of the one or more additional phosphorodiamidate morpholino multimers are formed as in (a).

[0013] In embodiments, the first phosphorodiamidate morpholino multimer and each of the additional phosphorodiamidate morpholino multimers consist of the same nucleotide sequence.

[0014] In embodiments, the first morpholino nucleotide monomer is protected at the 5’-OH.

[0015] In embodiments, the method further includes functionalizing the first phosphorodiamidate morpholino multimer of step (a) or the second phosphorodiamidate morpholino multimer of step (b).

[0016] In embodiments, the second phosphorodiamidate morpholino multimer of step (b) is functionalized and the method further includes coupling the functionalized second phosphorodiamidate morpholino multimer in (b) to the first phosphorodiamidate morpholino multimer of (a) to from the extended PMO.

[0017] In embodiments, the first phosphorodiamidate morpholino multimer of step (a) is functionalized and the method further includes attaching the functionalized first phosphorodiamidate morpholino multimer of (a) to a solid support.

[0018] In embodiments, the solid support includes a polystyrene resin.

[0019] In embodiments, the first phosphorodiamidate morpholino multimer of (a) is conjugated to the solid support at the 5’ end.

[0020] In embodiments, the method further includes cleaving the PMO comprising the extended PMO from the solid support.

[0021] In embodiments, the method further includes coupling the extended PMO with one or more additional phosphorodiamidate morpholino (PMO) monomers at the 3’ end, the 5’ end, or both the 3’ end and the 5’ end of the extended PMO.

[0022] In embodiments, the additional PMO monomer is functionalized and attached to a solid support.

[0023] In embodiments, the phosphorodiamidate morpholino oligomer (PMO) includes the first phosphorodiamidate morpholino multimer and at least one additional phosphorodiamidate morpholino multimer and includes the following sequence: Xp(M1-M2-M3-M4)xYq, wherein: M1and M2are each independently C, A, U, or G; M3and M4are each independently C, A, U, G or are absent; each X and each Y are each independently C, A, U or G; p and q are each independently an integer from 0 to 3; and x is an integer from 1 to 10.

[0024] In embodiments, the phosphorodiamidate morpholino oligomer (PMO) includes a dinucleotide repeat and includes the following sequence: Xp(M1-M2)xYq, wherein: M1and M2are each independently C, A, U, or G; each X and each Y are each independently C, A, U or G; p and q are each independently an integer from 0 to 1; and x is an integer from 1 to 10.

[0025] In embodiments, the phosphorodiamidate morpholino oligomer (PMO) includes a trinucleotide repeat and includes the following sequence: Xp(M1-M2-M3)xYq, wherein: M1, M2, and M3are each independently C, A, U, or G; each X and each Y are each independently C, A, U or G; p and q are each independently an integer from 0 to 2; and x is an integer from 1 to 10.

[0026] In embodiments, the phosphorodiamidate morpholino oligomer (PMO) includes a tetranucleotide sequence and includes the following sequence: Xp(M1-M2-M3-M4)xYq, wherein: M1, M2, M3, and M4are each independently C, A, U, or G; each X and each Y are each independently C, A, U or G; p and q are each independently an integer from 0 to 3; and x is an integer from 1 to 10.

[0027] In embodiments, the phosphorodiamidate morpholino oligomer (PMO) includes from 3 to 20, 3 to 15, 3 to 10, 3 to 8 or 4 to 6 nucleotide repeats.

[0028] In embodiments, the phosphorodiamidate morpholino oligomer (PMO) includes 3, 4, 5, 6, 7, 8, 9 or 10 nucleotide repeats.

[0029] In embodiments, the phosphorodiamidate morpholino oligomer (PMO) includes a nucleotide sequence that is complementary to a (CUG) expanded repeat in a target RNA sequence.

[0030] In embodiments, the phosphorodiamidate morpholino oligomer (PMO) includes a nucleotide sequence that is complementary to a (CAG) expanded repeat in a target RNA sequence.

[0031] In embodiments, the phosphorodiamidate morpholino oligomer (PMO) includes the following sequence: Xp(CAG)xYq, wherein: each X and each Y are each independently C, A or G; p and q are each independently an integer from 0 to 2; and x is an integer from 1 to 10.

[0032] In embodiments, the phosphorodiamidate morpholino oligomer (PMO) includes a sequence of (CAG)x, G(CAG)x, AG(CAG)x, (CAG)xC, (CAG)xCA, G(CAG)xC, G(CAG)xCA, AG(CAG)xC, AG(CAG)xCA, or combinations thereof, wherein x is an integer from 1 to 10.

[0033] In embodiments, the phosphorodiamidate morpholino oligomer (PMO) includes Xp(CUG)xYq, wherein: each X and each Y are each independently C, U or G; p and q are each independently an integer from 0 to 2; and x is an integer from 1 to 10.

[0034] In embodiments, the phosphorodiamidate morpholino oligomer (PMO) includes a sequence of (CUG)x, G(CUG)x, UG(CUG)x, (CUG)xC, (CUG)xCU, G(CUG)xC, G(CUG)xCU, UG(CUG)xC, UG(CUG)xCU, or combinations thereof, wherein x is an integer from 1 to 10.

[0035] In embodiments, the phosphorodiamidate morpholino oligomer (PMO) includes a nucleotide sequence that is complementary to a (GAA) expanded repeat in a target RNA sequence.

[0036] In embodiments, the phosphorodiamidate morpholino oligomer (PMO) includes the following sequence: Xp(UUC)xYq, wherein: each X and each Y are each independently U or C; p and q are each independently an integer from 0 to 2; and x is an integer from 1 to 10.

[0037] In embodiments, the phosphorodiamidate morpholino oligomer (PMO) includes a sequence of (UUC)x, C(UUC)x, UC(UUC)x, (UUC)xU, (UUC)xUU, C(UUC)xU, C(UUC)xUU, UC(UUC)xU, UC(UUC)xUU, or combinations thereof, wherein x is an integer from 1 to 10.

[0038] In embodiments, the phosphorodiamidate morpholino oligomer (PMO) includes a nucleotide sequence that is complementary to a (CGG) expanded repeat in a target RNA sequence.

[0039] In embodiments, phosphorodiamidate morpholino oligomer (PMO) includes a sequence of Xp(CCG)xYq, wherein: each X and each Y are each independently C or G; p and q are each independently an integer from 0 to 2; and x is an integer from 1 to 10.

[0040] In embodiments, the phosphorodiamidate morpholino oligomer (PMO) includes a sequence of (CCG)x, G(CCG)x, CG(CCG)x, (CCG)xC, (CCG)xCC, G(CCG)xC, G(CCG)xCC, CG(CCG)xC, CG(CCG)xCC, or combinations thereof, wherein x is an integer from 1 to 10.

[0041] In embodiments, the phosphorodiamidate morpholino oligomer (PMO) includes a nucleotide sequence that is complementary to a (GCG) expanded repeat in a target RNA sequence.

[0042] In embodiments, the phosphorodiamidate morpholino oligomer (PMO) includes a sequence of Xp(CGC)xYq, wherein: each X and each Y are each independently C or G; p and q are each independently an integer from 0 to 2; and x is an integer from 1 to 10.

[0043] In embodiments, the phosphorodiamidate morpholino oligomer (PMO) includes a sequence of (CGC)x, C(CGC)x, GC(CGC)x, (CGC)xC, (CGC)xCG, C(CGC)xC, C(CGC)xCG, GC(CGC)xC, GC(CGC)xCG, or combinations thereof, wherein x is an integer from 1 to 10.

[0044] In embodiments, the phosphorodiamidate morpholino oligomer (PMO) includes a nucleotide sequence that is complementary to a (CCUG) expanded repeat in a target RNA sequence.

[0045] In embodiments, the phosphorodiamidate morpholino oligomer (PMO) includes a nucleotide sequence of Xp(CAGG)xYq, wherein: each X and each Y are each independently C, A or G; p and q are each independently an integer from 0 to 3; and x is an integer from 1 to 10.

[0046] In embodiments, the phosphorodiamidate morpholino oligomer (PMO) includes a sequence of (CAGG)x, G(CAGG)x, GG(CAGG)x, AGG(CAGG)x, (CAGG)xC, (CAGG)xCA, (CAGG)xCAG, G(CAGG)xC, G(CAGG)xCA, G(CAGG)xCAG, GG(CAGG)xC, GG(CAGG)xCA, GG(CAGG)xCAG, AGG(CAGG)xC, AGG(CAGG)xCA, AGG(CAGG)xCAG, or combinations thereof, wherein x is an integer from 1 to 10.

[0047] In embodiments, the PMO includes a phosphoro-terminus, and wherein the phosphoro- terminus is a phosphorodiamidate.

[0048] In embodiments, the PMO includes a phosphoro-terminus, and wherein the phosphoro- terminus is a phosphoric acid.

[0049] In embodiments, the PMO includes a phosphoro-terminus, and wherein the phosphoro- terminus is a phosphonamidite.

[0050] In embodiments, a method for elongating a growing chain during synthesis of a PMO is provided. The method can include reacting a deprotected morpholino oligonucleotide unit attached to a solid support a morpholino multimer including the following structure:, whereinR1and R2are each independently H or substituted or unsubstituted alkyl, aryl, alkynyl, alkenyl, cycloalkyl, heterocyclyl, or heteroaryl; Mnis a PMO monomer; (Mn)m-1is a PMO multimer having m-1 PMO nucleotides; n is the position of the Mnmonomer in the (Mn)m-1multimer sequence; and m is the number of Mnmonomers in the (Mn)m multimer. In embodiments, method can include where LG is chloro group. In embodiments, method can include where R1and R2are methyl.

[0052] The disclosure also provides methods for treating a subject using a PMO of the disclosure, wherein the PMO is prepared according to a method as described herein. The method of treating can include obtaining information about the subject, and the information can be used for determining the most appropriate way to treat the subject. In embodiments, the information is obtained by diagnosing a subject, for example, by performing genetic testing of the subject, where information obtained by the genetic testing is used to diagnose and facilitate treatment of the subject using a PMO of the disclosure.

[0053] In embodiments, the information obtained about the subject, or the diagnosis of the subject, relates to the subject expressing one or more isoforms of a gene. In embodiments, the information obtained about the subject, or the diagnosis of the subject, relates to the subject expressing one or more single nucleotide polymorphisms (SNPs) of a gene. The one or more isoforms, or one or more SNPs, may or may not correlate with a disease state of a subject for which the PMO is administered to treat the subject. For example, information can be obtained, or diagnosis can be performed, to provide information about a gene isoform and / or a SNP, wherein the gene is associated or implicated in Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), Huntington disease (HD), Fragile X Syndrome, Friedreich’s Ataxia, oculopharyngealmuscular dystrophy (OPMD), dentatorubral-pallidoluysian atrophy (DRPLA), Spinocerebellar ataxia (SCA), Spinal and bulbar muscular atrophy (SBMA), myotonic dystrophy 1 (DM1), or myotonic dystrophy 2 (DM2). BRIEF DESCRIPTION OF THE FIGURES

[0054] FIG.1 is a schematic diagram illustrating a conventional solid state synthesis scheme for producing a 21-mer PMO product, taking 20 cycles. Mnis a PMO monomer, with n being the location of the monomer in the PMO sequence in a 6’ to 3’ direction. For example, M1is the 6’- most PMO nucleotide, M2is adjacent and coupled to the M1, M3is adjacent and coupled to M2, and so on. Each cycle of the conventional solid state synthesis scheme adds a single PMO monomer (Mn). The circle in FIG.1 represents a solid support.

[0055] FIG.2 is a schematic diagram illustrating three reaction schemes in which multimers are added during solid state PMO synthesis in accordance with embodiments of the present disclosure, which result the production of 20-mers (top and bottom) or a 21-mer (middle) in substantially less cycles relative to the convention process illustrated in FIG.1. The circle represents a solid support; and M1, M2, M3, and M4are PMO monomers, each of which may be the same or different.

[0056] FIG. 3 is a schematic diagram illustrating a general overview of an embodiment of a method described herein. The shaded circle represents a solid support. Mnis a morpholino nucleotide monomer (e.g., C, A, G, U, or T); (Mn)mis a morpholino nucleotide multimer; m can be an integer from 2 to 20, such as 2 to 11, or 2 to 4; and x is the number of reaction cycles in which a morpholino nucleotide multimer (Mn)mis added to a PMO chain growing from the solid support to produce the PMO attached to the solid support. While FIG. 3 shows a morpholino nucleotide multimer (Mn)minitially coupled to the solid support, it will be appreciated that a morpholino nucleotide monomer (Mn) can be initially coupled to the solid support.

[0057] FIG. 4 is a schematic diagram illustrating a general overview of an embodiment of a method described herein. The shaded circle represents a solid support. Mnis a morpholino nucleotide monomer (e.g., C, A, G, U, or T); (Mn)mis a morpholino nucleotide multimer; m can be an integer from 2 to 20, such as 2 to 11, or 2 to 4; and x is the number of reaction cycles in which a morpholino nucleotide multimer (Mn)mis added to a PMO chain growing from the solid support. In embodiments, a morpholino nucleotide monomer (Mn) is added to a monomer coupledto the solid support prior to adding a morpholino nucleotide multimer (Mn)m. z is the number of reaction cycles in which a morpholino nucleotide monomer (Mn) is added to a PMO chain growing from the solid support. In embodiments, a morpholino nucleotide(Mn) is added to a growing PMO chain coupled to the solid support after to adding the morpholino nucleotide multimers [(Mn)m]x. y is the number of reaction cycles in which a morpholino nucleotide monomer (Mn) is added to a PMO chain growing from the solid support. While FIG.4 shows a morpholino nucleotide monomer (Mn) initially coupled to the solid support, it will be appreciated that a morpholino nucleotide multimer (Mn)mcan be initially coupled to the solid support.

[0058] FIG.5 is a schematic diagram illustrating an embodiment of a method for forming a PMO comprising multimer repeats on a solid support. Mnand (Mn)mare as described above regarding FIGS.1 and 3-4; The circle represents a solid support; PG is a protecting group, and x is an integer. A 3’-protected morpholino nucleotide multimer (Mn)mis coupled to the solid support. The process includes x-1 cycles of deprotecting, neutralizing, and coupling of a functionalized, protected morpholino nucleotide multimer to produce a solid-supported PMO product ([(Mn)m]x-PG), which may be cleaved from the solid support and deprotected to produce the PMO product ([(Mn)m]x).

[0059] FIG. 6, with partial views FIGS. 6A-6C, provides a schematic diagram illustrating an overview of a method for producing a multimeric repeat PMO. The approximate relationship between the partial views is provided by FIG.6. In the embodiment shown, the PMO is a CAG trimeric repeat PMO. However, it is envisioned that the method can be used for other multimeric repeat PMOs. A 6’-functionalized, 3’ protected morpholino nucleotide multimer is coupled to a solid support, such as an aminomethyl polystyrene resin, as shown in FIG.6A. The resulting solid- supported morpholino nucleotide multimer is 3’ protected. Additional 6’-functionalized, 3’ protected morpholino nucleotide multimers, for example trimers having a CAG sequence, can be coupled to the 3’ end of the solid-supported morpholino nucleotide multimer (or 3’ end of an extended PMO chain) through multiple (x) rounds of (i) deprotecting the 3’ end of a morpholino nucleotide multimer, neutralizing, and coupling, or (ii) deprotecting the 3’ end of a morpholino nucleotide multimer, neutralizing, coupling, and oxidative amination to produce a 3’-protected, solid supported PMO having x+1 number of repeats, as shown in FIG. 6B. Cleavage from the solid support and deprotection results in a PMO product having x+1 number of repeats, as shown in FIG.6C.

[0060] FIG. 7 with partial views FIGS. 7A-7C, provides a schematic diagram illustrating an overview of a method of producing a multimeric repeat PMO. The approximate relationship between the partial views is provided by FIG. 7. In the embodiment shown, the PMO is a CTG trimeric repeat PMO. A 6’-functionalized, 3’ protected morpholino nucleotide trimer having a CTG sequence is coupled to a solid support, such as an aminomethyl polystyrene resin as shown in FIG. 7A. The resulting solid-supported CTG morpholino nucleotide trimer is 3’ protected. Additional 6’-functionalized, 3’ protected morpholino nucleotide trimers having a CTG sequences can be coupled to the 3’ end of the solid-supported CTG morpholino nucleotide trimer (or 3’ end of an extended PMO chain) through multiple (x) rounds of (i) deprotecting the 3’ end of a CTG morpholino nucleotide trimer, neutralizing, and coupling, or (ii) deprotecting the 3’ end of a CTG morpholino nucleotide trimer, neutralizing, coupling, and oxidative amination to produce a 3’- protected, solid supported PMO having x+1 number of CTG repeats, as shown in FIG. 7B. Cleavage from the solid support and deprotection results in a PMO product having CTG repeats, as shown in FIG.7C.

[0061] FIGS.8A-C are schematic diagrams showing three different strategies for forming PMOs according to the methods disclosed herein. FIG.8A illustrates the first strategy (Strategy 1), which is also referred to herein as the “phosphonamidate coupling” strategy. Mnand (Mn)mare as described above regarding FIGS.1 and 3-4; the circle represents a solid support; PG is a protecting group; LG is a leaving group; R1and R2are each independently H or substituted or unsubstituted alkyl, aryl, alkynyl, alkenyl, cycloalkyl, heterocyclyl, or heteroaryl. FIG.8B illustrates the second strategy (Strategy 2), which is also referred to herein as the “H-phosphonate coupling” strategy. Mnand (Mn)mare as described above regarding FIGS. 1 and 3-4; the circle represents a solid support; PG is a protecting group; R1and R2are each independently H or substituted or unsubstituted alkyl, aryl, alkynyl, alkenyl, cycloalkyl, heterocyclyl, or heteroaryl. FIG. 8C illustrates the third strategy (Strategy 3), which is also referred to herein as the “phosphoramidite coupling” strategy. Mnand (Mn)mare as described above regarding FIGS. 1 and 3-4; the circle represents a solid support; PG is a protecting group; R1and R2are each independently H or substituted or unsubstituted alkyl, aryl, alkynyl, alkenyl, cycloalkyl, heterocyclyl, or heteroaryl. “B’ in the figures refers to any nucleobase, A, G, T, C, or U.

[0062] FIG. 9A is a schematic drawing illustrating a pathway for functionalizing a trimer for coupling to a solid surface. As shown, the functionalization results in a 6’-N-Hydroxy-5-norbornene-2,3-dicarboximide (HONB) succinic ester PMO multimer. The multimer shown in the figures is a PMO trimer having three nucleotide bases, “B”, where the sequence of the trimer can include any combination of any three of A, G, C, T, or U. While a trimer is shown, it is understood that other multimers (e.g., dimers, trimers, tetramers, etc.,) can be made with different nucleic acid sequences that also include a 6’-N-Hydroxy-5-norbornene-2,3-dicarboximide (HONB) succinic ester modification.

[0063] FIG. 9B is a schematic drawing illustrating a pathway for functionalizing a trimer for coupling to a solid surface. As shown, the functionalization results in a 6’-N-Hydroxy-5- norbornene-2,3-dicarboximide (HONB) succinic ester CAG PMO trimer. It is envisioned that other multimers (e.g., dimers, trimers, tetramers, etc.) can be made with different nucleic acid sequences that also include a 6’-N-Hydroxy-5-norbornene-2,3-dicarboximide (HONB) succinic ester modification. FIG.9B shows the reaction conditions that can be used to functionalize a PMO having a CAG trimer repeat. The pathway in FIG.9B corresponds to the general pathway in FIG. 9A, including reagents and reaction conditions that may be employed. DMAP = 4- Dimethylaminopyridine, DCC = dicyclohexylcarbodiimide, TEA = triethylamine, DCM = dichloromethane, EDC = 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, and DBU = 1,8- diazabicyclo[5.4.0]undec-7-ene.

[0064] FIG. 9C is a schematic drawing illustrating a pathway for functionalizing a trimer for coupling to a solid surface. As shown, the functionalization results in a 6’-N-Hydroxy-5- norbornene-2,3-dicarboximide (HONB) succinic ester CTG PMO trimer. It is envisioned that other multimers (e.g., dimers, trimers, tetramers, etc.) can be made with different nucleic acid sequences that also include a 6’-N-Hydroxy-5-norbornene-2,3-dicarboximide (HONB) succinic ester modification. FIG. 9C shows the reaction conditions that can be used to functionalize a PMO having a CTG trimer repeat. The pathway in FIG.9C corresponds to the general pathway in FIG. 9A, including reagents and reaction conditions that may be employed. DMAP = 4- Dimethylaminopyridine, DCC = dicyclohexylcarbodiimide, TEA = triethylamine, DCM = dichloromethane, EDC = 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, and DBU = 1,8- diazabicyclo[5.4.0]undec-7-ene.

[0065] FIG. 10A is a schematic drawing illustrating various approaches for functionalizing a 3’ protected PMO multimer according to Strategy 1. Although the multimer shown in the figures is a CAG PMO trimer, it will be appreciated that this is only one example of a functionalized PMOmultimer. It is envisioned that this method can be used with other multimers (e.g., dimers, trimers, tetramers, etc.) with different nucleic acid sequences. The functionalization pathway results in a 6’-N-dimethyl phosphoramidochloridate PMO multimer. It will be appreciated that this is one example of a functionalized PMO multimer that may be employed in accordance with the methods of Strategy 1.

[0066] FIG. 10B is a schematic drawing illustrating various approaches for functionalizing a 3’ protected PMO multimer according to Strategy 1. Although the multimer shown in the figures is a CTG PMO trimer, it will be appreciated that this is only one example of a functionalized PMO multimer. It is envisioned that this method can be used with other multimers (e.g., dimers, trimers, tetramers, etc.) with different nucleic acid sequences. The functionalization pathway results in a 6’-N-dimethyl phosphoramidochloridate PMO multimer. It will be appreciated that this is one example of a functionalized PMO multimer that may be employed in accordance with the methods of Strategy 1.

[0067] FIG. 10C is a schematic drawing illustrating various approaches for functionalizing a 3’ protected PMO multimer according to Strategy 2. Although the multimer shown in the figures is a CAG PMO trimer, it will be appreciated that this is only one example of a functionalized PMO multimer. It is envisioned that this method can be used with other multimers (e.g., dimers, trimers, tetramers, etc.) with different nucleic acid sequences. The functionalization pathway results in a 5’-H-phosphonate PMO multimer. It will be appreciated that this is one example of a functionalized PMO multimer that may be employed in accordance with the methods of Strategy 2.

[0068] FIGS.10D-E are schematic drawings showing some reaction conditions that may be used to functionalize a PMO multimer according to Strategy 1. Although the trimer shown in the figures is either a CAG PMO trimer (FIG.10D) or a CTG PMO trimer (FIG.10E), it will be appreciated that this is only one example of a functionalized PMO multimer. It is envisioned that this method can be used with other multimers (e.g., dimers, trimers, tetramers, etc.) with different nucleic acid sequences. The pathways in FIG.10D (Strategy 1) and FIG.10E (Strategy 1) correspond to the pathway in FIG.10A, including reagents and reaction conditions that may be employed. DMAP = 4-Dimethylaminopyridine, DCC = dicyclohexylcarbodiimide, TEA = triethylamine, DCM = dichloromethane, EDC = 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, and DBU = 1,8- diazabicyclo[5.4.0]undec-7-ene.

[0069] FIG. 10F is a schematic drawing showing some reaction conditions that may be used to functionalize a PMO multimer according to Strategy 2. Although the trimer shown in the figures is a CAG PMO trimer, it will be appreciated that this is only one example of a functionalized PMO multimer. It is envisioned that this method can be used with other multimers (e.g., dimers, trimers, tetramers, etc.) with different nucleic acid sequences. The pathway in 10F (Strategy 2) corresponds to the pathway in FIG.10C, with reagents and reaction conditions that may be employed. DMAP = 4-Dimethylaminopyridine, DCC = dicyclohexylcarbodiimide, TEA = triethylamine, DCM = dichloromethane, EDC = 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, and DBU = 1,8- diazabicyclo[5.4.0]undec-7-ene.

[0070] FIG. 11 is a schematic drawing illustrating an example of a Strategy 1 reaction scheme used to generate a solid-supported PMO having a (BBB)7 repeat sequence, where “BBB” can be any trimer repeat sequence having any three of A, T, G, C, or U. In embodiments, the reaction scheme shown in FIG. 11 can be used to generate a PMO having a (CAG)7trimer repeat (inset left) or a (CTG)7 trimer repeat (inset right). DCM = dichloromethane, TFA = trifluoroacetic acid, TFE = trifluoroethanol, IPA = Propan-2-ol, DIEA = di-isopropyl ethylamine, NEM = N- ethylmorpholine, DMI = dimethylimidazolidinone, Bz = benzoyl, and Trt = trityl. The inset structure (6’-Chlorophosphoramidate CAG PMO Trimer) is a functionalized, deprotected CAG PMO trimer used in the coupling reaction of each cycle.

[0071] FIG.12A is a schematic drawing illustrating an example of a Strategy 2 reaction scheme used to generate a solid-supported PMO having a (CAG)7repeat sequence. ACN = acetonitrile, Py = pyridine, Bz = benzoyl, and Trt = trityl.

[0072] FIG.12B the structure shown (6’-H-Phosphonate CAG PMO Trimer) is a functionalized, deprotected CAG PMO trimer used in the coupling reaction of each cycle according to Strategy 2 shown in FIG.12A.

[0073] FIG.13A is a schematic drawing illustrating an example of a Strategy 3 reaction scheme used to generate a solid-supported PMO having a (CAG)7repeat sequence. ACN = acetonitrile, DCI = 4,5-dicyanoimidazole, THF = tetrahydrofuran, Py = pyridine, Bz = benzoyl, and Trt = trityl. The inset structure (6’-Phosphoramidite CAG PMO Trimer) is a functionalized, deprotected CAG PMO trimer used in the coupling reaction of each cycle.

[0074] FIG.13B the structure shown (6’-Phosphoramidite CAG PMO Trimer) is a functionalized, deprotected CAG PMO trimer used in the coupling reaction of each cycle according to Strategy 3 shown in FIG.13A.

[0075] FIG. 14 with partial views FIGS. 14A-14E, provides a schematic diagram illustrating a reaction scheme for producing a 3’ protected CAG trimer in accordance with Example 1. The approximate relationship between the partial views is provided by FIG. 14.. TBS = tert- butyldimethylsilyl, DCM = dichloromethane, DBU = 1,8-diazabicyclo[5.4.0]undec-7-ene, TsOH = p-toluenesulfonic acid, and TEA = triethylamine.

[0076] FIG.15 is a schematic drawing illustrating a reaction scheme and conditions for coupling an N-deprotected phosphorodiamidate C-A-dimer product obtained in Example 1D with N-trityl protected chlorophosphoramidate G-monomer as described in Example 1E.

[0077] FIG.16 is a schematic drawing illustrating a reaction scheme and conditions for 6’-OTBS deprotection of a PMO multimer, including for example, the CAG-trimer obtained in Example 1E, as described in Example 1F.

[0078] FIG. 17 is a schematic drawing illustrating a reaction scheme and conditions for preparation of activated 6’-acid-PMO multimer, including for example, the 6’-acid-CAG-trimer as described in Example 2.

[0079] FIG. 18 is a schematic drawing illustrating a reaction scheme and conditions for preparation of a PMO multimer resin, including for example, a CAG resin as described in Example 3A.

[0080] FIG. 19 is a schematic drawing illustrating a reaction scheme and conditions for functionalization of 6’-OH PMO multimer, for example, a 6’-OH CAG-trimer in Example 4.

[0081] FIG.20 is a schematic drawing illustrating a reaction scheme and conditions for synthesis of PMO multimer, for example, a CAG multimer with purity analysis of each cycle in Example 6.

[0082] FIG. 21 is a schematic drawing illustrating a reaction scheme and conditions for general reaction scheme for conventional monomer synthesis with 20 reaction cycles as described in Comparative Example 8, where “BBB” represents a trimer repeat having a sequence of any three of A, T, G, C, or U..

[0083] FIG. 22 is a schematic drawing illustrating a reaction scheme and conditions for preparation of CTG resin in Example 9A.

[0084] FIG. 23 is a schematic drawing illustrating a reaction scheme and conditions for phosphorylation of 6’-OH morpholino substrate in Example 11.

[0085] FIG. 24 is a schematic drawing illustrating a reaction scheme and conditions for H- phosphonate coupling and oxidative amination in Example 12.

[0086] FIG. 25 is a schematic drawing illustrating a reaction scheme and conditions for phosphorylation of 6’-OH morpholino substrate in Example 13.

[0087] FIG. 26 is a schematic drawing illustrating a reaction scheme and conditions for phosphoramidite coupling and oxidative amination in Example 14. DETAILED DESCRIPTION

[0088] Methods of making phosphorodiamidate morpholino oligonucleotides (PMO) are described herein. The methods comprise adding PMO multimers to a growing chain during synthesis of the PMO. In embodiments, the methods further comprise adding one or more PMO monomer to the growing chain during synthesis of the PMO. In embodiments, the PMOs are synthesized on a solid support.

[0089] The addition of PMO multimers, as opposed to PMO monomers, reduces the number of synthesis cycles and can reduce the overall reaction time, improve purity of the resulting PMO, and / or reduce the amount of reagents used.

[0090] The reaction time for synthesizing a PMO using standard processes in which a single PMO monomer is added during each cycle of solid-state synthesis can be long, often taking from 3 to 5 hours per cycle. Accordingly, the synthesis of a 21-mer PMO can take about 60 to 100 hours.

[0091] FIG.1 illustrates a conventional solid-state synthesis scheme for producing a 21-mer PMO product, which takes 20 cycles. During each cycle a functionalized, protected PMO monomer (nucleotide) is coupled to a deprotected monomer (Mn) or the 3’ PMO monomer on a growing PMO chain attached to the solid support. The functionalized, protected PMO monomer (nucleotide) is added to the 3’ end of the extended PMO, the protecting group is removed (deprotection), the resulting extended PMO is neutralized, and an the functionalized, protected PMO monomer (nucleotide) can be added in the next cycle.

[0092] FIG.2 illustrates some examples of PMO synthesis processes of the present disclosure, in which multimers are incorporated into a growing chain during PMO synthesis. The incorporation of multimers, as opposed to monomers, can result in a substantial reduction in the number of cyclesto produce the PMO product. As shown in FIG.2, the production of a 20-mer using dimers can be accomplished in nine cycles, the production of a 21-mer using trimers can be accomplished in 6 cycles, and the production of a 20-mer using tetramers can be accomplished in 4 cycles. Accordingly, PMOs having a length the same as, or similar to, the PMO illustrated in FIG.1 can be produced in substantially fewer cycles when multimers are employed.

[0093] Reduced number of cycles can substantially reduce overall reaction time. Reduced number of cycles can improve purity of PMOs having the intended sequence. Fewer reaction cycles can also reduce the total material used and operation costs of the process. Therefore, the methods for PMO synthesis presented in the current disclosure can provide for a faster and simpler synthesis, save money by using fewer resources, provide yield improvement, result in a purer final product, reduce environmental impact, and / or provide a more reliable and controlled synthesis process.

[0094] While the reaction schemes shown in FIG. 2 include only incorporation of multimers at each cycle, it will be appreciated that during some cycles monomers may be incorporated into the growing PMO chain, for example, one or more monomers can be included at the 6’ and / or the 3’ end of the PMO chain. Overview of Reaction Schemes and Methods for Producing PMOs employing PMO multimers

[0095] FIGS.3-4 illustrate overviews of methods described herein. In FIG.3, the shaded circles represent a solid support; Mnis a morpholino nucleotide monomer (e.g., C, A, G, U, or T); (Mn)mis a phosphorodiamidate morpholino nucleotide multimer (or “morpholino nucleotide multimer”), where m can be an integer from 2 to 20, such as 2 to 11, or 2 to 4; x is the number of reaction cycles in which a morpholino nucleotide multimer (Mn)mis added to a PMO chain growing from the solid support to produce the PMO attached to the solid support; and n is the position of a given monomer M in the multimer (Mn)msequence. For example, M1is the monomer at the 6’ end of the multimer; M2is immediately 3’ to and coupled to M1; M3, if present, is immediately 3’ to and coupled to M2; M4, if present, is immediately 3’ to and coupled to M3, and so on.

[0096] FIG.3 illustrates the reaction of a solid-supported morpholino nucleotide multimer (Mn)mwith a free morpholino nucleotide multimer (Mn)mfor x cycles to produce a solid-supported PMO comprising x+1 morpholino nucleotide multimers (Mn)m. In embodiments, each morpholino nucleotide multimer (Mn)mhas the same sequence, and the process shown in FIG. 3 results in a PMO having a nucleotide repeat sequence. While FIG.3 shows a morpholino nucleotide multimer(Mn)minitially coupled to the solid support, it will be appreciated that a morpholino nucleotide monomer (Mn) can be initially coupled to the solid support.

[0097] In FIG. 4, the shaded circles represents a solid support; Mnis a morpholino nucleotide monomer (e.g., C, A, G, U, or T); (Mn)mis a morpholino nucleotide multimer, where m can be an integer from 2 to 20, such as 2 to 11, or 2 to 4; and x is the number of reaction cycles in which a morpholino nucleotide multimer (Mn)mis added to a PMO chain growing from the solid support. In embodiments, each morpholino nucleotide multimer (Mn)mhas the same sequence, and the process shown in FIG.4 results in a PMO having a nucleotide repeat sequence. In embodiments, a morpholino nucleotide monomer (Mn) is added to a monomer coupled to the solid support prior to adding a morpholino nucleotide multimer (Mn)m. If adding a morpholino nucleotide monomer (Mn) prior to adding a morpholino nucleotide multimer (Mn)m, z represents the number of reaction cycles in which a morpholino nucleotide monomer (Mn) is added to a PMO chain growing from the solid support. In embodiments, a morpholino nucleotide monomer (Mn) is added to a growing PMO chain coupled to the solid support after adding the morpholino nucleotide multimers [(Mn)m]x. If adding a morpholino nucleotide monomer (Mn) after adding morpholino nucleotide multimers [(Mn)m]x, y represents the number of reaction cycles in which a morpholino nucleotide monomer (Mn) is added to a PMO chain growing from the solid support. Adding one or more morpholino nucleotide monomers (Mn) to one or both ends may provide an offset of a nucleotide repeat sequence in the PMO when each added morpholino nucleotide multimer (Mn)mhas the same sequence. While FIG. 4 shows a morpholino nucleotide monomer (Mn) initially coupled to the solid support, it will be appreciated that a morpholino nucleotide multimer (Mn)mcan be initially coupled to the solid support.

[0098] Any total number of reaction cycles(x) for the addition of morpholino nucleotide multimers (Mn)mas shown in FIGS.3-4 may be performed. In embodiments, x is an integer from 1 to 50. In embodiments, x is an integer from 1 to 40. In embodiments, x is an integer from 1 to 30. In embodiments, x is an integer from 1 to 20. In embodiments, x is an integer from 1 to 15. In embodiments, x is 1. In embodiments, x is 2. In embodiments, x is 3. In embodiments, x is 4. In embodiments, x is 5. In embodiments, x is 6. In embodiments, x is 7. In embodiments, x is 8. In embodiments, x is 9. In embodiments, x is 10. In embodiments, x is 11. In embodiments, x is 12. In embodiments, x is 13. In embodiments, x is 14. In embodiments, x is 15.

[0099] Any total number of reaction cycles (z) for the addition of a morpholino nucleotide monomer (Mn) as shown in FIG.4 can be performed. In embodiments, z is an integer from 0 to 12.. In embodiments, z is an integer from 0 to 10. In embodiments, z is an integer from 0 to 3. In embodiments, z is an integer from 0 to 2. In embodiments, z is 0 or 1. In embodiments, z is 0.. In embodiments, z is 1. In embodiments, z is 2. In embodiments, z is 3.

[0100] Any total number of reaction cycles (y) for the addition of a morpholino nucleotide monomer (Mn) as shown in FIG.4 can be performed. In embodiments, y is an integer from 0 to 3. In embodiments, y is an integer from 0 to 2. In embodiments, y is 0 or 1. In embodiments, y is 0. In embodiments, y is 1. In embodiments, y is 2. In embodiments, y is 3.

[0101] A morpholino nucleotide monomer Mnwithin a PMO can have the following structure: ,

[0102] where Y is H or N(R1)independently H or substituted or unsubstituted alkyl, aryl, alkynyl, alkenyl, cycloalkyl, heterocyclyl, or heteroaryl; and where B is nucleobase, such as adenine (A), cytosine (C), guanine (G), thymine (T), or uracil (U). In embodiments, Y is N(CH3)2. The nucleobase B is attached to the 1’ position of the morpholino nucleotide monomer Mn, the nitrogen of the morpholino ring is at the 3’ position, and the phosphoro group is bound to the 6’ carbon. In embodiments, Y is N(R1)(R2), and R1and R2are each independently substituted or unsubstituted alkyl. In embodiments, Y is N(R1)(R2) and, R1and R2are each independently unsubstituted alkyl. In embodiments, Y is N(R1)(R2), and R1and R2are each methyl.

[0103] Each morpholino nucleotide monomer Mnof a morpholino nucleotide multimer (Mn)mor PMO shown in FIGS.3-4 can independently be an adenine (A) morpholino nucleotide monomer, a cytosine (C) morpholino nucleotide monomer, a guanine (G) morpholino nucleotide monomer, a thymine (T) morpholino nucleotide monomer, or a uracil (U) morpholino nucleotide monomer. For purposes of convenience, “A” may be used herein to refer to an adenine (A) morpholino nucleotide monomer, “C” may be used herein to refer to a cytosine (C) morpholino nucleotidemonomer, “G” may be used herein to refer to a guanine (G) morpholino nucleotide monomer, “T” may be used herein to refer to a thymine (T) morpholino nucleotide monomer, and “U” may be used herein to refer to a uracil (U) morpholino nucleotide monomer.

[0104] In embodiments, the sequence of the morpholino nucleotide multimer (Mn)madded in at least one subsequent cycle is different from the sequence of the morpholino nucleotide multimer (Mn)madded in the immediately preceding cycle. In embodiments, the sequence of all the morpholino nucleotide multimers (Mn)mis the same in each cycle in which a morpholino nucleotide multimer (Mn)mis added. For purposes of the present disclosure, a sequence of a PMO or a portion thereof is a sequence in the 6’ to 3’ direction unless otherwise stated.

[0105] In embodiments, the added PMO multimers (Mn)mform tandem repeats of the same PMO multimer. The tandem repeats can be, for example, dimer repeats, trimer repeats, tetramer repeats, pentamer repeats, hexamer repeats, or dodecamer repeats. In embodiments, the tandem repeats can be dimer repeats. In embodiments, the tandem repeats can be trimer repeats. In embodiments, the tandem repeats can be tetramer repeats. In embodiments, the PMOs described herein can be used for the treatment of nucleotide repeat disorders. Various repeat expansion diseases can be found in Paulson H. Repeat expansion diseases. Handb Clin Neurol. 2018;147:105-123. doi: 10.1016 / B978-0-444-63233-3.00009-9. PMID: 29325606; PMCID: PMC6485936.

[0106] In embodiments, the morpholino nucleotide monomer Mnor a morpholino nucleotide multimer (M)mof FIGS.3-4 is coupled to the solid support at the 6’ end. In embodiments, each cycle of addition of a morpholino nucleotide monomer Mnor a morpholino nucleotide multimer (Mn)mof FIGS. 3-4 to the growing PMO chain comprises coupling a 6’ functionalized, 3’ protected morpholino nucleotide monomer Mnor 6’ functionalized, 3’ protected morpholino nucleotide multimer (Mn)mto the PMO chain at the free 3’ end (the end not tethered to the solid support) to result in an extended PMO chain with a protected 3’ end. The 3’ N-protected extended PMO can be deprotected prior to adding another 6’ functionalized, 3’ protected morpholino nucleotide monomer Mnor 6’ functionalized, 3’ protected morpholino nucleotide multimer (Mn)mto the PMO chain in the next cycle.

[0107] The morpholino nucleotide monomer Mnor morpholino nucleotide multimer (Mn)mcan include any suitable 6’ functional group and 3’ protecting group, and the reagents and reaction conditions for the coupling and deprotection steps may vary depending on the functional group and protecting group employed.

[0108] For example, and with reference to FIG.5, an example of a reaction scheme to produce a PMO product comprising multimer repeats is shown. A solid supported multimer in which a 6’ end of a PMO multimer (Mn)mis attached to a solid support is coupled with a free 6’ functionalized, 3’ protected multimer through a number (x-1) reaction cycles of deprotection, neutralization, and coupling to produce a solid-supported, protected PMO Product [(Mn)m]x-PG, having x repeating multimers (Mn)m. In FIG. 5, each Mnwithin a PMO multimer (Mn)mis independently C, A, U, T, or G, and m is an integer of 2 or greater. In embodiments, m is 2 to 11. In embodiments, m is 2. In embodiments, m is 3. In embodiments, m is 4.

[0109] In FIG.5, PG is a protecting group. Any suitable protecting group may be used. Examples of suitable protecting groups are described elsewhere herein.

[0110] The 6’ functional group of the free functionalized, protected multimer can have a structure of P1, wherein ,LG is a leaving group; Y is independently hydrogen or N(R1)(R2), wherein if Y is H, then R1and R2are not present; and R1and R2,if present, and R3are each independently H or substituted or unsubstituted alkyl, aryl, alkynyl, alkenyl, cycloalkyl, heterocyclyl or heteroaryl.

[0111] While FIG.5 shows PMO multimers being coupled to the solid support and being added at each repeating cycle, it will be appreciated that PMO monomers can be added to the solid support or at any one of the repeating cycles (e.g., as shown in, for example, FIG.4).

[0112] As shown in FIG.5, the protecting group PG can be removed, and the PMO can be cleaved from the solid support to produce a free PMO product.

[0113] For purposes of illustration, FIG.6 shows an overview of a method of producing a repeat multimer PMO. Although FIG. 6 shows a method of producing a CAG repeat PMO, it isenvisioned that the method can be applied to other repeat multimer PMOs (e.g., dimer, trimer, tetramer, etc., having different nucleic acid sequences). As shown in FIG.6A, a 6’-functionalized, 3’ protected morpholino nucleotide multimer is coupled to a solid support, such as an aminomethyl polystyrene resin. The resulting solid-supported multimer is 3’ protected. Additional 6’- functionalized, 3’ protected morpholino nucleotide multimer can be coupled to the 3’ end of the solid-supported morpholino nucleotide multimer (or 3’ end of an extended PMO chain) through multiple (x) rounds of (i) deprotecting the 3’ end of a morpholino nucleotide multimer, neutralizing, and coupling, or (ii) deprotecting the 3’ end of a morpholino nucleotide multimer, neutralizing, coupling, and oxidative amination to produce a 3’-protected, solid supported PMO having x+1 number of multimeric repeats, as shown in FIG.6B. Cleavage from the solid support and deprotection results in a PMO product having x +1 number of multimeric repeats, as shown in FIG.6C. While FIG.6 shows synthesis of a CAG repeat PMO, it will be appreciated that the process shown in FIG. 6 may be used to produce a PMO having any other suitable sequence, including, for example, PMOs having other trinucleotide repeats, having dinucleotide repeats, or having tetranucleotide repeat sequences. The steps in FIG.6 of (i) deprotecting, neutralizing, and coupling, or (ii) deprotecting, neutralizing, coupling, and oxidative amination may be performed according to the strategies depicted in FIGS.8A-C.

[0114] For purposes of illustration, FIG.7 shows an overview of a method of producing a repeat multimer PMO. Although FIG. 7 shows a method of producing a CTG repeat PMO, it is envisioned that the method can be applied to other repeat multimer PMOs (e.g., dimer, trimer, tetramer, etc., having different nucleic acid sequences, see e.g., FIG.8). As shown in FIG.6A, a 6’-functionalized, 3’ protected morpholino nucleotide multimer is coupled to a solid support, such as an aminomethyl polystyrene resin. The resulting solid-supported multimer is 3’ protected. Additional 6’-functionalized, 3’ protected morpholino nucleotide multimer can be coupled to the 3’ end of the solid-supported morpholino nucleotide multimer (or 3’ end of an extended PMO chain) through multiple (x) rounds of (i) deprotecting the 3’ end of a morpholino nucleotide multimer, neutralizing, and coupling, or (ii) deprotecting the 3’ end of a morpholino nucleotide multimer, neutralizing, coupling, and oxidative amination to produce a 3’-protected, solid supported PMO having x+1 number of multimeric repeats, as show in FIG. 7B. Cleavage from the solid support and deprotection results in a PMO product having x +1 number of multimeric repeats, as shown in FIG. 7C. While FIG. 7 shows synthesis of a CTG repeat PMO, it will beappreciated that the process shown in FIG. 7 may be used to produce a PMO having any other suitable sequence, including, for example, PMOs having other trinucleotide repeats, having dinucleotide repeats, or having tetranucleotide repeat sequences. The steps in FIG. 7 of (i) deprotecting, neutralizing, and coupling, or (ii) deprotecting, neutralizing, coupling, and oxidative amination may be performed according to the strategies depicted in FIGS.8A-C.

[0115] FIGS.8A-C are schematic diagrams showing three different strategies for forming PMOs according to the methods disclosed herein. FIG.8A illustrates the first strategy (Strategy 1), which is also referred to herein as the “phosphonamidate coupling” strategy. Mnand (Mn)mare as described above regarding FIGS.1 and 3-4; the circle represents a solid support; PG is a protecting group; LG is a leaving group; R1and R2are each independently H or substituted or unsubstituted alkyl, aryl, alkynyl, alkenyl, cycloalkyl, heterocyclyl, or heteroaryl. In embodiments, R1and R2are each independently substituted or unsubstituted alkyl. In embodiments, R1and R2are each independently unsubstituted alkyl. In embodiments, R1and R2are each methyl. FIG.8B illustrates the second strategy (Strategy 2), which is also referred to herein as the “H-phosphonate coupling” strategy. Mnand (Mn)mare as described above regarding FIGS.1 and 3-4; the circle represents a solid support; PG is a protecting group; R1and R2are each independently H or substituted or unsubstituted alkyl, aryl, alkynyl, alkenyl, cycloalkyl, heterocyclyl, or heteroaryl. In embodiments, R1and R2are each independently substituted or unsubstituted alkyl. In embodiments, R1and R2are each independently unsubstituted alkyl. In embodiments, R1and R2are each methyl. FIG.8C illustrates the third strategy (Strategy 3), which is also referred to herein as the “phosphoramidite coupling” strategy. Mnand (Mn)mare as described above regarding FIGS. 1 and 3-4; the circle represents a solid support; PG is a protecting group; R1, R2, R3and are each independently H or substituted or unsubstituted alkyl, aryl, alkynyl, alkenyl, cycloalkyl, heterocyclyl, or heteroaryl; and Y is H or N(R1)(R2), wherein when Y is H, R1and R2are not present. In embodiments, Y is N(R1)(R2), and R1, R2, R3are each independently substituted or unsubstituted alkyl. In embodiments, Y is N(R1)(R2), and R1, R2, R3are each independently substituted alkyl. In embodiments, R1and R2are each isopropyl.

[0116] In FIGS.8A-C, a deprotected solid-supported PMO multimer is shown coupled with a 6’ functionalized, 3’ protected PMO multimer, followed by x-1 cycles of deprotecting, neutralization, and coupling of the functionalized, protected PMO multimer to a growing PMO chain on the solidsupport. The reaction schemes in FIGS.8A-C correspond to the functional groups (P1) described above.

[0117] In FIG.8A, the PMO multimer is shown functionalized with a phosphonamidite group and coupling with the deprotected solid-supported multimer or the deprotected growing chain results in a PMO product. Coupling of a N-deprotected growing PMO chain with a phosphonamidite functionalized multimer may be repeated a desired number (e.g., x-1 as shown in FIG. 8A) of times to obtain the PMO product with a specific number of repeats (e.g., x as shown in FIG.8A).

[0118] In FIG.8B, the PMO monomer is functionalized with a H-phosphonate group. Coupling of an N-deprotected morpholino with the H-phosphonate functionalized multimer provides an H- phosphonamidate product which may be further functionalized by oxidative amination to furnish the phosphorodiamidate morpholino oligomer product. Coupling of a N-deprotected growing PMO chain with a H-phosphonamidate functionalized multimer may be repeated a desired number (e.g., x-1 as shown in FIG. 8B) of times to obtain the PMO product with a specific number of repeats (e.g., x as shown in FIG.8B).

[0119] In FIG.8C, the PMO monomer is functionalized with a phosphoramidite group. Coupling of an N-deprotected morpholino with the phosphoramidite functionalized multimer provides a phosphoramidite product which may be further functionalized by oxidative amination to furnish the phosphorodiamidate morpholino oligomer product. Coupling of a N-deprotected growing PMO chain with a phosphoramidite functionalized multimer may be repeated a desired number (e.g., x-1 as shown in FIG. 8C) of times to obtain the PMO product with a specific number of repeats (e.g., x as shown in FIG.8C).

[0120] For the reaction schemes shown in the FIGS. herein, once a desired PMO length is synthesized, the protecting group PG can be removed, and the resulting PMO product can be cleaved from the solid support.

[0121] While FIGS. 8A-C show PMO multimers being coupled to the solid support and being added at each repeating cycle, it will be appreciated that PMO monomers can be added to the solid support or at any one of the repeating cycles, for example, as shown in FIG.4. Solid Support and Coupling PMO Monomer or Multimer to Solid Support

[0122] As used herein, “solid support” is any support suitable for use in solid phase reactions for PMO synthesis. The solid support can be sparingly soluble in solvents and reagents that are usedfor PMO synthesis. Non-limiting examples of solvents and reagents that can be used for PMO synthesis include dichloromethane, acetonitrile, tetrazole, N-methylimidazole, pyridine, acetic anhydride, lutidine, and trifluoroacetic acid. The solid support can be chemically stable to the reagents used in PMO synthesis.

[0123] Non-limiting examples of solid supports that can be used in the methods described herein include swellable polystyrene (e.g., aminomethyl polystyrene resin 1% divinylbenzene crosslinked (200-400 mesh) (2.4-3.0 mmol / g) (manufactured by Tokyo Chemical Industry), aminomethylated polystyrene resin HCl [divinylbenzene 1%, 100-200 mesh] (manufactured by Peptide Institute, Inc.)), non-swellable polystyrene (e.g., Primer Support (manufactured by GE Healthcare)), PEG chain-attached polystyrene (e.g., NH2-PEG resin (manufactured by Watanabe Chemical Co.), TentaGel resin), controlled pore glass (controlled pore glass; CPG) (manufactured by, e.g., CPG), oxalyl-controlled pore glass (cf., e.g., Alul et al., Nucleic Acids Research, Vol.19, 1527 (1991)), TentaGel support-aminopolyethylene glycol-derivatized support (for example, Wright et al., cf., Tetrahedron Letters, Vol.34, 3373 (1993)), a copolymer of Poros-polystyrene / divinylbenzene, and the like. In embodiments, the solid support comprises an aminomethyl polystyrene resin. In embodiments, solid support comprises an aminomethyl controlled pore glass resin.

[0124] In embodiments, the solid support comprises a linker. A linker can be a chemical structure that is used in solid-phase synthesis to covalently attach the first monomer, multimer, or building block to the solid support. The linker can serve as a bridge anchoring the growing molecule to the support throughout the synthesis process. Linkers can be designed to be stable during the synthetic steps, including repetitive cycles of reagent addition and washing, while also enabling controlled release of the final product through cleavage under specific conditions. The use of linkers may help facilitate an efficient stepwise assembly of complex molecules like morpholino oligomers (PMOs), while maintaining high yields and product purity.

[0125] Any suitable linker can be used in PMO synthesis. A suitable linker may be chosen based on compatibility with specific reaction conditions and cleavage requirements. Non-limiting examples of suitable linkers include acid-labile linkers, such as trityl and succinyl-based linkers, which can cleave under mild acidic conditions, and base-labile linkers, such as aminomethyl linkers, which can release products upon exposure to mild bases. Additional examples of suitable linkers include photo-labile linkers, which can release the product when exposed to UV light. Specific linkers, like the trityl-linker and methylaminomethyl-linker, can be used to balancechemical stability during synthesis with efficient cleavage and minimal residual contamination. The linker can be chosen based on a number of factors, including the ease of purification, product quality, and the compatibility of downstream processes. In embodiments, the linker comprises a UnyLinker.

[0126] A UnyLinker is a universal linker specifically designed for solid-phase oligonucleotide synthesis, including PMOs. See, e.g., Ravikumar et al. (May 16, 2008), Organic Process Research & Development, 12(3): 399-410 (doi: 10.1021 / op8000178). A UnyLinker has a rigid, bicyclic structure that can provide stability during synthesis while allowing efficient cleavage under mild conditions. Compatible with a wide range of chemistries, including DNA, RNA, and modified oligonucleotides, it may offer superior coupling efficiency and may streamline synthesis workflows. Loading of Solid Support

[0127] The 6’ PMO monomer or multimer may be loaded onto the solid support in any suitable manner. One suitable method for loading a solid support (resin) in a PMO synthesis process carried out using a column-based method, utilizing a syringe-type solid-phase synthesis column connected to an apparatus for controlled nitrogen flow or vacuum extraction, is briefly described. The procedure can include a sequence of resin treatment and washing steps, combining mixing of the resin with solutions and subsequent extraction of these treatments. Mixing can be achieved by allowing nitrogen to flow through the column, ensuring the solutions thoroughly permeate the resin for the specified duration. Following mixing, extraction can be performed by stopping the nitrogen flow, applying vacuum, and removing the solutions. All solution volumes should be proportional to the resin weight.B is a nucleobase; the circle represents a solid support;Mnis a PMO monomer; (Mn)m is a PMO multimer having m PMO nucleotides; n is the position of the Mnmonomer in the (Mn)mmultimer sequence; m is the number of Mnmonomers in the (Mn)m multimer; Py is pyridine; HBTU is 3-[Bis(dimethylamino)methyliumyl]-3H-benzotriazol-1-oxide hexafluorophosphate; HOBT is1H-1,2,3-Benzotriazol-1-ol; NEM is N-ethylmorpholine; NMP is N-methyl-2-pyrrolidinone.

[0128] In embodiments, m can be an integer from 2 to 20, such as 2 to 11, or 2 to 4. In embodiments, m is 2. In embodiments, m is 3. In embodiments, m is 4.

[0129] In embodiments, B can be any nucleotide base (i.e., “nucleobase”) including A, G, C, T, or U. In embodiments, B can be A. In embodiments, B can be G. In embodiments, B can be C. In embodiments, B can be T. In embodiments, B can be U.

[0130] In the reaction scheme above, a 6’-OH multimer (or monomer) substrate is activated by reacting with succinic anhydride in the presence of DMAP in polar aprotic solvents (for example, dichloromethane, N,N-dimethylformamide, pyridine, etc.) to give the acid product. DCC (dicyclohexylcarbodiimide), DIC, or other esterification reactions as known in the art, or other di- carboxylic acid-containing molecules can also be used instead of succinic anhydride.

[0131] The second step of the reaction scheme above can begin with the swelling of the aminomethyl polystyrene resin in a polar aprotic solvent (for example, N-methyl-2- pyrrolidonedichloromethane, N,N-dimethylformamide, pyridine, etc.) that effectively solvates the resin and facilitates subsequent chemical reactions. After swelling, the resin can be washed with dichloromethane (DCM) to remove residual swelling solvent, followed by a solution of diisopropylethylamine (DIPEA) in a mixture of isopropanol and DCM. This washing sequence removes impurities and prepares the resin for coupling reactions.

[0132] To functionalize the resin, a mixture containing a protected trimer acid derivative, HBTU, HOBt, and N-ethylmorpholine (NEM) in NMP can be added. HBTU is a coupling reagent that activates the carboxylic acid group, facilitating its reaction with the resin-bound amine. HOBt serves as a co-reagent to minimize side reactions and improve coupling efficiency. NEM acts as abase to neutralize the by-products of the coupling reaction, ensuring a smooth reaction. The mixture can be heated to, for example, 35°C and stirred to achieve coupling. After the reaction is complete, the resin may be washed with NMP to remove residual reagents, followed by DCM to clean. Any coupling reagent known in the art for amide bond formation / peptide (for example, HATU, HBTU / HOBT, PyAOP, etc.) synthesis can be used. Any weakly nucleophilic / weakly basic amine (DIEA, NEM, pyridine, lutidine, methylated imidazole, etc.) can be employed.

[0133] Following the coupling reaction, the resin may be treated with a capping solution to block any unreacted functional groups. Additional washing steps can be conducted to thoroughly clean the resin, which may then be dried using nitrogen flow and a high vacuum to achieve a constant weight. The final product is expected to show an increase in resin weight, reflecting successful functionalization.

[0134] In embodiments, a solid support may be loaded with a monomer, a dimer, a trimer, a tetramer, or another suitable multimer. In embodiments, a solid support may be loaded with a monomer. In embodiments, a solid support may be loaded with a dimer. In embodiments, a solid support may be loaded with a trimer. In embodiments, a solid support may be loaded with a tetramer. Unylinker loading

[0135] A Unylynker can be loaded by reacting the 6’-OH PMO substrate (monomer or multimer) with 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite in the presence of diisopropylethylamine in dichloromethane (Conditions A), or with 2-cyanoethyl N,N,N′,N′- tetraisopropylphosphorodiamidite in the presence of 5-ethylthiotetrazole in dichloromethane and acetonitrile (Conditions B), as shown below., wherein: B is a nucleobase; Mnis a PMO monomer; (Mn)m is a PMO multimer having m PMO nucleotides; n is the position of the Mnmonomer in the (Mn)mmultimer sequence; and m is the number of Mnmonomers in the (Mn)mmultimer.

[0136] In embodiments, m can be an integer from 2 to 20, such as 2 to 11, or 2 to 4. In embodiments, m is 2. In embodiments, m is 3. In embodiments, m is 4.

[0137] In embodiments, B can be any nucleotide base (i.e., “nucleobase”) including A, G, C, T, or U. In embodiments, B can be A. In embodiments, B can be G. In embodiments, B can be C. In embodiments, B can be T. In embodiments, B can be U. Functionalization for resin loading

[0138] FIG. 9A shows one pathway for functionalizing a PMO multimer for loading on a solid support. The pathway shown in FIG.9A can be used to functionalize an PMO multimer having a sequence of “BBB”, where each B can be any of A, T, G, C, or U. It will be appreciated that the reaction scheme may be employed with PMO multimers having different overall lengths, different PMO multimers (e.g., dimers, trimers, tetramers, etc.,), and different multimer sequences. In embodiments, the reaction scheme may be used with PMO multimers having a CAG sequence, as shown in FIG.9B. In embodiments, the reaction scheme may be used with PMO multimers having a CTG sequence, as shown in FIG.9C. FIG.9A shows a functionalization that results in a 6’-N- Hydroxy-5-norbornene-2,3-dicarboximide (HONB) succinic ester CAG PMO trimer..

[0139] FIG. 9B shows reaction conditions that can be used to functionalize a PMO multimer having a CAG trimer repeat. As shown, a 6’-OH, 3’ protected CAG PMO is reacted with succinic anhydride in DMAP or DCC, TEA, DCM at 20 C for two hours, and the reaction product is reacted with N-Hydroxy-5-norbornene-2,3-dicarboxylic acid imide in DMAP, EDC, DCM at 20- 55 C for 5 hours to produce a 6’-N-hydroxy-5-norbornene-2,3-dicarboximide (HONB) succinic ester CAG PMO trimer, which then may be coupled with a resin. It will be appreciated that reaction temperatures and times shown in FIG.9B may be varied.

[0140] FIG. 9C shows reaction conditions that can be used to functionalize a PMO multimer having a CTG trimer repeat. As shown, a 6’-OH, 3’ protected CTG PMO is reacted with succinic anhydride in DMAP or DCC, TEA, DCM at 20 C for two hours, and the reaction product is reacted with N-Hydroxy-5-norbornene-2,3-dicarboxylic acid imide in DMAP, EDC, DCM at 20- 55 C for 5 hours to produce a 6’-N-hydroxy-5-norbornene-2,3-dicarboximide (HONB) succinic ester CTG PMO trimer, which then may be coupled with a resin. It will be appreciated that reaction temperatures and times shown in FIG.9C may be varied.

[0141] It will be appreciated that resin loading methods described above are merely examples of methods for loading a solid support with a PMO monomer or multimer and that any other suitable resin and loading method may be employed. (I) Strategy 1: Phosphonamidate Coupling

[0142] As indicated above, for example, with reference to FIG.8A, methods for the preparation of PMOs described herein include phosphonamidate coupling according to Strategy 1. As shown in the Examples that follow, Strategy 1 (phosphonamidate coupling) resulted in substantially higher yields than Strategies 2 and 3, which are discussed in more detail below.

[0143] Strategy 1 (phosphonamidate coupling) is generally illustrated in the reaction scheme below:wherein: each B is independently a nucleobase; LG is a leaving group; R1and R2are each independently H or substituted or unsubstituted alkyl, aryl, alkynyl, alkenyl, cycloalkyl, heterocyclyl, or heteroaryl; R4is a hydrogen or a protecting group; the circle represents a solid support; Mnis a PMO monomer; (Mn)mis a PMO multimer having m PMO nucleotides; n is the position of the Mnmonomer in the (Mn)m multimer sequence; m is the number of Mnmonomers in the (Mn)m multimer; and x is the total number of (Mn)mmultimers following in x-1 additional cycles.

[0144] In embodiments, R1and R2are each independently substituted or unsubstituted alkyl. In embodiments, R1and R2are each independently unsubstituted alkyl. In embodiments, R1and R2are each methyl.

[0145] In embodiments, m can be an integer from 2 to 20, such as 2 to 11, or 2 to 4. In embodiments, m is 2. In embodiments, m is 3. In embodiments, m is 4.

[0146] In embodiments, x is an integer from 1 to 50. In embodiments, x is an integer from 1 to 40. In embodiments, x is an integer from 1 to 30. In embodiments, x is an integer from 1 to 20. In embodiments, x is an integer from 1 to 15. In embodiments, x is 1. In embodiments, x is 2. In embodiments, x is 3. In embodiments, x is 4. In embodiments, x is 5. In embodiments, x is 6. In embodiments, x is 7. In embodiments, x is 8. In embodiments, x is 9. In embodiments, x is 10. Inembodiments, x is 11. In embodiments, x is 12. In embodiments, x is 13. In embodiments, x is 14. In embodiments, x is 15.

[0147] As used herein, "protecting group" refers to chemical moieties that block some or all reactive moieties of another compound and prevent such moieties from participating in chemical reactions until the protective group is removed. Protecting groups suitable for the present disclosure include those disclosed in Protective Groups in Organic Synthesis (Greene and Wuts, 2nded.), which is hereby incorporated by reference in its entirety. In embodiments, the morpholino ring nitrogen protecting group is an arylmethyl protecting group. In embodiments, the arylmethyl protecting group is a triarylmethyl protecting group. The arylmethyl protecting group can be a substituted arylmethyl protecting group.

[0148] As used herein, the term “nucleobase” refers to the portion of a nucleoside or nucleotide that is capable of hydrogen bonding to a nucleobase of another nucleic acid. A nucleobase can comprise any atom or group of atoms capable of hydrogen bonding. In embodiments, the nucleobase is a nitrogenous base. A natural nucleobase is a nucleobase that is unmodified from its naturally occurring form found in RNA or DNA and includes, for example, cytosine (C), guanine (G), adenine (A), thymine (T) (in DNA) or uracil (U) (in RNA). Modified DNA nucleobases include 5-methylcytidine (5mC). Modified RNA nucleobases include, but are not limited to, pseudouridine (Ψ), dihydrouridine (D), inosine (I), ribothymidine (rT), and 7-methylguanosine (m7G). In embodiments, one or more PMOs comprise a modified nucleobase.

[0149] LG can be any suitable leaving group. The leaving group may be any functional group that may leave the phosphorus atom under suitable reaction conditions. The leaving ability of a leaving group is influenced by both the electronic properties and the steric properties of the leaving group. The leaving group may be able to depart and / or be able to stabilize the transition state or the reaction intermediate. In a typical coupling reaction, the leaving group may allow the phosphorus atom to retain sufficient electrophilicity, facilitating its attraction for nucleophiles, such as the ring nitrogen of a morpholine. In embodiments, the leaving group is an electron withdrawing group. In embodiments, the electron-withdrawing leaving group is a halide. In embodiments, the leaving group is a non-halide functional group. In embodiments, the leaving group is an electron- withdrawing non-halide functional group. In embodiments, the leaving group is a halide. In embodiments, the leaving group is chloride.

[0150] In embodiments, the Strategy 1 reaction scheme can be used to generate a solid-supported PMO having a (BBB)7 repeat sequence as shown in FIG. 11, , where “BBB” can be any trimer repeat sequence having any three of A, T, G, C, or U. In embodiments, the reaction scheme shown in FIG. 11 can be used to generate a PMO having a (CAG)7 trimer repeat. In embodiments, the reaction scheme shown in FIG.11 can be used to generate a PMO having a (CTG)7 trimer repeat. It will be understood that the reaction scheme shown in FIG.11 may be used with PMO multimers having a sequence other than CAG or CTG.

[0151] As shown, in FIG.11, a solid supported 6’-OH, 3’ protected PMO trimer is reacted with a 6’-chlorophosphoramidate CAG PMO trimer (inset left) for six cycles to produce the solid- supported 21-mer having a (CAG)7trimer repeat . Each cycle includes deprotecting (detritylating) the 3’ end of the PMO coupled to the solid support, neutralizing, and coupling. Detritylation in includes reacting with 4-cyanopyridine in dichloromethane (DCM), trifluoroacetic acid (TFA), trifluoroethanol (TFE), and EtOH. Neutralization includes washing with propan-2-ol (IPA), di- isopropyl ethylamine (DIEA), dichloromethane (DCM). Coupling includes reacting the monomer in the presence of 0.4 M N-ethylmorpholine (NEM) in dimethylimidazolidinone (DMI). It will be understood that other suitable reaction conditions and reagents may be employed.

[0152] As shown, in FIG.11, a solid supported 6’-OH, 3’ protected PMO trimer is reacted with a 6’-chlorophosphoramidate CTG PMO trimer (inset RIGHT) for six cycles to produce the solid- supported 21-mer having a (CTG)7trimer repeat . Each cycle includes deprotecting (detritylating) the 3’ end of the PMO coupled to the solid support, neutralizing, and coupling. Detritylation in includes reacting with 4-cyanopyridine in dichloromethane (DCM), trifluoroacetic acid (TFA), trifluoroethanol (TFE), and EtOH. Neutralization includes washing with propan-2-ol (IPA), di- isopropyl ethylamine (DIEA), dichloromethane (DCM). Coupling includes reacting the monomer in the presence of 0.4 M N-ethylmorpholine (NEM) in dimethylimidazolidinone (DMI). It will be understood that other suitable reaction conditions and reagents may be employed. Deprotection of the Solid-Supported Multimer (or Monomer)

[0153] Prior to phosphonamidate coupling, a 3’ N-protecting group on the solid-supported multimer (or monomer if employed), can be removed to deprotect the solid-supported multimer (or monomer, as the case may be) so that the solid-supported multimer (or monomer) may reactwith the phosphonamidite morpholino monomer or multimer. Deprotection may occur generally as shown below:, wherein: B is a nucleobase; R1and R2are each independently H or substituted or unsubstituted alkyl, aryl, alkynyl, alkenyl, cycloalkyl, heterocyclyl, or heteroaryl; R4is a hydrogen or a protecting group; the circle represents a solid support; Mnis a PMO monomer; (Mn)mis a PMO multimer having m PMO nucleotides; n is the position of the Mnmonomer in the (Mn)m multimer sequence; and m is the number of Mnmonomers in the (Mn)m multimer.

[0154] In embodiments, R1and R2are each independently substituted or unsubstituted alkyl. In embodiments, R1and R2are each independently unsubstituted alkyl. In embodiments, R1and R2are each methyl.

[0155] In embodiments, m can be an integer from 2 to 20, such as 2 to 11, or 2 to 4. In embodiments, m is 2. In embodiments, m is 3. In embodiments, m is 4.

[0156] In embodiments, B can be any nucleotide base (i.e., “nucleobase”) including A, G, C, T, or U. In embodiments, B can be A. In embodiments, B can be G. In embodiments, B can be C. In embodiments, B can be T. In embodiments, B can be U.

[0157] In embodiments, complete deprotection is achieved. Failure to completely remove the protecting group may lead to shortened PMO sequences that may contaminate the desired PMO product. Morpholino ring nitrogen protecting groups can be removed via reaction with any suitabledeprotection reagents under appropriate reaction conditions. Suitable deprotection strategies can include acidic, basic, and nucleophilic cleavage. In embodiments, the morpholino ring nitrogen protecting groups are removed under acidic conditions. In embodiments, acidic deprotection is used, particularly for acid-labile protecting groups like tert-butoxycarbonyl (Boc) and arylmethyl protecting groups. In embodiments, the arylmethyl protecting groups include trityl or substituted trityl protecting groups. In embodiments, the trityl moiety-based protecting groups suitable for morpholino ring nitrogen protection between coupling steps include unsubstituted trityl, 4- methyltrityl, 4,4’-dimethyltrityl, and 4,4’,4’’-trimethyltrityl, or 4-methoxytrityl protecting groups. In embodiments, other acids, such as trifluoroacetic acid (TFA) or p-toluenesulfonic acid (TsOH), are employed to cleave these groups efficiently under mild conditions. In embodiments, basic conditions are employed for the deprotection, particularly for base-labile groups like fluorenylmethoxycarbonyl (Fmoc). In embodiments, protecting group cleavage is achieved using a strong base, such as piperidine or DBU, in an organic solvent. Basic conditions may be well- suited for the selective removal of certain protecting groups while preserving acid-sensitive functionalities in the molecule. Such deprotection methods can be particularly advantageous in solid-phase synthesis, where orthogonal deprotection strategies may be desired.

[0158] In embodiments, the protecting group is an arylmethyl protecting group, and the arylmethyl protecting group is deprotected using acidic cleavage. Trityl groups may be highly acid-labile and may be easily removed with TFA in organic solvents such as dichloromethane (DCM). During deprotection, the trityl group may be cleaved, producing the free amine and a trityl alcohol byproduct. Deprotection of trityl groups may also be performed with other acids, such as, TsOH. This reagent may provide a milder acidic environment compared to TFA and may be effective in solvents like methanol or dichloromethane.

[0159] In embodiments, the protecting group is a triaryl methyl protecting group, and deprotection of the triaryl methyl protecting groups includes exposing the triaryl methyl-protected ring nitrogen to a reagent solution comprising a heterocyclic amine salt in a trifluoroethanol-containing solvent. The salt can be, for example, a salt of a heterocyclic amine, having a pKa in the range of 1-4 in its protonated form, with an acid selected from a sulfonic acid, trifluoroacetic acid, and hydrochloric acid. The heterocyclic amine is selected from an electron withdrawing group-substituted pyridine, thiazole, pyridazine, pyrazole, triazole, and electron withdrawing group-substituted substituted derivatives of these. Such electron withdrawing groups may include halogen, cyano, aldehyde,keto, carboxyester, and carboxamide. In embodiments, the heterocyclic amine is an electron withdrawing group-substituted pyridine, such as a chloro- or cyano-substituted pyridine. The amine salt is a salt, a sulfonic acid, such as an alkylsulfonate, (fluoroalkyl)sulfonate, or p- toluenesulfonate, or a trifluoroacetate.

[0160] The deprotecting reagent(s) used in the deprotection step can be diluted with any suitable solvent, for example, water, methanol, ethanol, isopropyl alcohol, acetonitrile, tetrahydrofuran, DMF, N,N-dimethylimidazolidone, N-methylpiperidone, or a mixture thereof. In embodiments, an amount of deprotecting agent used is at least 1 mol equivalent and up to 10000 mol equivalents, at least 5 mol equivalents and up to 1000 mol equivalents, at least 5 mol equivalents and up to 500 mol equivalents, at least 10 mol equivalents and up to 100 mol equivalents, or at least 10 mol equivalents and up to 50 mol equivalents, based on 1 mol of the protecting group-containing compound. In embodiments, an amount of deprotecting agent used is within the range of at least 1 mol equivalent and up to 10000 mol equivalents based on 1 mol of the protecting group- containing compound.

[0161] In embodiments, one or more of the steps of the methods of PMO synthesis disclosed herein are carried out at temperatures higher or lower than room temperature (25 °C). In embodiments, the deprotection reaction mixture is exposed to, or may be at, temperatures of at least -80 °C and up to 150 °C, at least -50 °C and up to 120 °C, at least -10 °C and up to 100 °C, at least 0 °C and up to 90 °C, at least 10 °C and up to 75 °C, at least 25 °C and up to 75 °C, or at least 50 °C and up to 60 °C. In embodiments, the deprotection reaction mixture is exposed to, or may be at, temperatures in a range of 15 °C to 75 °C, in a range of 40 °C to 70 °C, or in a range of 50 °C to 60 °C. In embodiments, the deprotection reaction mixture is exposed to, or may be at, temperatures of at least -80 °C, at least -50 °C, at least -10 °C, at least 0 °C, at least 10 °C, at least 25 °C, at least 35 °C, at least 50 °C, at least 60 °C, at least 75 °C, at least 90 °C, at least 100 °C, at least 120 °C, or at least 140 °C. In embodiments, the deprotection reaction mixture is exposed to, or may be at, temperatures of up to 150 °C, up to 120 °C, up to 100 °C, up to 90 °C, up to 75 °C, up to 75 °C, up to 60 °C, up to 50 °C, up to 25 °C, up to 10 °C, up to 0 °C, up to -10 °C, up to -50 °C, or up to -78 °C. In embodiments, the deprotection reaction mixture may be exposed to, or may be at, temperatures of at least -10 °C and up to 100 °C, at least 0 °C and up to 90 °C, at least 10 °C and up to 75 °C, at least 25 °C and up to 75 °C, or at least 50 °C and up to 60 °C. In embodiments, the deprotection reaction mixture may be exposed to, or may be at, temperatures in a range of 15°C to 75 °C, in a range of 40 °C to 70 °C, or in a range of 50 °C to 60 °C. In embodiments, the deprotection reaction mixture is exposed to, or may be at, temperatures of at least -10 °C, at least 0 °C, at least 10 °C, at least 25 °C, at least 35 °C, at least 50 °C, at least 60 °C, at least 75 °C, or at least 90 °C. In embodiments, the deprotection reaction mixture is to, or may be at, temperatures of up to 100 °C, up to 90 °C, up to 75 °C, up to 75 °C, up to 60 °C, up to 50 °C, up to 25 °C, up to 10 °C, up to 0 °C.

[0162] The reaction time for the deprotection reaction may vary depending upon the compound to be deprotected, reaction temperature, deprotection reagent, etc. In embodiments, the reaction time for the deprotection reaction is at least 10 minutes and up to 30 hours, at least 30 minutes and up to 24 hours, at least 1 hour and up to 20 hours, at least 2 hours and up to 15 hours, at least 5 hours and up to 10 hours, or at least 5 hours and up to 20 hours. In embodiments, the reaction time for the deprotection reaction is in a range of 10 minutes to 30 hours, in a range of 30 minutes to 24 hours, in a range of 1 hour to 20 hours, in a range of 2 hours to 15 hours, in a range of 5 hours to 10 hours, or in a range of 5 hours to 20 hours. In embodiments, the reaction time for the deprotection reaction is at least 10 minutes and up to 10 hours, at least 30 minutes and up to 8 hours, at least 1 hour and up to 6 hours, at least 2 hours and up to 5 hours, at least 3 hours and up to 4, or at least 5 hours. In embodiments, the reaction time for the deprotection reaction is in a range of 10 minutes to 10 hours, in a range of 30 minutes to 8 hours, in a range of 1 hour to 6 hours, in a range of 2 hours to 5 hours, or in a range of 3 hours to 4 hours.

[0163] In embodiments, deprotection occurs generally as shown below: ,B is a nucleobase; the circle represents a solid support; Mnis a PMO monomer;(Mn)m is a PMO multimer having m PMO nucleotides; n is the position of the Mnmonomer in the (Mn)m multimer sequence; and m is the number of Mnmonomers in the (Mn)mmultimer.

[0164] In embodiments, m can be an integer from 2 to 20, such as 2 to 11, or 2 to 4. In embodiments, m is 2. In embodiments, m is 3. In embodiments, m is 4.

[0165] In embodiments, B can be any nucleotide base (i.e., “nucleobase”) including A, G, C, T, or U. In embodiments, B can be A. In embodiments, B can be G. In embodiments, B can be C. In embodiments, B can be T. In embodiments, B can be U.

[0166] As illustrated above, the morpholino ring nitrogen protecting group can be a triarylmethyl protecting group. The arylmethyl protecting group may be a substituted triarylmethyl protecting group. Examples of triarylmethyl protecting groups include unsubstituted trityl (shown above), 4- methyltrityl, 4,4’-dimethyltrityl, and 4,4’,4’’-trimethyltrityl, or 4-methoxytrityl protecting groups.

[0167] The triarylmethyl protecting groups can be removed with TFA in organic solvents such as dichloromethane (DCM). Deprotection of trityl groups may also be performed with other acids, such as, TsOH.

[0168] In embodiments, the deprotection of the morpholino ring nitrogen protecting groups includes exposing the triarylmethyl-protected ring nitrogen to a reagent solution comprising a heterocyclic amine salt in a trifluoroethanol-containing solvent, the salt being a salt of a heterocyclic amine, having a pKa in the range of 1-4 in its protonated form, with an acid selected from a sulfonic acid, trifluoroacetic acid, and hydrochloric acid. In embodiments, the heterocyclic amine is an electron withdrawing group-substituted pyridine, such as a chloro- or cyano- substituted pyridine. The amine salt can be a salt of a sulfonic acid, such as an alkylsulfonate, (fluoroalkyl)sulfonate, or p-toluenesulfonate, or a trifluoroacetate.

[0169] For a more selective deprotection process, 4-cyanopyridinium trifluoroacetate may be used to remove trityl groups. This reagent, formed in situ by mixing 4-cyanopyridine with TFA, may offer a mild yet highly effective alternative for cleaving trityl groups at room temperature. The method may minimize side reactions and may be particularly valuable for deprotecting nitrogen while preserving sensitive functionalities in PMOs.

[0170] In embodiments, the salt is 4-cyanopyridinium trifluoroacetate (CYTFA). In embodiments, the deprotection reaction solvent includes dichloromethane and trifluoroethanol in volume ratio in the range of 90:10 to 25:75. In embodiments, the deprotection reaction solvent includesdichloromethane and trifluoroethanol in volume ratio of 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, 50:50, 45:55, 40:60, 35:65, 30:70, or 25:75. In embodiments, the deprotection reaction solvent includes dichloromethane and trifluoroethanol in volume ratio of 80:20. In embodiments, the deprotection reaction solvent includes dichloromethane, trifluoroethanol, and ethanol in volume ratio of 80:20:1 for DCM / TFE / EtOH.

[0171] The use of the TFE as a deprotection reaction solvent is believed to enhance the selectivity of the detritylation reaction over amidate formation (hydrolysis) and phosphorodiamidate cleavage. TFE is a potent hydrogen bonding solvent and decreases the reactivity of nucleophiles in solution. Hence, it slows the attack on phosphorus necessary for P-N bond cleavage. TFE also promotes SN1type solvolysis reactions. An increasing TFE concentration is believed to both suppress nucleophilic attack on the phosphorodiamidate linkage and promote detritylation.

[0172] Unsubstituted pyridinium salts are not sufficiently acidic for optimal deprotection, but the use of pyridinium species containing electron withdrawing groups (such as, halogen, carbonyl, cyano) allows rapid cleavage of the protecting group. In embodiments, the pyridinium salt is present in the deprotection reaction in a range of 1 wt.-% to 15 wt.-%. In embodiments, the pyridinium salt is present in the deprotection reaction in an amount of at least 1 wt.-%, at least 2 wt.-%, at least 3 wt.-%, at least about 4 wt.-%, at least 5 wt.-%, at least 6 wt.-%, at least 7 wt.-%, at least 8 wt.-%, at least 9 wt.-%, at least 10 wt.-%, at least 11 wt.-%, at least 12 wt.-%, at least 13 wt.-%, or at least 14 wt.-%. In embodiments, the pyridinium salt is present in the deprotection reaction in a n amount of up to 15 wt.-%, up to about 14 wt.-%, up to 13 wt.-%, up to 12 wt.-%, up to 11 wt.-%, up to 10 wt.-%, up to 9 wt.-%, up to 8 wt.-%, up to 7 wt.-%, up to 6 wt.-%, up to about 5 wt.-%, up to 4 wt.-%, up to 3 wt.-%, or up to 2 wt.-%. In embodiments, the pyridinium salt is present in the deprotection reaction in an amount of 2 wt.-%,

[0173] Acids useful in forming the pyridinium salts include sulfonic acids, such as methanesulfonic, trifluoromethanesulfonic, and p-toluenesulfonic acid, trifluoroacetic acid, and hydrochloric acid. Although a carboxylic acid, trifluoroacetic acid does not cap the growing PMO chain if present during the coupling reaction, and its carboxylate is not sufficiently nucleophilic to promote amidate formation. Particularly preferred are trifluoroacetic and especially methanesulfonic acid. The pyridines useful in forming the pyridinium salts include halogen substituted pyridines, especially the less expensive chloropyridines, of which 3-chloropyridine is preferred, and cyanopyridines, for which 4-cyanopyridine is preferred. The 3- and 4-cyanoopyridines are readily available, inexpensive bulk chemicals. In general, the efficacy of the salts correlates inversely with the pKa of the pyridinium species.

[0174] Also useful are nicotinic acid derivatives (i.e. esters, such as ethyl nicotinate, and nicotinamide), as well as their ketone and aldehyde congeners. Generally, however, these are less potent reagents than the cyanopyridinium salts.

[0175] It will be appreciated that salts of heterocycles other than pyridines can function as selective detritylation reagents under the conditions described, provided the pKa of the protonated form is similar to that of substituted pyridines of the invention. Examples may be found in the many tables of pKa for heterocycles found in the literature. Examples include thiazole (pKa 2.53), pyridazine (pKa 2.33), pyrazole (pKa 2.47), triazole (pKa 2.30), and substituted derivatives thereof, especially derivatives substituted with EWG as described above.

[0176] In embodiments, the pyridinium salt in the deprotection reaction is 3-chloropyridinium methanesulfonate (CPM) or 4-cyanopyridinium trifluoroacetate (CYTFA). In embodiments, the pyridinium salt in the deprotection reaction is 4-cyanopyridinium trifluoroacetate (CYTFA). In embodiments, the deprotection reaction includes solutions of 2% (w / v) of CYTFA in 20% trifluoroethanol / DCM (v / v) containing 0.9% ethanol (v / v). Neutralization

[0177] After the deprotection reaction, the deprotected morpholino substituent may be subjected to neutralization by being brought into contact with neutralization agents. In embodiments, the neutralizing agents include a monoalkyl, a dialkyl, a trialkyl amine (for example, diisopropylethylamine), or sterically hindered amine bases. In embodiments, the neutralizing agent base is N,N-diisopropylethylamine or N-ethylmorpholine.. In embodiments, the neutralizing agent base is N,N-diisopropylethylamine. In embodiments, the neutralizing agent base is N- ethylmorpholine. In embodiments, the neutralization agent is mixed with a solvent such as a small molecule alcohol including methanol, ethanol, or isopropyl alcohol. Other solvents known in the art may be used. In embodiments, dichloromethane is used. Dichloromethane provides can swell certain solid-support resins. In embodiments, a mixture of solvents may be used to prepare neutralizing solutions. In embodiments, the neutralization agent includes dichloromethane and trifluoroethanol.

[0178] In embodiments, the deprotected morpholino substituent is treated with at least 2-fold and up to 30-fold volume of neutralization agent solution or mixture. The deprotected morpholino substituent can be treated with at least 3-fold and up to 28-fold, at least 4-fold and up to 25-fold, at least 5-fold and up to 20-fold, at least 6-fold and up to 15-fold, or at least 7-fold and up to 9- fold volume of neutralization agent solution or mixture. In embodiments, the deprotected morpholino substituent is treated with at least 2-fold and up to 10-fold volume of neutralization agent solution or mixture. The deprotected morpholino substituent can be treated with at least 3- fold and up to 18-fold, at least 4-fold and up to 15-fold, at least 5-fold and up to 12-fold, at least 6-fold and up to 10-fold, or at least 7-fold and up to 9-fold volume of neutralization agent solution or mixture.

[0179] In embodiments, the deprotected morpholino substituent is treated with a neutralization agent solution or mixture for from 1 to 30 minutes, from 2 to 25 minutes, from 3 to 20 minutes, from 4 to 15 minutes, or from 5 to 10 minutes. In embodiments, the deprotected morpholino substituent is treated with a neutralization agent solution or mixture for from 1 to 20 minutes, from 2 to 15 minutes, from 3 to 12 minutes, from 4 to 10 minutes, or from 5 to 10 minutes.

[0180] In embodiments, the deprotected morpholino substituent is treated with a neutralization agent solution or mixture until a desirable pH of the solution is achieved. In embodiments, the desirable pH of the solution is at least 3 and up to 12, at least 4 and up to 11, at least 5 and up to 10, at least 5 and up to 9, at least 5 and up to 8, at least 6 and up to 8, or at least 6 and up to 7. In embodiments, the desirable pH of the solution is 7. Coupling

[0181] The deprotection of the morpholine nitrogen, and neutralization may be followed by coupling of the deprotected solid-supported multimer and a 6’- phosphonamidate functionalized non-solid-supported multimer as generally illustrated in the reaction scheme below:a LG is a leaving group; R1and R2are each independently H or substituted or unsubstituted alkyl, aryl, alkynyl, alkenyl, cycloalkyl, heterocyclyl, or heteroaryl; R4is a hydrogen or a protecting group; the circle represents a solid support; Mnis a PMO monomer; (Mn)m is a PMO multimer having m PMO nucleotides; n is the position of the Mnmonomer in the (Mn)mmultimer sequence; and m is the number of Mnmonomers in the (Mn)mmultimer.

[0182] In embodiments, m can be an integer from 2 to 20, such as 2 to 11, or 2 to 4. In embodiments, m is 2. In embodiments, m is 3. In embodiments, m is 4.

[0183] In embodiments, R1and R2are each independently substituted or unsubstituted alkyl. In embodiments, R1and R2are each independently unsubstituted alkyl. In embodiments, R1and R2are each methyl.

[0184] The coupling reaction may include the use of a base and a solvent.

[0185] The 6’-functionalized non-solid-supported multimer may include any suitable leaving group (LG). The leaving group may be any functional group that may leave the phosphorus atom under suitable reaction conditions. The leaving ability of a leaving group is influenced by both the electronic properties and the steric properties of the leaving group. The leaving group may be able to depart and / or be able to stabilize the transition state or the reaction intermediate. In a typical coupling reaction, the leaving group may allow the phosphorus atom to retain sufficientelectrophilicity, facilitating its attraction for nucleophiles, such as the ring nitrogen of a morpholine. In embodiments, the leaving group is an electron withdrawing group. In embodiments, the electron-withdrawing leaving group is a halide. In embodiments, the leaving group is a non-halide functional group. In embodiments, the leaving group is an electron- withdrawing non-halide functional group.

[0186] In embodiments, the leaving group is a halide. The leaving ability of a halide leaving group is influenced by both the electronic properties and the steric properties of the halide. Halides generally display a better leaving ability down the halogen group in the periodic table, i.e., generally the leaving ability of the halides follows the trend of iodo being a good leaving group and fluoro being the worst leaving group among the halides (I > Br > Cl >> F). For example, fluoro group may be less desirable as a leaving group due to its high electronegativity and small size, making it less likely to provide optimum results in this reaction without strong activation. Furthermore, phosphorus atom's electrophilicity increases when bound to halogens due to their electron-withdrawing nature. Larger halogens like bromo and iodo may polarize the P-halogen bond more effectively than smaller fluoro, making phosphorus more electrophilic. However, the size of the halogen may affect transition state stability owing to the steric factors and. For example, larger halogens like iodo and bromo may create more steric hindrance, potentially slowing the reaction compared to chloro or fluoro. In embodiments, the leaving group is a chloro. In embodiments, the leaving group is a fluoro.

[0187] The choice of the halide leaving group may achieve an optimal balance of the steric properties and electronic properties. For example, using bromo to form phosphoramidobromidate may lead to efficient reactions, but the increased size and polarizability of the bromo functionality might alter the kinetics of the reaction, or the processes involved in isolating products. Similarly, the use of an iodo functionality may expedite the reaction process, but the iodo group’s high polarizability might result in unintended side reactions, especially if other nucleophiles or bases are present in the mixture. A fluoro leaving group may present challenges under standard conditions, necessitating stronger bases or catalysts to activate the phosphorus-fluorine bond effectively.

[0188] In embodiments, the leaving group may be a non-halide functional group. In embodiments, the leaving group is an electron-withdrawing non-halide functional group. In embodiments, the electron-withdrawing non-halide functional group may include, for example, substituents such assulfonates (for example, tosylate or mesylate groups). In embodiments, the leaving group is an activated ester (for example, a nitrophenyl ester, a pentafluorophenyl ester, a succinamide ester, or a hydroxybenzotriazole ester). While the presence of electron-withdrawing groups may not be a strict necessity for facilitating a successful reaction, their presence can enhance reaction efficiency. Electron-withdrawing groups may stabilize the transition state and ease the departure of the leaving group.

[0189] In embodiments, the leaving group is chloride. The use of chloride as a leaving group provides a balance of reactivity and stability. In embodiments, the leaving group is bromide or iodide. Bromide and iodide leaving groups may provide higher reactivity with faster reactions but can make increase instability due to their bigger size. In embodiments, the leaving group is a sulfonate. In embodiments, the sulfonate leaving group is a tosylate. In embodiments, the sulfonate leaving group is a mesylate. In embodiments, the sulfonate leaving group is a triflate. In embodiments, the leaving group is an activated ester. In embodiments, the activated ester leaving group is a nitrophenyl ester. In embodiments, the activated ester leaving group is a pentafluorophenyl ester. In embodiments, the activated ester leaving group is a succinamide ester. In embodiments, the activated ester leaving group is a hydroxybenzotriazole ester. In embodiments, the leaving group is an imidazole-based leaving group. In embodiments, the leaving group is a pyrazole-based leaving group.

[0190] In embodiments, the coupling reaction is performed by treating the deprotected solid- supported multimer and the 6’-functionalized non-solid-supported multimer with a coupling solution. In embodiments, the coupling solution may include from 1 equivalent to 50 equivalents of the 6’-functionalized non-solid-supported multimer with respect to the solid-supported multimer. In embodiments, the coupling solution includes from 2 to 45 equivalents, from 3 to 40 equivalents, from 4 to 35 equivalents, from 5 to 30 equivalents, from 6 to 25 equivalents, from 7 to 20 equivalents, from 8 to 15 equivalents, from 9 to 10 equivalents, from 7 to 9 equivalents, from 3 to 5 equivalents, from 2 to 5 equivalents, from 3 to 4 equivalents, 4 equivalents, or 5 equivalents of the 6’-functionalized non-solid-supported multimer with respect to the solid-supported multimer. In embodiments, the coupling solution includes from 1 to 30 equivalents, from 2 to 25 equivalents, from 3 to 20 equivalents, from 4 to 15 equivalents, from 5 to 12 equivalents, or from 6 to 10 equivalents of the 6’-functionalized non-solid-supported multimer with respect to the solid- supported multimer. In embodiments, the coupling solution includes at least 1 equivalent, at least2 equivalents, at least 3 equivalents, at least 4 equivalents, at least 5 equivalents, at least 6 equivalents, at least 7 equivalents, at least 8 equivalents, at least 9 equivalents, at least 10 equivalents, at least 12 equivalents, at least 15 equivalents, at least 20 equivalents, at least 25 equivalents, at least 30 equivalents, at least 35 equivalents, at least 40 equivalents, or at least 45 equivalents of the 6’-functionalized non-solid-supported multimer with respect to the solid- supported multimer. In embodiments, the coupling solution includes up to 50 equivalents, up to 45 equivalents, up to 40 equivalents, up to 35 equivalents, up to 30 equivalents, up to 25 equivalents, up to 20 equivalents, up to 15 equivalents, up to 12 equivalents, up to 10 equivalents, up to 9 equivalents, up to 8 equivalents, up to 7 equivalents, up to 6 equivalents, up to 5 equivalents, up to 4 equivalents, up to 3 equivalents, or up to 2 equivalents of the 6’-functionalized non-solid- supported multimer with respect to the solid-supported multimer. In embodiments, the coupling solution may include from 1 equivalent to 30 equivalents of the 6’-functionalized non-solid- supported multimer with respect to the solid-supported multimer.

[0191] In embodiments, the coupling solution includes from 0.01 M to 5 M concentration of the 6’-functionalized non-solid-supported multimer. In embodiments, the coupling solution may include from 0.01 M to 5 M, from 0.02 M to 4.5 M, from 0.03 M to 4 M, from 0.04 M to 3.5 M, from 0.05 M to 3 M, from 0.1 M to 2.5 M, from 0.1 M to 2 M, from 0.2 M to 1.5 M, from 0.3 M to 1 M, from 0.4 M to 0.5 M, or from 0.1 M to 0.2 M concentration of the 6’-functionalized non- solid-supported multimer. In embodiments, the coupling solution includes at least 0.01 M, at least 0.02 M, at least 0.03 M, at least 0.04 M, at least 0.05 M, at least 0.1 M, at least 0.1 M, at least 0.2 M, at least 0.3 M, at least 0.4 M, at least 0.5 M, at least 0.6 M, at least 0.7 M, at least 0.8 M, at least 0.9 M, at least 1 M, at least 1.5 M, at least 2 M, at least 2.5 M, at least 3 M, at least 3.5 M, at least 4 M, or at least 4.5 M concentration of the 6’-functionalized non-solid-supported multimer. In embodiments, the coupling solution includes up to 5 M, up to 4.5 M, up to 4 M, up to 3.5 M, up to 3 M, up to 2.5 M, up to 2 M, up to 1.5 M, up to 1 M, up to 0.9 M, up to 0.8 M, up to 0.7 M, up to 0.6 M, up to 0.5 M, up to 0.4 M, up to 0.3 M, up to 0.2 M, up to 0.1 M, up to 0.05 M, up to 0.04 M, up to 0.03 M, or up to 0.02 M concentration of the 6’-functionalized non-solid-supported multimer.

[0192] In embodiments, a base may be added to the coupling reaction. The added base may help deprotonate the 6’-OH group of the substrate. The base may help neutralize acids (such as HCl) produced during the activation of functional groups and maintain the pH of the reaction mixture.In embodiments, the base used in the coupling reaction is a non-nucleophilic base. In embodiments, a non-nucleophilic bases is employed. Nucleophilic bases may attack the electrophilic phosphorus or react with active intermediates in the reaction mixture and form side- products. In embodiments, the base used in the coupling reaction is a mild base. Stronger bases may cause side reactions such as base-catalyzed hydrolysis, degradation of sensitive functional groups, or racemization. In embodiments, the base used in the coupling reaction is a mild and non- nucleophilic base. In embodiments, the base used in the coupling reaction is not a mild and non- nucleophilic base. In embodiments, the base used in the coupling reaction is a mild or non- nucleophilic base. In embodiments, the base used in the coupling reaction is not be a mild or non- nucleophilic base.

[0193] In embodiments, the bases used in the coupling reaction are one or more of N-ethyl morpholine (NEM), diisopropylethylamine (DIPEA), triethylamine (TEA), N-methylmorpholine (NMM), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), pyridine, or imidazole. In embodiments, the base used in the coupling reaction is N-ethyl morpholine (NEM).

[0194] In embodiments, the bases used in the coupling reaction are in a concentration from 0.01 M to 5 M. In embodiments, the bases used in the coupling reaction are in a concentration from 0.01 M to 5 M, from 0.02 M to 4.5 M, from 0.03 M to 4 M, from 0.04 M to 3.5 M, from 0.05 M to 3 M, from 0.1 M to 2.5 M, from 0.1 M to 2 M, from 0.2 M to 1.5 M, from 0.3 M to 1 M, from 0.4 M to 0.5 M, or from 0.1 M to 0.2 M. In embodiments, the bases used in the coupling reaction are in a concentration of at least 0.01 M, at least 0.02 M, at least 0.03 M, at least 0.04 M, at least 0.05 M, at least 0.1 M, at least 0.1 M, at least 0.2 M, at least 0.3 M, at least 0.4 M, at least 0.5 M, at least 0.6 M, at least 0.7 M, at least 0.8 M, at least 0.9 M, at least 1 M, at least 1.5 M, at least 2 M, at least 2.5 M, at least 3 M, at least 3.5 M, at least 4 M, or at least 4.5 M. In embodiments, the bases used in the coupling reaction are in a concentration of up to 5 M, up to 4.5 M, up to 4 M, up to 3.5 M, up to 3 M, up to 2.5 M, up to 2 M, up to 1.5 M, up to 1 M, up to 0.9 M, up to 0.8 M, up to 0.7 M, up to 0.6 M, up to 0.5 M, up to 0.4 M, up to 0.3 M, up to 0.2 M, up to 0.1 M, up to 0.0.05 M, up to 0.04 M, up to 0.03 M, or up to 0.02 M.

[0195] In embodiments, one or more of the steps of the methods of PMO synthesis disclosed herein are carried out at temperatures higher or lower than room temperature (25 °C). The temperature that the reactants are exposed to may impact the efficiency of the reaction and fidelity and / or stability of the reaction product. In embodiments, the coupling reaction mixture is exposedto, or may be at, temperatures of at least -80 °C and up to 150 °C, at least -50 °C and up to 120 °C, at least -10 °C and up to 100 °C, at least 0 °C and up to 90 °C, at least 10 °C and up to 75 °C, at least 25 °C and up to 75 °C, or at least 50 °C and up to 60 °C. In embodiments, the coupling reaction mixture is exposed to, or may be at, temperatures of at least -80 °C, at least -50 °C, at least -10 °C, at least 0 °C, at least 10 °C, at least 25 °C, at least 35 °C, at least 50 °C, at least 60 °C, at least 75 °C, at least 90 °C, at least 100 °C, at least 120 °C, or at least 140 °C. In embodiments, the coupling reaction mixture is exposed to, or may be at, temperatures of up to 150 °C, up to 120 °C, up to 100 °C, up to 90 °C, up to 75 °C, up to 75 °C, up to 60 °C, up to 50 °C, up to 25 °C, up to 10 °C, up to 0 °C, up to -10 °C, up to -50 °C, or up to -80 °C.

[0196] The reaction time for the deprotection reaction may vary depending upon the compound to be deprotected, reaction temperature, etc. In embodiments, the reaction time for the deprotection reaction may be at least 10 minutes and up to 30 hours, at least 30 minutes and up to 24 hours, at least 1 hour and up to 20 hours, at least 2 hours and up to 15 hours, at least 3 hours and up to 5 hours, at least 3 hours and up to 10 hours, or at least 3 hours and up to 20 hours. In embodiments, the reaction time for the deprotection reaction may be in a range of 10 minutes to 30 hours, 30 minutes to 24 hours, 1 hour to 20 hours, 2 hours to 15 hours, 3 hours to 5 hours, or 4 hours.

[0197] The inclusion of various additives can improve the reaction efficiency in the synthesis of PMOs. In embodiments, an additive is added to the coupling reaction. In embodiments, the additives include inorganic salts. In embodiments, the inorganic salt additive is lithium bromide.

[0198] In embodiments, the additive is included in the coupling reaction in an amount from 1 equivalent to 25 equivalents with respect to the solid-supported multimer. In embodiments, the additive is included in the coupling reaction in an amount from 1 to 25 equivalents, from 2 to 25 equivalents, from 3 to 20 equivalents, from 4 to 15 equivalents, from 4 to 12 equivalents, from 4 to 10 equivalents, from 4 to 6 equivalents, from 5 to 10 equivalents, from 6 to 8 equivalents, or 5 equivalents with respect to the solid-supported multimer. In embodiments, the additive is included in the coupling reaction in an amount of at least 1 equivalent, at least 2 equivalents, at least 3 equivalents, at least 4 equivalents, at least 5 equivalents, at least 6 equivalents, at least 7 equivalents, at least 8 equivalents, at least 9 equivalents, at least 10 equivalents, at least 12 equivalents, at least 15 equivalents, at least 20 equivalents, or at least 25 equivalents with respect to the solid-supported multimer. In embodiments, the additive is included in the coupling reaction in an amount of up to 25 equivalents, up to 20 equivalents, up to 15 equivalents, up to 12equivalents, up to 10 equivalents, up to 9 equivalents, up to 8 equivalents, up to 7 equivalents, up to 6 equivalents, up to 5 equivalents, up to 4 equivalents, up to 3 equivalents, or up to 2 equivalents with respect to the solid-supported multimer. In embodiments, the additive is included in the coupling reaction in an amount of up to 4 equivalents with respect to the solid-supported multimer. In embodiments, the additive is included in the coupling reaction in an amount of up to 5 equivalents with respect to the solid-supported multimer. In embodiments, the additive is included in the coupling reaction in an amount of up to 6 equivalents with respect to the solid-supported multimer.

[0199] In embodiments, the coupling reaction employs any suitable solvent known to one with skill in the art. In embodiments, the coupling reaction employs any suitable polar aprotic solvent. In embodiments, the solvent is dimethylformamide (DMF), acetonitrile (MeCN), N-methyl-2- pyrrolidinone (NMP), dimethylimidazolidinone (DMI), tetrahydrofuran (THF), or 1,4-dioxane, or the like. In embodiments, the coupling reaction is performed in DMI as solvent. While it is understood the volume of the solvent used would depend on the target concentration of the reactants in the reaction mixture, in embodiments, the coupling reaction employs the solvent in at least volume equivalent and up to 50 volume equivalents relative to the weight of the substrate. In embodiments, the coupling reaction employs the solvent in at least 1 volume equivalent and up to 50 volume equivalents, at least 10 volume equivalent and up to 40 volume equivalents, or at least 20 volume equivalent and up to 30 volume equivalents relative to the weight of the substrate. In embodiments, the coupling reaction employs the solvent in at least 1 volume equivalent, at least 5 volume equivalents, at least 10 volume equivalents, at least 15 volume equivalents, at least 20 volume equivalents, at least 25 volume equivalents, at least 30 volume equivalents, at least 35 volume equivalents, at least 40 volume equivalents, or at least 45 volume equivalents relative to the weight of the substrate. In embodiments, the coupling reaction employs the solvent in up to 50 volume equivalents, up to 45 volume equivalents, up to 40 volume equivalents, up to 35 volume equivalents, up to 30 volume equivalents, up to 25 volume equivalents, up to 20 volume equivalents, up to 15 volume equivalents, up to 10 volume equivalents, or up to 5 volume equivalents relative to the weight of the substrate. In embodiments, the coupling reaction employs the solvent in at least 15 volume equivalents and up to 25 volume equivalents, or at least 18 volume equivalents and up to 22 volume equivalents, relative to the weight of the substrate. Inembodiments, the coupling reaction employs the solvent in 20 volume equivalents relative to the weight of the substrate. Functionalization (Phosphorylation)

[0200] Prior to coupling with a solid-supported multimer, the non-solid-supported multimer may be functionalized at the 6’-position. In embodiments, the 6’-OH group of the non-solid-supported multimer may be phosphorylated. One example of phosphoryl functionalization is shown in the scheme below:, wherein: B is a nucleobase; LG is a leaving group; R1and R2are each independently H or substituted or unsubstituted alkyl, aryl, alkynyl, alkenyl, cycloalkyl, heterocyclyl, or heteroaryl; R4is a hydrogen or a protecting group; Mnis a PMO monomer; (Mn)mis a PMO multimer having m PMO nucleotides; n is the position of the Mnmonomer in the (Mn)m multimer sequence; and m is the number of Mnmonomers in the (Mn)m multimer.

[0201] In embodiments, m can be an integer from 2 to 20, such as 2 to 11, or 2 to 4. In embodiments, m is 2. In embodiments, m is 3. In embodiments, m is 4.

[0202] In embodiments, the phosphorylating agents include phosphorus oxychloride (POCl3), phosphorus pentachloride (PCl5), bis-(2,2,2-trichloroethyl) phosphorochloridate, dialkyl phosphoramidites, pyrophosphoryl chloride (P2O5ClO4), or dimethylaminophosphoryl dichloride. Additional phosphorylating agents can be found in Wei, Tetrahedron 69 (2013) 3615e363.

[0203] In embodiments, the phosphorylating agent in the functionalization reaction is included in an amount from 0.1 equivalents to 10 equivalents with respect to the 6’-OH- non-solid-supported multimer. In embodiments, the phosphorylating agent in the functionalization reaction is included in an amount from 0.1 equivalents to 10 equivalents, from 0.2 equivalents to 8 equivalents, from 0.3 equivalents to 7 equivalents, from 0.4 equivalents to 6 equivalents, from 0.5 equivalents to 5 equivalents, from 0.6 equivalents to 4 equivalents, from 0.7 equivalents to 3 equivalents, from 0.8 equivalents to 2.2 equivalents, from 0.9 equivalents to 2 equivalents, from 1 equivalent to 1.5 equivalents, or from 1.1 equivalents to 2.2 equivalents with respect to the 6’-OH-non-solid- supported multimer. In embodiments, the phosphorylating agent in the functionalization reaction is included in an amount of at least 0.1 equivalents, at least 0.2 equivalents, at least 0.3 equivalents, at least 0.4 equivalents, at least 0.5 equivalents, at least 0.6 equivalents, at least 0.7 equivalents, at least 0.8 equivalents, at least 0.9 equivalents, at least 1 equivalent, at least 1.1 equivalents, at least 1.2 equivalents, at least 1.3 equivalents, at least 1.4 equivalents, at least 1.5 equivalents, at least 2 equivalents, at least 2.1 equivalents, at least 2.2 equivalents, at least 2.3 equivalents, at least 2.4 equivalents, at least 2.5 equivalents, at least 3 equivalents, at least 3.5 equivalents, at least 4 equivalents, at least 4.5 equivalents, at least 5 equivalents, at least 6 equivalents, at least 7 equivalents, at least 8 equivalents, or at least 9 equivalents with respect to the 6’-OH-non-solid- supported multimer. In embodiments, the phosphorylating agent in the functionalization reaction is included in an amount of up to 10 equivalents, up to 9 equivalents, up to 8 equivalents, up to 7 equivalents, up to 6 equivalents, up to 5 equivalents, up to 4.5 equivalents, up to 4 equivalents, up to 3.5 equivalents, up to 3 equivalents, up to 2.5 equivalents, up to 2 equivalents, up to 1.5 equivalents, up to 1 equivalent, up to 0.9 equivalents, up to 0.8 equivalents, up to 0.7 equivalents, up to 0.6 equivalents, up to 0.5 equivalents, up to 0.4 equivalents, up to 0.3 equivalents, or up to 0.2 equivalents with respect to the 6’-OH-non-solid-supported multimer.

[0204] In embodiments, the 6’-OH group of the non-solid-supported multimer is aminated following the phosphorylation step. In embodiments, the aminating agents may include, for example, primary amines (such as, methylamine, ethylamine, n-butylamine, or benzylamine),secondary amines (such as, dimethylamine, diethylamine, dipropylamine, piperidine, or morpholine), tertiary amines (such as, triethylamine, tributylamine), cyclic amines (such as, pyrrolidine, piperazine, or imidazole), aromatic amines (such as, aniline or substituted anilines such as para-toluidine, or 2,4-dimethylaniline). In embodiments, the aminating agent in the functionalization reaction is dimethylamine.

[0205] In embodiments, the aminating agent in the functionalization reaction is included in an amount from 0.1 equivalents to 10 equivalents with respect to the 6’-OH- non-solid-supported multimer. In embodiments, the aminating agent in the functionalization reaction may be included in an amount from 0.1 equivalents to 10 equivalents, from 0.2 equivalents to 8 equivalents, from 0.3 equivalents to 7 equivalents, from 0.4 equivalents to 6 equivalents, from 0.5 equivalents to 5 equivalents, from 0.6 equivalents to 4 equivalents, from 0.7 equivalents to 3 equivalents, from 0.8 equivalents to 2.2 equivalents, from 0.9 equivalents to 2 equivalents, from 1 equivalent to 1.5 equivalents, or from 1.1 equivalents to 2.2 equivalents with respect to the 6’-OH-non-solid- supported multimer. In embodiments, the aminating agent in the functionalization reaction is included in an amount of at least 0.1 equivalents, at least 0.2 equivalents, at least 0.3 equivalents, at least 0.4 equivalents, at least 0.5 equivalents, at least 0.6 equivalents, at least 0.7 equivalents, at least 0.8 equivalents, at least 0.9 equivalents, at least 1 equivalent, at least 1.1 equivalents, at least 1.2 equivalents, at least 1.3 equivalents, at least 1.4 equivalents, at least 1.5 equivalents, at least 2 equivalents, at least 2.1 equivalents, at least 2.2 equivalents, at least 2.3 equivalents, at least 2.4 equivalents, at least 2.5 equivalents, at least 3 equivalents, at least 3.5 equivalents, at least 4 equivalents, at least 4.5 equivalents, at least 5 equivalents, at least 6 equivalents, at least 7 equivalents, at least 8 equivalents, or at least 9 equivalents with respect to the 6’-OH-non-solid- supported multimer. In embodiments, the aminating agent in the functionalization reaction is included in an amount of up to 10 equivalents, up to 9 equivalents, up to 8 equivalents, up to 7 equivalents, up to 6 equivalents, up to 5 equivalents, up to 4.5 equivalents, up to 4 equivalents, up to 3.5 equivalents, up to 3 equivalents, up to 2.5 equivalents, up to 2 equivalents, up to 1.5 equivalents, up to 1 equivalent, up to 0.9 equivalents, up to 0.8 equivalents, up to 0.7 equivalents, up to 0.6 equivalents, up to 0.5 equivalents, up to 0.4 equivalents, up to 0.3 equivalents, or up to 0.2 equivalents with respect to the 6’-OH-non-solid-supported multimer. In embodiments, the aminating agent in the functionalization reaction is included in an amount equimolar to the phosphorylating agent.

[0206] The reaction for the functionalization of the non-solid-supported multimer can include the use of a strong non-nucleophilic base as a ‘proton sponge.’ The strongly basic nature of the proton sponge may help deprotonate the hydroxyl group of the 6’-hydroxy multimer. In embodiments, the proton sponge in the functionalization of the non-solid-supported multimer is 1,8- bis(dimethylamino)naphthalene. Additional reagents can include other organic bases such as 1,1,3,3-tetramethylguanidine, 1,8-diazabicyclo(5.4.0)undec-7-ene (DBU). In embodiments, the additional reagents can include aromatic amines such as dialkylaminobenzene or 1,8- Bis(dimethylamino)naphthalene. In embodiments, the additional reagents can include heteroaromatic amines and N-alkyl heteroaromatic amines such as imidazoles, N-alkyl benzimidazoles, alkylpyridines. In embodiments, the additional reagents can include tertiary amines such as1,2,2,6,6-pentamethylpiperidine, N,N-Diisopropylethylamine (DIPEA), DABCO. In embodiments, the additional reagents can include phosphazenes such as 2-tert-butylimino-2- diethylamino-1,3-dimethylperhydro-1,3,2-diazaphosphorine (BEMP) or t-Bu-P4.

[0207] In embodiments, the proton sponge in the functionalization reaction is included in an amount from 0.1 equivalents to 5 equivalents with respect to the 6’-OH- non-solid-supported multimer. In embodiments, the proton sponge in the functionalization reaction is included in an amount from 0.1 equivalents to 5 equivalents, from 0.2 equivalents to 4 equivalents, from 0.3 equivalents to 3 equivalents, from 0.4 equivalents to 2 equivalents, from 0.5 equivalents to 1 equivalent, from 0.6 equivalents to 0.9 equivalents, or from 0.7 equivalents to 0.8 equivalents with respect to the 6’-OH-non-solid-supported multimer. In embodiments, the proton sponge in the functionalization reaction is included in an amount of at least 0.1 equivalents, at least 0.2 equivalents, at least 0.3 equivalents, at least 0.4 equivalents, at least 0.5 equivalents, at least 0.6 equivalents, at least 0.7 equivalents, at least 0.8 equivalents, at least 0.9 equivalents, at least 1 equivalent, at least 1.1 equivalents, at least 1.2 equivalents, at least 1.3 equivalents, at least 1.4 equivalents, at least 1.5 equivalents, at least 2 equivalents, at least 2.1 equivalents, at least 2.2 equivalents, at least 2.3 equivalents, at least 2.4 equivalents, at least 2.5 equivalents, at least 3 equivalents, at least 3.5 equivalents, at least 4 equivalents, at least 4.5 equivalents with respect to the 6’-OH-non-solid-supported multimer. In embodiments, the proton sponge in the functionalization reaction is included in an amount of up to 5 equivalents, up to 4.5 equivalents, up to 4 equivalents, up to 3.5 equivalents, up to 3 equivalents, up to 2.5 equivalents, up to 2 equivalents, up to 1.5 equivalents, up to 1 equivalent, up to 0.9 equivalents, up to 0.8 equivalents,up to 0.7 equivalents, up to 0.6 equivalents, up to 0.5 equivalents, up to 0.4 equivalents, up to 0.3 equivalents, or up to 0.2 equivalents with respect to the 6’-OH-non-solid-supported multimer.

[0208] In embodiments, the reaction for the functionalization of the non-solid-supported multimer includes the use of additional bases. Additional bases can help neutralize any acidic byproducts of the reaction and buffer the reaction. In embodiments, the additional base in the functionalization of the non-solid-supported multimer is 2,6-lutidine.

[0209] In embodiments, the additional base in the functionalization reaction is included in an amount from 0.5 equivalents to 20 equivalents with respect to the 6’-OH- non-solid-supported multimer. In embodiments, the additional base in the functionalization reaction is included in an amount from 0.5 equivalents to 20 equivalents, from 0.8 equivalents to 15 equivalents, from 1 equivalent to 10 equivalents, from 1.5 equivalents to 8 equivalents, from 2 equivalents to 7 equivalents, from 2.4 equivalents to 6 equivalents, from 2.5 equivalents to 5 equivalents, or from 3 equivalents to 4 equivalents with respect to the 6’-OH-non-solid-supported multimer. In embodiments, the additional base in the functionalization reaction is included in an amount of at least 0.5 equivalents, at least 0.6 equivalents, at least 0.7 equivalents, at least 0.8 equivalents, at least 0.9 equivalents, at least 1 equivalent, at least 1.1 equivalents, at least 1.2 equivalents, at least 1.3 equivalents, at least 1.4 equivalents, at least 1.5 equivalents, at least 2 equivalents, at least 2.1 equivalents, at least 2.2 equivalents, at least 2.3 equivalents, at least 2.4 equivalents, at least 2.5 equivalents, at least 3 equivalents, at least 3.5 equivalents, at least 4 equivalents, at least 4.5 equivalents, at least 5 equivalents, at least 5.5 equivalents, at least 6 equivalents, at least 6.5 equivalents, at least 7 equivalents, at least 10 equivalents, or at least 15 equivalents with respect to the 6’-OH-non-solid-supported multimer. In embodiments, the additional base in the functionalization reaction is included in an amount of up to 20 equivalents, up to 15 equivalents, up to 10 equivalents, up to 6 equivalents, up to 5 equivalents, up to 4.5 equivalents, up to 4 equivalents, up to 3.5 equivalents, up to 3 equivalents, up to 2.5 equivalents, up to 2 equivalents, up to 1.5 equivalents, up to 1 equivalent, up to 0.9 equivalents, up to 0.8 equivalents, up to 0.7 equivalents, or up to 0.6 equivalents with respect to the 6’-OH-non-solid-supported multimer.

[0210] The reaction time for the functionalization reaction may be in the range of 30 minutes to 20 hours. The reaction time for the functionalization reaction can be at least 30 minutes and up to 20 hours, at least 45 minutes and up to 12 hours, at least 1 hour and up to 10 hours, at least 2 hours and up to 8 hours, at least 2 hours and up to 5 hours, or at least 2 hours and up to 4 hours. Inembodiments, the reaction time for the functionalization reaction is at least 30 minutes, at least 45 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 10 hours, or at least 15 hours. In embodiments, the reaction time for the functionalization reaction is up to 20 hours, up to 15 hours, up to 12 hours, up to 10 hours, up to 7 hours, up to 6 hours, up to 5 hours, up to 4 hours, up to 3 hours, up to 2 hours, up to 1 hour, up to 45 minutes, or up to 30 minutes.

[0211] In embodiments, the 6’-OH group of the non-solid-supported multimer may be phosphorylated as shown in the scheme below:, wherein: B is a nucleobase; R4is a hydrogen or a protecting group; Mnis a PMO monomer; (Mn)mis a PMO multimer having m PMO nucleotides; n is the position of the Mnmonomer in the (Mn)m multimer sequence; and m is the number of Mnmonomers in the (Mn)m multimer.

[0212] In embodiments, m can be an integer from 2 to 20, such as 2 to 11, or 2 to 4. In embodiments, m is 2. In embodiments, m is 3. In embodiments, m is 4.

[0213] In embodiments, B can be any nucleotide base (i.e., “nucleobase”) including A, G, C, T, or U. In embodiments, B can be A. In embodiments, B can be G. In embodiments, B can be C. In embodiments, B can be T. In embodiments, B can be U.

[0214] In embodiments, the phosphorus oxychloride (POCl3) is included in an amount from 1 equivalents to 2.5 equivalents with respect to the 6’-OH- non-solid-supported multimer.

[0215] In embodiments, the 6’-OH group of the non-solid-supported multimer is aminated with dimethylamine following the phosphorylation step. In embodiments, dimethylamine is included in an amount from 1 equivalents to 2.5 equivalents with respect to the 6’-OH- non-solid-supported multimer.

[0216] In embodiments, the reaction for the functionalization of the non-solid-supported multimer includes 1,8-bis(dimethylamino)naphthalene as a ‘proton sponge.’ In embodiments, ,8- bis(dimethylamino)naphthalene is included in an amount from 0.5 equivalents to 1.5 equivalents with respect to the 6’-OH- non-solid-supported multimer.

[0217] In embodiments, the reaction for the functionalization of the non-solid-supported multimer includes 2,6-lutidine. In embodiments, the 2,6-lutidine is included in an amount from 2 equivalents to 7 equivalents with respect to the 6’-OH- non-solid-supported multimer.

[0218] FIG.10A shows one pathway for functionalizing a CAG PMO trimer according to Strategy 1. It will be appreciated that the reaction scheme may be employed with PMO multimers having sequences other than CAG. The functionalization results in a 6’-N-dimethyl phosphoramidochloridate CAG PMO trimer. It will be appreciated that this is one example of a functionalized CAG PMO trimer that may be employed in accordance with the methods of Strategy 1 described herein.

[0219] FIG.10B shows one pathway for functionalizing a CTG PMO trimer according to Strategy 1. It will be appreciated that the reaction scheme may be employed with PMO multimers having sequences other than CTG. The functionalization results in a 6’-N-dimethyl phosphoramidochloridate CTG PMO trimer. It will be appreciated that this is one example of a functionalized CTG PMO trimer that may be employed in accordance with the methods of Strategy 1 described herein.

[0220] FIG.10D shows examples of reaction conditions that can be used to functionalize the CAG PMO trimer according to Strategy 1. As shown, a 6’-OH, 3’ protected CAG PMO is reacted with POCl3 in dimethylamine, dichloromethane (DCM) at 0 to 10 C for four hours to produce a 6’-N- dimethyl phosphoramidochloridate CAG PMO trimer. It will be appreciated that reaction temperatures and times shown in FIG.10D may be varied.

[0221] FIG.10E shows examples of reaction conditions that can be used to functionalize the CTG PMO trimer according to Strategy 1. As shown, a 6’-OH, 3’ protected CTG PMO is reacted with POCl3in dimethylamine, dichloromethane (DCM) at 0 to 10 C for four hours to produce a 6’-N- dimethyl phosphoramidochloridate CTG PMO trimer. It will be appreciated that reaction temperatures and times shown in FIG.10E may be varied.

[0222] It will be appreciated that functionalization methods described above are merely examples of methods for functionalizing a PMO multimer in accordance with Strategy 1 and that other suitable processes may be employed. (II) Strategy 2: H-Phosphonate Coupling

[0223] As indicated above, for example, with reference to FIG.8B, methods for the preparation of PMOs described herein can include an H-phosphonate substrate. Coupling of an N-deprotected morpholino with the H-phosphonate substrate provides an H-phosphonamidate product which may be further functionalized by oxidative amination to furnish the phosphorodiamidate morpholino oligomer PMO product. Coupling and functionalization may be repeated a desired number of times to obtain the multimer product with a specific number of repeats.

[0224] The oxidative amination reaction of H-phosphonamidate is thought to consist of a tautomerization to a tricoordinated phosphoramidite intermediate, an oxidative halogenation reaction with CCl4, and a subsequent amination of the resultant intermediate.

[0225] In embodiments, the H-phosphonate precursor for coupling with the solid-supported N- deprotected multimer is prepared by the phosphorylation of 6’-OH morpholino nucleoside generally as shown below:, wherein:B is a nucleobase; R4is a hydrogen or a protecting group; Mnis a PMO monomer; (Mn)m is a PMO multimer having m PMO nucleotides; n is the position of the Mnmonomer in the (Mn)m multimer sequence; and m is the number of Mnmonomers in the (Mn)mmultimer.

[0226] In embodiments, m can be an integer from 2 to 20, such as 2 to 11, or 2 to 4. In embodiments, m is 2. In embodiments, m is 3. In embodiments, m is 4.

[0227] In embodiments, B can be any nucleotide base (i.e., “nucleobase”) including A, G, C, T, or U. In embodiments, B can be A. In embodiments, B can be G. In embodiments, B can be C. In embodiments, B can be T. In embodiments, B can be U.

[0228] Base abstraction of the 6’-hydroxyl proton activates the molecule for a nucleophilic attack on the electrophilic phosphorus of the phosphorylating agent. The phosphorylation reaction can be performed with any phosphorylating reagent known in the art, including, for example, diphenylphosphite, phosphorus oxychloride, phosphorus trichloride, dichlorophosphite, diethylphosphite, diisopropylphosphite, and the like. The reaction can be performed in the presence of a weak base such as pyridine. Additional phosphorylating agents can be found in Wei, Tetrahedron 69 (2013) 3615e363. Suitable solvents can include those that are aprotic and dipolar, including but not to be limited to, dichloromethane, acetonitrile, THF, chloroform. Toluene. Phosphorylation can be followed by hydrolysis of the intermediate to furnish the H-phosphonate substrate, generally as shown below:each B is independently a nucleobase;R1and R2are each independently H or substituted or unsubstituted alkyl, aryl, alkynyl, alkenyl, cycloalkyl, heterocyclyl, or heteroaryl; R4is a hydrogen or a protecting group; the circle represents a solid support; Mnis a PMO monomer; (Mn)mis a PMO multimer having m PMO nucleotides; n is the position of the Mnmonomer in the (Mn)mmultimer sequence; and m is the number of Mnmonomers in the (Mn)m multimer.

[0229] In embodiments, m can be an integer from 2 to 20, such as 2 to 11, or 2 to 4. In embodiments, m is 2. In embodiments, m is 3. In embodiments, m is 4.

[0230] The 6’-H-phosphonate substrate can be coupled with solid supported N-deprotected morpholino substrate to furnish the H-phosphonamidate product under the coupling conditions known in the art and / or described herein. In embodiments, the 6’-H-phosphonate substrate is present in excess of the N-deprotected morpholino substrate in the coupling reaction. In embodiments, the 6’-H-phosphonate substrate is present in an amount of at least 1 equivalent and up to 10 equivalents with respect to the N-deprotected morpholino substrate in the coupling reaction. In embodiments, the 6’-H-phosphonate substrate is present in an amount of at least 1 equivalent and up to 10 equivalents, at least 3 equivalents and up to 7 equivalents, or at least 5 equivalents with respect to the N-deprotected morpholino substrate in the coupling reaction. In embodiments, the 6’-H-phosphonate substrate is present in an amount of at least 1 equivalent, at least 2 equivalents, at least 3 equivalents, at least 4 equivalents, at least 5 equivalents, at least 6 equivalents, at least 7 equivalents, at least 8 equivalents, or at least 9 equivalents with respect to the N-deprotected morpholino substrate in the coupling reaction. In embodiments, the 6’-H- phosphonate substrate is present in an amount of up to 10 equivalents, up to 9 equivalents, up to 8 equivalents, up to 7 equivalents, up to 6 equivalents, up to 5 equivalents, up to 4 equivalents, up to 3 equivalents, or up to 2 equivalents with respect to the N-deprotected morpholino substrate in the coupling reaction. In embodiments, the 6’-H-phosphonate substrate is present in an amount of up to 5 equivalents with respect to the N-deprotected morpholino substrate in the coupling reaction.

[0231] The coupling reaction can be assisted by a suitable coupling agent. In embodiments, the coupling agent is benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBop), bis-(2-oxo-3-oxazolidinyl)phosphinic chloride (BOPCl), tetramethyluroniumderivatives (e.g., HATU, HBTU), dicyclohexylcarbodiimide (DCC), N,N'- diisopropylcarbodiimide (DIC), 1,1'-carbonyldiimidazole (CDI), 1-Ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC), 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene (MTBD), or the like. In embodiments, the coupling agent is present in an amount of at least 1 equivalent and up to 20 equivalents with respect to the N-deprotected morpholino substrate in the coupling reaction. embodiments, the coupling agent is present in an amount of at least 1 equivalent and up to 20 equivalents, at least 2 equivalents and up to 18 equivalents, at least 5 equivalents and up to 15 equivalents, at least 8 equivalents and up to 12 equivalents, at least 9 equivalents and up to 11 equivalents, or 10 equivalents with respect to the N-deprotected morpholino substrate in the coupling reaction. In embodiments, the coupling agent is present in an amount of at least 1 equivalent, at least 2 equivalents, at least 5 equivalents, at least 8 equivalents, at least 10 equivalents, at least 12 equivalents, at least 15 equivalents, or at least 18 equivalents with respect to the N-deprotected morpholino substrate in the coupling reaction. In embodiments, the coupling agent is present in an amount of up to 20 equivalents, up to 18 equivalents, up to 15 equivalents, up to 12 equivalents, up to 10 equivalents, up to 8 equivalents, up to 5 equivalents, or up to 2 equivalents with respect to the N-deprotected morpholino substrate in the coupling reaction.

[0232] The coupling reaction can be carried out in a suitable solvent or mixture of solvents. For example, the coupling reaction may be carried out in a polar aprotic solvent, or a basic solvent, or a mixture of thereof. While it is understood the volume of the solvent used would depend on the target concentration of the reactants in the reaction mixture, in embodiments, the coupling reaction may employ the solvent in at least 1 volume equivalent and up to 50 volume equivalents with respect to the N-deprotected morpholino substrate in the coupling reaction. In embodiments, the coupling reaction employs the solvent in at least 1 volume equivalent and up to 50 volume equivalents, at least 10 volume equivalents and up to 40 volume equivalents, or at least 20 volume equivalents and up to 30 volume equivalents with respect to the N-deprotected morpholino substrate in the coupling reaction. In embodiments, the coupling reaction employs the solvent in at least 1 volume equivalent, at least 5 volume equivalents, at least 10 volume equivalents, at least 15 volume equivalents, at least 20 volume equivalents, at least 25 volume equivalents, at least 30 volume equivalents, at least 35 volume equivalents, at least 40 volume equivalents, or at least 45 volume equivalents with respect to the N-deprotected morpholino substrate in the coupling reaction. In embodiments, the coupling reaction employs the solvent in up to 50 volumeequivalents, up to 45 volume equivalents, up to 40 volume equivalents, up to 35 volume equivalents, up to 30 volume equivalents, up to 25 volume equivalents, up to 20 volume equivalents, up to 15 volume equivalents, up to 10 volume equivalents, or up to 5 volume with respect to the N-deprotected morpholino substrate in the coupling reaction. In embodiments, the coupling reaction employs the solvent in 20 volume equivalents with respect to the N-deprotected morpholino substrate in the coupling reaction.

[0233] Coupling of N-deprotected morpholino substrate with the H-phosphonate substrate provides an H-phosphonamidate product which may be further functionalized by oxidative amination to furnish the phosphorodiamidate morpholino oligomer product. Suitable reagents for oxidative amination include a halogenating reagent and an aminating reagent known to one skilled in the art. In embodiments, the halogenating agent in the oxidative amination of the H- phosphonamidate to furnish a phosphorodiamidate includes, for example, CCl4, CBr4, CBrCl3, I2, or the like. In embodiments, the halogenating agent in the oxidative amination of the H- phosphonamidate to furnish a phosphorodiamidate is CBrCl3.

[0234] In embodiments, the halogenating agent in the oxidative amination of the H- phosphonamidate to furnish a phosphorodiamidate is at least 1 equivalent and up to 50 equivalents with respect to the H-phosphonamidate substrate in the reaction. In embodiments, the halogenating agent in the oxidative amination of the H-phosphonamidate to furnish a phosphorodiamidate is at least 1 equivalent and up to 50 equivalents, at least 10 equivalents and up to 40 equivalents, or at least 20 equivalents and up to 30 equivalents with respect to the H-phosphonamidate substrate in the reaction. In embodiments, the halogenating agent in the oxidative amination of the H- phosphonamidate to furnish a phosphorodiamidate is at least 1 equivalent, at least 5 equivalents, at least 10 equivalents, at least 15 equivalents, at least 20 equivalents, at least 25 equivalents, at least 30 equivalents, at least 35 equivalents, at least 40 equivalents, or at least 45 equivalents with respect to the H-phosphonamidate substrate in the reaction. In embodiments, the halogenating agent in the oxidative amination of the H-phosphonamidate to furnish a phosphorodiamidate is up to 50 equivalents, up to 45 equivalents, up to 40 equivalents, up to 35 equivalents, up to 30 equivalents, up to 25 equivalents, up to 20 equivalents, up to 15 equivalents, up to 10 equivalents, or up to 5 with respect to the H-phosphonamidate substrate in the reaction. In embodiments, the halogenating agent in the oxidative amination of the H-phosphonamidate to furnish aphosphorodiamidate is 20 equivalents with respect to the H-phosphonamidate substrate in the reaction.

[0235] In embodiments, the aminating agent in the oxidative amination of the H-phosphonamidate to furnish a phosphorodiamidate includes, for example, primary amines (such as, methylamine, ethylamine, n-butylamine, or benzylamine), secondary amines (such as, dimethylamine, diethylamine, dipropylamine, piperidine, or morpholine), tertiary amines (such as, triethylamine, tributylamine), cyclic amines (such as, pyrrolidine, piperazine, or imidazole), aromatic amines (such as, aniline or substituted anilines such as para-toluidine, or 2,4-dimethylaniline). In embodiments, the aminating agent in the oxidative amination of the H-phosphonamidate to furnish a phosphorodiamidate is dimethylamine.

[0236] In embodiments, the aminating agent in the oxidative amination of the H-phosphonamidate to furnish a phosphorodiamidate is at least 10 equivalents and up to 80 equivalents with respect to the H-phosphonamidate substrate in the reaction. In embodiments, the aminating agent in the oxidative amination of the H-phosphonamidate to furnish a phosphorodiamidate is at least 10 equivalents and up to 80 equivalents, at least 20 equivalents and up to 60 equivalents, or at least 30 equivalents and up to 50 equivalents with respect to the H-phosphonamidate substrate in the reaction. In embodiments, the aminating agent in the oxidative amination of the H- phosphonamidate to furnish a phosphorodiamidate is at least 10 equivalents, at least 20 equivalents, at least 30 equivalents, at least 40 equivalents, at least 50 equivalents, at least 60 equivalents, or at least 70 equivalents with respect to the H-phosphonamidate substrate in the reaction. In embodiments, the aminating agent in the oxidative amination of the H- phosphonamidate to furnish a phosphorodiamidate is up to 80 equivalents, up to 70 equivalents, up to 60 equivalents, up to 50 equivalents, up to 40 equivalents, up to 30 equivalents, up to 20 equivalents, or up to 15 equivalents with respect to the H-phosphonamidate substrate in the reaction. In embodiments, the aminating agent in the oxidative amination of the H- phosphonamidate to furnish a phosphorodiamidate is 40 equivalents with respect to the H- phosphonamidate substrate in the reaction.

[0237] FIG.10C shows one pathway for functionalizing a CAG PMO trimer according to Strategy 2. It will be appreciated that the reaction scheme may be employed with PMO multimers having sequences other than CAG. In the pathway shown, the functionalization results in a 5’-H- phosphonate CAG PMO trimer. It will be appreciated that this is one example of a functionalizedCAG PMO trimer that may be employed in accordance with the methods of Strategy 2 described herein.

[0238] FIG.10F shows one pathway for functionalizing a CAG PMO trimer according to Strategy 2. As shown, a 6’-OH, 3’ protected CAG PMO is reacted with PCl3 and imidazole in triethylamine (TEA) or 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), dichloromethane (DCM) at -78 C for 30 minutes to produce a 5’-H-phosphonate CAG PMO trimer. It will be appreciated that reaction temperatures and times shown in FIG.10E may be varied.

[0239] It will be appreciated that functionalization methods described above are merely examples of methods for functionalizing a PMO multimer in accordance with Strategy 2 and that other suitable processes may be employed.

[0240] One example of a Strategy 2 reaction scheme used to generate a solid-supported PMO having a (CAG)7 repeat sequence is shown in FIG. 12A. It will be understood that the reaction scheme shown in FIG.12A may be used with PMO multimers having a sequence other than CAG. As shown, a solid supported 6’-OH, 3’ protected CAG PMO trimer is reacted with a 6’-H- phosphonate CAG PMO trimer (FIG.12B) for six cycles to produce the solid-supported 21-mer. Each cycle includes deprotecting (detritylating) the 3’ end of the PMO coupled to the solid support, neutralizing, coupling in the presence of PyNTP in acetonitrile (ACN) / pyridine (Py) at 0 C for 20 minutes, and oxidative amination. Oxidative amination includes reacting with CCl4 in dimethylamine in water, acetonitrile (ACN) / pyridine (Py) at 0 C for 1 minute. It will be understood that other suitable reaction conditions and reagents may be employed. (III) Strategy 3: Phosphoramidite Coupling

[0241] As indicated above, for example, with reference to FIG. 6 and FIG. 7, methods for the preparation of PMOs described herein can include a phosphorodiamidate substrate. Coupling of an N-deprotected morpholino with a 6’-phosphoramidite substrate provides a phosphoramidite multimer which may be further functionalized by oxidizing and aminating it to furnish the phosphorodiamidate morpholino oligomer product. Coupling and functionalization may be repeated a desired number of times to obtain the multimer product with a specific number of repeats.

[0242] In embodiments, the 6’-phosphoramidite precursor for coupling with the solid-supported N-deprotected multimer may be prepared by the phosphorylation of 6’-OH morpholino nucleoside, generally as shown below:, wherein: B is a nucleobase; Y is independently hydrogen or N(R1)(R2), wherein if Y is H, then R1and R2are not present; R1and R2,if present, and R3are each independently H or substituted or unsubstituted alkyl, aryl, alkynyl, alkenyl, cycloalkyl, heterocyclyl or heteroaryl; R4is a hydrogen or a protecting group; Mnis a PMO monomer; (Mn)m is a PMO multimer having m PMO nucleotides; n is the position of the Mnmonomer in the (Mn)mmultimer sequence; and m is the number of Mnmonomers in the (Mn)mmultimer.

[0243] In embodiments, m can be an integer from 2 to 20, such as 2 to 11, or 2 to 4. In embodiments, m is 2. In embodiments, m is 3. In embodiments, m is 4.

[0244] In embodiments, B can be any nucleotide base (i.e., “nucleobase”) including A, G, C, T, or U. In embodiments, B can be A. In embodiments, B can be G. In embodiments, B can be C. In embodiments, B can be T. In embodiments, B can be U.

[0245] The phosphorylation reaction can be performed with a phosphorylating reagent in the presence of an activator. The phosphorylation reaction can include any phosphorylating agentsknown in the art, including, for example, tetraisopropylphosphorodiamidite, bis-(2-cyanoethyl) N,N-diisopropylphosphoramidite, 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one, bis(trichlorophenyl)phosphorochloridate, tetraethyl pyrophosphorodiamidite, phosphoryl chloride (POCl3), diethyl chlorophosphate, phenyl dichlorophosphate, cyclic trimetaphosphate, tetrametaphosphate, or benzylphosphorodiamidite derivatives, and the like. In embodiments, the phosphorylating agent in the phosphorylation reaction is tetraisopropylphosphorodiamidite. In embodiments, the phosphorylating agent in the phosphorylation reaction is from at least 1 equivalent to up to 5 equivalents with respect to the 6’-OH morpholino nucleoside substrate. In embodiments, the phosphorylating agent in the phosphorylation reaction is at least 1 equivalent, 1.5 equivalents, 2 equivalents, 2.5 equivalents, 3 equivalents, 3.5 equivalents, 4 equivalents, or 4.5 equivalents with respect to the 6’-OH morpholino nucleoside substrate. In embodiments, the phosphorylating agent in the phosphorylation reaction is up to 5 equivalents, up to 4.5 equivalents, up to 4 equivalents, up to 3.5 equivalents, up to 3 equivalents, up to 2.5 equivalents, up to 2 equivalents, or up to 1.5 equivalents with respect to the 6’-OH morpholino nucleoside substrate. In embodiments, the phosphorylating agent in the phosphorylation reaction is 1.5 equivalents with respect to the 6’-OH morpholino nucleoside substrate.

[0246] In embodiments, the phosphorylation reaction is performed in the presence of an activator. The phosphorylation reaction can include any activators known in the art, including, for example, 4,5-dicyanoimidazole (DCI), diisopropylammonium tetrazolide, 1H-tetrazole, 5-(ethylthio)-1H- tetrazole (ETT), benzylthiotetrazole (BTT), benzylmercapto-1H-tetrazole (BMT), 4,5- dichloroimidazole, 4,5-dimethylimidazole, pyridinium chloride / acetonitrile (PyrCl / MeCN), TFA.Py, or the like. In embodiments, the activator in the phosphorylation reaction is diisopropylammonium tetrazolide. In embodiments, the activator in the phosphorylation reaction is TFA.Py. In embodiments, the activator in the phosphorylation reaction is 4,5-dicyanoimidazole (DCI). In embodiments, the activator in the phosphorylation reaction is from at least 1 equivalent to up to 5 equivalents with respect to the 6’-OH morpholino nucleoside substrate. In embodiments, the activator in the phosphorylation reaction is at least 1 equivalent, at least 1.3 equivalents, 1.5 equivalents, 2 equivalents, 2.5 equivalents, 3 equivalents, 3.5 equivalents, 4 equivalents, or 4.5 equivalents with respect to the 6’-OH morpholino nucleoside substrate. In embodiments, the activator in the phosphorylation reaction is up to 5 equivalents, up to 4.5 equivalents, up to 4 equivalents, up to 3.5 equivalents, up to 3 equivalents, up to 2.5 equivalents, up to 2 equivalents,up to 1.3 equivalents, or up to 1.5 equivalents with respect to the 6’-OH morpholino nucleoside substrate. In embodiments, the activator in the phosphorylation reaction is 1.3 equivalents with respect to the 6’-OH morpholino nucleoside substrate. Additional reagents can be found in Wei, Tetrahedron 69 (2013) 3615e363.

[0247] The 6’-phosphoramidite substrate may be coupled with solid supported N-deprotected morpholino substrate to furnish the phosphoramidite product, generally as shown below: ,each B is independently a nucleobase; Y is independently hydrogen or N(R1)(R2), wherein if Y is H, then R1and R2are not present; R1and R2are each independently H or substituted or unsubstituted alkyl, aryl, alkynyl, alkenyl, cycloalkyl, heterocyclyl or heteroaryl; R4is a hydrogen or a protecting group; Mnis a PMO monomer; (Mn)mis a PMO multimer having m PMO nucleotides; n is the position of the Mnmonomer in the (Mn)m multimer sequence; and m is the number of Mnmonomers in the (Mn)m multimer.

[0248] In embodiments, m can be an integer from 2 to 20, such as 2 to 11, or 2 to 4. In embodiments, m is 2. In embodiments, m is 3. In embodiments, m is 4.

[0249] The 6’-phosphoramidite substrate can be coupled with solid supported N-deprotected morpholino substrate to furnish the phosphoramidite product under the coupling conditions known in the art and / or described herein. In embodiments, the 6’-phosphoramidite substrate is present inexcess of the N-deprotected morpholino substrate in the coupling reaction. In embodiments, the 6’-phosphoramidite substrate is present in an amount of at least 1 equivalent and up to 10 equivalents with respect to the N-deprotected morpholino substrate in the coupling reaction. In embodiments, the 6’-phosphoramidite substrate is present in an amount of at least 1 equivalent and up to 10 equivalents, at least 3 equivalents and up to 7 equivalents, or at least 5 equivalents with respect to the N-deprotected morpholino substrate in the coupling reaction. In, the 6’- phosphoramidite substrate is present in an amount of at least 1 equivalent, at least 2 equivalents, at least 3 equivalents, at least 4 equivalents, at least 5 equivalents, at least 6 equivalents, at least 7 equivalents, at least 8 equivalents, or at least 9 equivalents with respect to the N-deprotected morpholino substrate in the coupling reaction. In embodiments, the 6’-phosphoramidite substrate is present in an amount of up to 10 equivalents, up to 9 equivalents, up to 8 equivalents, up to 7 equivalents, up to 6 equivalents, up to 5 equivalents, up to 4 equivalents, up to 3 equivalents, or up to 2 equivalents with respect to the N-deprotected morpholino substrate in the coupling reaction. In embodiments, the 6’-phosphoramidite substrate is present in an amount of up to 5 equivalents with respect to the N-deprotected morpholino substrate in the coupling reaction.

[0250] The coupling reaction can be assisted by a suitable activator. In embodiments, the activator may be 4,5-dicyanoimidazole (DCI), diisopropylammonium tetrazolide, 1H-tetrazole, 5- (ethylthio)-1H-tetrazole (ETT), benzylthiotetrazole (BTT), benzylmercapto-1H-tetrazole (BMT), 4,5-dichloroimidazole, 4,5-dimethylimidazole, pyridinium chloride / acetonitrile (PyrCl / MeCN), TFA.Py, or the like. In embodiments, the activator in the phosphorylation reaction is 5-(ethylthio)- 1H-tetrazole (ETT). In embodiments, the activator in the phosphorylation reaction is benzylthiotetrazole (BTT). In embodiments, the activator in the phosphorylation reaction is 4,5- dicyanoimidazole (DCI).

[0251] In embodiments, the activator is present in an amount of at least 1 equivalent and up to 20 equivalents with respect to the N-deprotected morpholino substrate in the coupling reaction. In embodiments, the activator is present in an amount of at least 1 equivalent and up to 20 equivalents, at least 2 equivalents and up to 18 equivalents, at least 5 equivalents and up to 15 equivalents, at least 8 equivalents and up to 12 equivalents, at least 9 equivalents and up to 11 equivalents, or 10 equivalents with respect to the N-deprotected morpholino substrate in the coupling reaction. In embodiments, the activator is present in an amount of at least 1 equivalent, at least 2 equivalents, at least 5 equivalents, at least 8 equivalents, at least 10 equivalents, at least 12 equivalents, at least15 equivalents, or at least 18 equivalents with respect to the N-deprotected morpholino substrate in the coupling reaction. In embodiments, the activator is present in an amount of up to 20 equivalents, up to 18 equivalents, up to 15 equivalents, up to 12 equivalents, up to 10 equivalents, up to 8 equivalents, up to 5 equivalents, or up to 2 equivalents with respect to the N-deprotected morpholino substrate in the coupling reaction.

[0252] In embodiments, the coupling reaction employs any suitable solvent known to one with skill in the art. In embodiments, the coupling reaction employs any suitable polar aprotic solvent. In embodiments, the solvent is dimethylformamide (DMF), acetonitrile (MeCN), N-methyl-2- pyrrolidinone (NMP), dimethylimidazolidinone (DMI), tetrahydrofuran (THF), 1,4-dioxane, or mixtures thereof. In embodiments, the coupling reaction employs ACN and THF (10V, 10:1) as the solvent in the coupling reaction. While it is understood the volume of the solvent used would depend on the target concentration of the reactants in the reaction mixture, in embodiments, the coupling reaction employs the solvent in at least 1 volume equivalent and up to 50 volume equivalents with respect to the N-deprotected morpholino substrate in the coupling reaction. In embodiments, the coupling reaction employs the solvent in at least 1 volume equivalent and up to 50 volume equivalents, at least 10 volume equivalents and up to 40 volume equivalents, or at least 20 volume equivalents and up to 30 volume equivalents with respect to the N-deprotected morpholino substrate in the coupling reaction. In embodiments, the coupling reaction employs the solvent in at least 1 volume equivalent, at least 5 volume equivalents, at least 10 volume equivalents, at least 15 volume equivalents, at least 20 volume equivalents, at least 25 volume equivalents, at least 30 volume equivalents, at least 35 volume equivalents, at least 40 volume equivalents, or at least 45 volume equivalents with respect to the N-deprotected morpholino substrate in the coupling reaction. In embodiments, the coupling reaction employs the solvent in up to 50 volume equivalents, up to 45 volume equivalents, up to 40 volume equivalents, up to 35 volume equivalents, up to 30 volume equivalents, up to 25 volume equivalents, up to 20 volume equivalents, up to 15 volume equivalents, up to 10 volume equivalents, or up to 5 volume with respect to the N-deprotected morpholino substrate in the coupling reaction. In embodiments, the coupling reaction employs the solvent in 10 volume equivalents with respect to the N-deprotected morpholino substrate in the coupling reaction.

[0253] Coupling of N-deprotected morpholino substrate with the 6’-phosphoramidite substrate provides a phosphoramidite product which may be further functionalized by oxidative aminationto furnish the phosphorodiamidate morpholino oligomer product. Suitable reagents for this reaction include a halogenating reagent and an aminating reagent known to one skilled in the art. In embodiments, the halogenating agent in the oxidative amination of the phosphoramidite to furnish a phosphorodiamidate includes, for example, CCl4, CBr4, CBrCl3, I2, or the like. In embodiments, the halogenating agent in the oxidative amination of the phosphoramidite to furnish a phosphorodiamidate is I2.

[0254] In embodiments, the halogenating agent in the oxidative amination of the phosphoramidite to furnish a phosphorodiamidate is at least 1 equivalent and up to 50 equivalents with respect to the phosphoramidite substrate in the reaction. In embodiments, the halogenating agent used in the oxidative amination of the phosphoramidite to furnish a phosphorodiamidate is in a concentration from 0.01 M to 5 M. In embodiments, the halogenating agent used in the oxidative amination of the phosphoramidite to furnish a phosphorodiamidate is in a concentration from 0.01 M to 5 M, from 0.02 M to 4.5 M, from 0.03 M to 4 M, from 0.04 M to 3.5 M, from 0.05 M to 3 M, from 0.1 M to 2.5 M, from 0.1 M to 2 M, from 0.2 M to 1.5 M, from 0.3 M to 1 M, from 0.4 M to 0.5 M, or from 0.1 M to 0.2 M. In embodiments, the halogenating agent used in the oxidative amination of the phosphoramidite to furnish a phosphorodiamidate is in a concentration of at least 0.01 M, at least 0.02 M, at least 0.03 M, at least 0.04 M, at least 0.05 M, at least 0.1 M, at least 0.1 M, at least 0.2 M, at least 0.3 M, at least 0.4 M, at least 0.5 M, at least 0.6 M, at least 0.7 M, at least 0.8 M, at least 0.9 M, at least 1 M, at least 1.5 M, at least 2 M, at least 2.5 M, at least 3 M, at least 3.5 M, at least 4 M, or at least 4.5 M. In embodiments, the halogenating agent used in the oxidative amination of the phosphoramidite to furnish a phosphorodiamidate is in a concentration of up to 5 M, up to 4.5 M, up to 4 M, up to 3.5 M, up to 3 M, up to 2.5 M, up to 2 M, up to 1.5 M, up to 1 M, up to 0.9 M, up to 0.8 M, up to 0.7 M, up to 0.6 M, up to 0.5 M, up to 0.4 M, up to 0.3 M, up to 0.2 M, up to 0.1 M, up to 0.0.05 M, up to 0.04 M, up to 0.03 M, or up to 0.02 M.

[0255] In embodiments, the aminating agent in the oxidative amination of the phosphoramidite to furnish a phosphorodiamidate includes, for example, primary amines (such as, methylamine, ethylamine, n-butylamine, or benzylamine), secondary amines (such as, dimethylamine, diethylamine, dipropylamine, piperidine, or morpholine), tertiary amines (such as, triethylamine, tributylamine), cyclic amines (such as, pyrrolidine, piperazine, or imidazole), or aromatic amines (such as, aniline or substituted anilines such as para-toluidine, or 2,4-dimethylaniline). Inembodiments, the aminating agent in the oxidative amination of the phosphoramidite to furnish a phosphorodiamidate is dimethylamine.

[0256] In embodiments, the aminating agent in the oxidative amination of the phosphoramidite to furnish a phosphorodiamidate is in a concentration from 0.1 M to 5 M. In embodiments, the aminating agent in the oxidative amination of the phosphoramidite to furnish a phosphorodiamidate is in a concentration from 0.1 M to 5 M, from 0.2 M to 4.5 M, from 0.3 M to 4 M, from 0.4 M to 3 M, from 0.5 M to 2 M, from 0.1 M to 1 M, from 0.1 M to 0.9 M, from 0.1 M to 0.8 M, from 0.1 M to 0.7 M, from 0.1 M to 0.6 M, from 0.1 M to 0.5 M, from 0.1 M to 0.4 M, from 0.1 M to 0.3 M, from 0.1 M to 0.2 M, from 0.2 M to 0.8 M, from 0.3 M to 0.7 M, or from 0.4 M to 0.5 M. In embodiments, the aminating agent in the oxidative amination of the phosphoramidite to furnish a phosphorodiamidate is in a concentration of at least 0.1 M, at least 0.2 M, at least 0.3 M, at least 0.4 M, at least 0.5 M, at least 0.6 M, at least 0.7 M, at least 0.8 M, at least 0.9 M, at least 1 M, at least 1.5 M, at least 2 M, at least 2.5 M, at least 3 M, at least 3.5 M, at least 4 M, or at least 4.5 M. embodiments, the aminating agent in the oxidative amination of the phosphoramidite to furnish a phosphorodiamidate is in a concentration of up to 5 M, up to 4.5 M, up to 3 M, up to 3.5 M, up to 3 M, up to 2.5 M, up to 2 M, up to 1.5 M, up to 1 M, up to 0.5 M, up to 0.4 M, up to 0.3 M, or up to 0.2 M. In embodiments, the aminating agent in the oxidative amination of the phosphoramidite to furnish a phosphorodiamidate is in a concentration of 2 M.

[0257] One example of a Strategy 3 reaction scheme used to generate a solid-supported PMO having a (CAG)7repeat sequence is shown in FIG. 13A. It will be understood that the reaction scheme shown in FIG.13A may be used with PMO multimers having a sequence other CAG. As shown, a solid supported 6’-OH, 3’ protected CAG PMO trimer is reacted with a 6’- chlorophosphoramidate CAG PMO trimer (FIG. 13B) for six cycles to produce the solid- supported 21-mer. Each cycle includes deprotecting (detritylating) the 3’ end of the PMO coupled to the solid support, neutralizing, coupling in 4,5-dicyanoimidazole (DCI), acetonitrile (CAN) at 35 C for 1 hour, and oxidative amination. Oxidative amination includes reacting with I2in tetrahydrofuran (THF), pyridine (Py), NMe2at 35 C for 10 minutes. It will be understood that other suitable reaction conditions and reagents may be employed.Examples of PMOs

[0258] Any suitable PMO can be made according to the methods described herein in which at least one multimer is incorporated into a growing chain of a PMO during PMO elongation. In embodiments, the PMO comprises two or more multimer repeats, such as a dimer repeat, a trimer repeat, or a tetramer repeat.

[0259] In embodiments, the PMO includes from 2 to 52 multimer repeats. In embodiments, the PMO includes from 2 to 45, from 2 to 40, from 2 to 35, from 2 to 30, from 2 to 25, from 2 to 24, from 2 to 23, from 2 to 22, from 2 to 21, from 2 to 20, from 2 to 19, from 2 to 18, from 2 to 17, from 2 to 16, from 2 to 15, from 3 to 30, from 3 to 25, from 3 to 20, from 3 to 15, or from 3 to 10 multimer repeats. In embodiments, the PMO includes up to 52 nucleotide repeats. In embodiments, the PMO includes up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 24, up to 23, up to 22, up to 21, up to 20, up to 19, up to 18, up to 17, up to 16, up to 15, up to 10, up to 8, up to 5, or up to 3 nucleotide repeats. In embodiments, the PMO includes greater than 2, greater than 4, greater than 5, greater than 6, greater than 8, greater than 10, greater than 12, greater than 15, greater than 16, greater than 17, greater than 18, greater than 19, greater than 20, greater than 21, greater than 22, greater than 23, greater than 24, greater than 25, greater than 28, greater than 30, greater than 35, greater than 40, greater than 45, or greater than 48 nucleotide repeats. In embodiments, the PMO comprises from 3 to 15 nucleotide repeats. In embodiments, the PMO comprises from 3 to 10 nucleotide repeats. In embodiments, the PMO comprises from 3 to 8 nucleotide repeats. In embodiments, the PMO comprises from 4 to 6 nucleotide repeats. In embodiments, the PMO comprises 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide repeats. In embodiments, the PMO comprises 2 nucleotide repeats. In embodiments, the PMO comprises 3 nucleotide repeats. In embodiments, the PMO comprises 4 nucleotide repeats. In embodiments, the PMO comprises 5 nucleotide repeats. In embodiments, the PMO comprises 6 nucleotide repeats. In embodiments, the PMO comprises 7 nucleotide repeats. In embodiments, the PMO comprises 8 nucleotide repeats. In embodiments, the PMO comprises 9 nucleotide repeats. In embodiments, the PMO comprises 10 nucleotide repeats. In embodiments, the nucleotide repeats in the PMO are contiguous. In embodiments, the nucleotide repeats in the PMO are not contiguous. In embodiments, the PMO comprises a nucleotide sequence that includes an incomplete nucleotide repeat on the 5’ end, the 3’ end, or both.

[0260] In embodiments, the PMO comprises from 1 to 26 dinucleotide repeats. In embodiments, the PMO comprises from 2 to 25, from 2 to 24, from 2 to 23, from 2 to 22, from 2 to 21, from 2 to 20, from 2 to 19, from 2 to 18, from 2 to 17, from 2 to 16, from 2 to 15, from 3 to 25, from 3 to 22, from 3 to 20, from 3 to 15, from 3 to 10, from 4 to 9, from 5 to 8, or from 6 to 7 dinucleotide repeats. In embodiments, the PMO comprises up to 26, up to 25, up to 24, up to 23, up to 22, up to 21, up to 20, up to 19, up to 18, up to 17, up to 16, up to 15, up to 10, up to 8, up to 5, up to 3, up to 2, or up to 1 dinucleotide repeats. In embodiments, the PMO comprises greater than 1, greater than 2, greater than 4, greater than 5, greater than 6, greater than 8, greater than 10, greater than 12, greater than 15, greater than 16, greater than 17, greater than 18, greater than 19, greater than 20, greater than 21, greater than 22, greater than 23, greater than 24, or greater than 25 dinucleotide repeats. In embodiments, the dinucleotide repeats in the PMO are contiguous. In embodiments, the dinucleotide repeats in the PMO are not contiguous. In embodiments, the PMO comprises a dinucleotide sequence that includes an incomplete dinucleotide repeat on the 5’ end, the 3’ end, or both.

[0261] In embodiments, the PMO comprises from 1 to 18 trinucleotide repeats. In embodiments, the PMO comprises from 2 to 17, from 2 to 16, from 2 to 15, from 3 to 18, from 3 to 15, from 3 to 12, from 3 to 10, from 3 to 9, from 4 to 9, from 5 to 8, or from 6 to 7 trinucleotide repeats. In embodiments, the PMO comprises up to 18, up to 17, up to 16, up to 15, up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 trinucleotide repeats. In embodiments, the PMO comprises greater than 1, greater than 2, greater than 4, greater than 5, greater than 6, greater than 8, greater than 10, greater than 12, greater than 15, greater than 16, or greater than 17 trinucleotide repeats. In embodiments, the PMO comprises 18 trinucleotide repeats. In embodiments, the trinucleotide repeats in the PMO are contiguous. In embodiments, the trinucleotide repeats in the PMO are not contiguous. In embodiments, the PMO comprises a trinucleotide sequence that includes an incomplete trinucleotide repeat on the 5’ end, the 3’ end, or both.

[0262] In embodiments, the PMOs described herein can include trinucleotide repeats having the sequence CAG. In embodiments, the PMO comprises from 2 to 17, from 2 to 16, from 2 to 15, from 3 to 18, from 3 to 15, from 3 to 12, from 3 to 10, from 3 to 9, from 4 to 9, from 5 to 8, or from 6 to 7 CAG trinucleotide repeats. In embodiments, the PMO comprises 2 CAG trinucleotide repeats. In embodiments, the PMO comprises 3 CAG trinucleotide repeats. In embodiments, thePMO comprises 4 CAG trinucleotide repeats. In embodiments, the PMO comprises 5 CAG trinucleotide repeats. In embodiments, the PMO comprises 6 CAG trinucleotide repeats. In embodiments, the PMO comprises 7 CAG trinucleotide repeats. In embodiments, the PMO comprises 8 CAG trinucleotide repeats. In embodiments, the PMO comprises 9 CAG trinucleotide repeats. In embodiments, the PMO comprises 10 CAG trinucleotide repeats.

[0263] In embodiments, the PMO consists of 2 CAG trinucleotide repeats. In embodiments, the PMO consists of 3 CAG trinucleotide repeats. In embodiments, the PMO consists of 4 CAG trinucleotide repeats. In embodiments, the PMO consists of 5 CAG trinucleotide repeats. In embodiments, the PMO consists of 6 CAG trinucleotide repeats. In embodiments, the PMO consists of 7 CAG trinucleotide repeats. In embodiments, the PMO consists of 8 CAG trinucleotide repeats. In embodiments, the PMO consists of 9 CAG trinucleotide repeats. In embodiments, the PMO consists of 10 CAG trinucleotide repeats.

[0264] In embodiments, the PMOs described herein can include trinucleotide repeats having the sequence CTG. In embodiments, the PMO comprises from 2 to 17, from 2 to 16, from 2 to 15, from 3 to 18, from 3 to 15, from 3 to 12, from 3 to 10, from 3 to 9, from 4 to 9, from 5 to 8, or from 6 to 7 CTG trinucleotide repeats. In embodiments, the PMO comprises 2 CTG trinucleotide repeats. In embodiments, the PMO comprises 3 CTG trinucleotide repeats. In embodiments, the PMO comprises 4 CTG trinucleotide repeats. In embodiments, the PMO comprises 5 CTG trinucleotide repeats. In embodiments, the PMO comprises 6 CTG trinucleotide repeats. In embodiments, the PMO comprises 7 CTG trinucleotide repeats. In embodiments, the PMO comprises 8 CTG trinucleotide repeats. In embodiments, the PMO comprises 9 CTG trinucleotide repeats. In embodiments, the PMO comprises 10 CTG trinucleotide repeats.

[0265] In embodiments, the PMO consists of 2 CTG trinucleotide repeats. In embodiments, the PMO consists of 3 CTG trinucleotide repeats. In embodiments, the PMO consists of 4 CTG trinucleotide repeats. In embodiments, the PMO consists of 5 CTG trinucleotide repeats. In embodiments, the PMO consists of 6 CTG trinucleotide repeats. In embodiments, the PMO consists of 7 CTG trinucleotide repeats. In embodiments, the PMO consists of 8 CTG trinucleotide repeats. In embodiments, the PMO consists of 9 CTG trinucleotide repeats. In embodiments, the PMO consists of 10 CTG trinucleotide repeats.

[0266] In embodiments, the PMO comprises from 1 to 13 tetranucleotide repeats. In embodiments, the PMO comprises from 2 to 13, from 2 to 12, from 2 to 11, from 3 to 13, from 3 to 12, from 3 to10, from 3 to 9, from 4 to 9, from 4 to 8, from 5 to 8, or from 6 to 7 tetranucleotide repeats. In embodiments, the PMO comprises up to 13, up to 12, up to 11, up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 tetranucleotide repeats. In embodiments, the PMO comprises greater than 1, greater than 2, greater than 3, greater than 4, greater than 5, greater than 6, greater than 7, greater than 8, greater than 9, greater than 10, greater than 11, or greater than 12 tetranucleotide repeats. In embodiments, the tetranucleotide repeats in the PMO are contiguous. In embodiments, the tetranucleotide repeats in the PMO are not contiguous. In embodiments, the PMO comprises a tetranucleotide sequence that includes an incomplete tetranucleotide repeat on the 5’ end, the 3’ end, or both.

[0267] The PMOs described herein can be used for the treatment of nucleotide repeat disorders. In embodiments, the PMO includes a multimer repeat sequence that is complementary to a sequence found in a genomic DNA sequence. In embodiments, the PMO includes a multimer repeat sequence that is complementary to a sequence found in an RNA sequence. In embodiments, the PMO includes a multimer repeat sequence that is complementary to a sequence found in an mRNA sequence. In embodiments, the PMO includes a multimer sequence that is complementary to a nucleotide sequence found in a pre-mRNA sequence. In embodiments, the PMO includes a nucleotide sequence that is complementary to a nucleotide sequence found in a mature mRNA sequence.

[0268] In embodiments, the PMO comprises a sequence Xp(M1-M2-M3-M4)xYq, wherein: M1and M2are each independently C, A, U, or G; M3and M4are each independently C, A, U, G or are absent; each X and each Y are each independently C, A, U or G; p and q are each independently an integer from 0 to 3; and x is an integer from 1 to 10. In embodiments, the PMO comprises a dinucleotide repeat comprising Xp(M1-M2)xYq, wherein: M1and M2are each independently C, A, U, or G; X and Y are each independently C, A, U or G; p and q are each independently 0 or 1; and x is an integer from 1 to 10. In embodiments, the PMO comprises a trinucleotide repeat comprising Xp(M1-M2-M3)xYq, wherein: M1, M2, and M3are each independently C, A, U, or G; X and Y are each independently C, A, U or G; p and q are each independently an integer from 0 to 2; and x is an integer from 1 to 10. In embodiments, the PMO comprises a tetranucleotide sequence comprising Xp(M1-M2-M3-M4)xYq, wherein: M1, M2, and M3, and M4are each independently C, A, U, or G; X and Y are each independently C, A, U or G; p and q are each independently an integer from 0 to 3; and x is an integer from 1 to 10. In embodiments, x is 1. In embodiments, x is2. In embodiments, x is 3. In embodiments, x is 4. In embodiments, x is 5. In embodiments, x is 6. In embodiments, x is 7. In embodiments, x is 8. In embodiments, x is 9. In embodiments, x is 10.

[0269] In embodiments, the PMO comprises from 3 to 20 nucleotide multimer repeats. In embodiments, the PMO comprises from 3 to 15 multimer nucleotide repeats. In embodiments, the PMO comprises from 3 to 10 multimer nucleotide repeats. In embodiments, the PMO comprises from 3 to 8 multimer nucleotide repeats. In embodiments, the PMO comprises from 4 to 6 multimer nucleotide repeats. In embodiments, the PMO comprises 3, 4, 5, 6, 7, 8, 9 or 10 multimer nucleotide repeats. In embodiments, the PMO comprises 2 multimer nucleotide repeats. In embodiments, the PMO comprises 3 multimer nucleotide repeats. In embodiments, the PMO comprises 4 multimer nucleotide repeats. In embodiments, the PMO comprises 5 multimer nucleotide repeats. In embodiments, the PMO comprises 6 multimer nucleotide repeats. In embodiments, the PMO comprises 7 multimer nucleotide repeats. In embodiments, the PMO comprises 8 multimer nucleotide repeats. In embodiments, the PMO comprises 9 multimer nucleotide repeats. In embodiments, the PMO comprises 10 multimer nucleotide repeats. In embodiments, the multimer nucleotide repeats in the PMO are contiguous. In embodiments, the multimer nucleotide repeats in the PMO are not contiguous. In embodiments, the PMO comprises a nucleotide sequence that includes an incomplete nucleotide repeat on the 5’ end, the 3’ end, or both the 5’ end and the 3’ end.

[0270] In embodiments, the PMO comprises from 3 to 20 dinucleotide repeats. In embodiments, the PMO comprises from 3 to 15 dinucleotide repeats. In embodiments, the PMO comprises from 3 to 10 dinucleotide repeats. In embodiments, the PMO comprises from 3 to 8 dinucleotide repeats. In embodiments, the PMO comprises from 4 to 6 dinucleotide repeats. In embodiments, the PMO comprises 3, 4, 5, 6, 7, 8, 9, or 10 dinucleotide repeats. In embodiments, the PMO comprises 2 dinucleotide repeats. In embodiments, the PMO comprises 3 dinucleotide repeats. In embodiments, the PMO comprises 4 dinucleotide repeats. In embodiments, the PMO comprises 5 dinucleotide repeats. In embodiments, the PMO comprises 6 dinucleotide repeats. In embodiments, the PMO comprises 7 dinucleotide repeats. In embodiments, the PMO comprises 8 dinucleotide repeats. In embodiments, the PMO comprises 9 dinucleotide repeats. In embodiments, the PMO comprises 10 dinucleotide repeats. In embodiments, the dinucleotide repeats in the PMO are contiguous. In embodiments, the dinucleotide repeats in the PMO are not contiguous. In embodiments, the PMOcomprises a dinucleotide repeat sequence that includes an incomplete dinucleotide repeat on the 5’ end, the 3’ end, or both the 5’ end and the 3’ end.

[0271] In embodiments, the PMO comprises from 3 to 20 trinucleotide repeats. In embodiments, the PMO comprises from 3 to 15 trinucleotide repeats. In embodiments, the PMO comprises from 3 to 10 trinucleotide repeats. In embodiments, the PMO comprises from 3 to 8 trinucleotide repeats. In embodiments, the PMO comprises from 4 to 6 trinucleotide repeats. In embodiments, the PMO comprises 3, 4, 5, 6, 7, 8, 9, or 10 trinucleotide repeats. In embodiments, the PMO comprises 2 trinucleotide repeats. In embodiments, the PMO comprises 3 trinucleotide repeats. In embodiments, the PMO comprises 4 trinucleotide repeats. In embodiments, the PMO comprises 5 trinucleotide repeats. In embodiments, the PMO comprises 6 trinucleotide repeats. In embodiments, the PMO comprises 7 trinucleotide repeats. In embodiments, the PMO comprises 8 trinucleotide repeats. In embodiments, the PMO comprises 9 trinucleotide repeats. In embodiments, the PMO comprises 10 trinucleotide repeats. In embodiments, the trinucleotide repeats in the PMO are contiguous. In embodiments, the trinucleotide repeats in the PMO are not contiguous. In embodiments, the PMO comprises a trinucleotide repeat sequence that includes an incomplete trinucleotide repeat on the 5’ end, the 3’ end, or both the 5’ end and the 3’ end.

[0272] In embodiments, the PMO comprises from 3 to 20 tetranucleotide repeats. In embodiments, the PMO comprises from 3 to 15 tetranucleotide repeats. In embodiments, the PMO comprises from 3 to 10 tetranucleotide repeats. In embodiments, the PMO comprises from 3 to 8 tetranucleotide repeats. In embodiments, the PMO comprises from 4 to 6 tetranucleotide repeats. In embodiments, the PMO comprises 3, 4, 5, 6, 7, 8, 9, or 10 tetranucleotide repeats. In embodiments, the PMO comprises 2 tetranucleotide repeats. In embodiments, the PMO comprises 3 tetranucleotide repeats. In embodiments, the PMO comprises 4 tetranucleotide repeats. In embodiments, the PMO comprises 5 tetranucleotide repeats. In embodiments, the PMO comprises 6 tetranucleotide repeats. In embodiments, the PMO comprises 7 tetranucleotide repeats. In embodiments, the PMO comprises 8 tetranucleotide repeats. In embodiments, the PMO comprises 9 tetranucleotide repeats. In embodiments, the PMO comprises 10 tetranucleotide repeats. In embodiments, the tetranucleotide repeats in the PMO are contiguous. In embodiments, the tetranucleotide repeats in the PMO are not contiguous. In embodiments, the PMO comprises a tetranucleotide repeat sequence that includes an incomplete tetranucleotide repeat on the 5’ end, the 3’ end, or both the 5’ end and the 3’ end.

[0273] In embodiments, methods of making and using PMOs for the treatment of trinucleotide repeat disorders are provided. Trinucleotide repeat disorders include, but are not limited to, the disorders shown in Table 1. Table 1: Trinucleotide repeat disorders Trinucleotide Disorder Gene repeat or 1)

[0274] Tetranucleotide repeat disorders include, but are not limited to, the disorders shown in Table 2. Table 2: Tetranucleotide repeat disorders Tetranucleotide Disorder GeneRepeat Expansion Diseases

[0275] More than 40 diseases, most of which primarily affect the nervous system, are caused by expansions of simple sequence repeats dispersed throughout the human genome. Paulson, H. (2018). Repeat Expansion Diseases. Hanb Clin Neurol. 147:105-123 (doi: 10.1016 / B978-0-444-63233-3.00009-9). Although expanded trinucleotide repeat diseases are the most common, tetra-, penta-, hexa, and dodeca-nucleotide repeat expansions have been identified as the cause of human disease. Huntington's disease (HD)

[0276] Huntington's disease (HD) is an autosomal-dominant, progressive neurodegenerative disorder. A CAG repeat expansion, which can be of various lengths, in the IT15 (HTT) gene is causative for HD, with greater number of repeats correlating with disease onset and severity. CAG repeats in the range of 36-39 increase the likelihood of HD during a normal life span, and of 40 or more are fully penetrant. Repeats of this CAG repeat expansion in HTT results in a polyglutamine stretch close to the N‐terminus of the HD protein huntingtin (htt), and this stretch is believed to result in a toxic gain of function when the polyglutamine-containing huntingtin is expressed in the cell. HTT comprises 67 exons giving two major transcripts of either ~ 10 kb or 13 kb depending upon the 3′ untranslated region (UTR). The mammalian HTT gene is approximately 180 kb in length, contains 67 exons, and provides mature transcripts of approximately 10.3 and 13.7 kb. The HTT isoform that is about 13.5 kb is highly expressed in the brain and ovary, while the shortest isoform of about 10.3 kb is predominately expressed in dividing cells in the testes, B-cells, and muscle. See, for example, Paulson, H., Chapter 9 - Repeat expansion diseases, Handbook of Clinical Neurology, 147: 105-123, 2018; and Walker, F. O., Huntington's disease, The Lancet, 369:218-228, 2007; Papadopoulou, A.S., et al. Extensive Expression Analysis of Htt Transcripts in Brain Regions from the zQ175 HD Mouse Model Using a QuantiGene Multiplex Assay. Sci Rep 9:16137, 2019; and Zubkova, A.E., and Yudkin, D.V., Regulation of HTT mRNA Biogenesis: The Norm and Pathology. Int. J. Mol. Sci.25:11493, 2024.

[0277] Sathasivam, K. et al. (Aberrant splicing of HTT generates the pathogenic exon 1 protein in Huntington disease. Proc Natl Acad Sci USA 110, 2366–2370, 2013) has shown that exon 1 of HTT does not always splice to exon 2, resulting in two small polyadenylated mRNAs (Httexon1). Sathasivam showed that aberrant splicing occurs in HD knock-in mouse models and is CAG repeat length–dependent. The level of these transcripts increased in a polyQ length– dependent manner when comparing the Q50, Q100, and Q150 lines (mouse Htt sequences only) or the Q80 and zQ175 lines (human-mouse chimeric Htt). Accordingly, Sathasivam showed thatthe occurrence of incomplete splicing increases with increasing CAG repeat length and the Httexon1 transcripts are translated to generate the highly pathogenic exon 1 HTT protein.

[0278] Isoforms of HTT are known in the art. For example, Ruzo, A., et al. (2015, Discovery of Novel Isoforms of Huntingtin Reveals a New Hominid-Specific Exon. PLoS ONE 10(5): e0127687) describes HTT mRNA splice isoforms expressed in normal and HTT-expanded human embryonic stem cell (hESC) lines and cortical neurons differentiated from hESCs. These isoforms include ones where HTT protein domains were eliminated to generate smaller HTT proteins, as well as one that incorporated an additional exon. HTT splice isoforms were identified including those having skipped exons (e.g., skipped exons 10, 22, 34-35, 58, and 66) in RNA-seq data. Forexample, the HTT- 10 (exon 10 skip) results in the deletion of amino acids 427-442, which is adomain target of posttranslational modifications, particularly for the phosphorylation of Ser434, which decreases caspase-dependent cleavage of HTT. Ruzo notes shorter forms of HTT are typically associated with enhanced toxicity. Also, the absence of Ser434 phosphorylation in the HTT-Δ10 isoform, thought to otherwise decreases toxicity indirectly by affecting proteolysis of HTT, is predicted to be more toxic than the full-length isoform. Ruzo also notes the HTT splice isoform 3 (HTT-Δ13) which lacks 16 amino acids (609-624) at the C-terminus of exon 13 affects the predicted armadillo-like repeat section of HTT and may affect HTT structure or protein-protein interactions. Other HTT isoforms include HTT splice isoform 2 (HTT-Δ12) which lacks 135 nucleotides from the 3’ end of exon 12, and that eliminates 45 amino acids amino acids (539 to 583) from the HTT protein, HTT splice isoform 4 (HTT-Δ46) resulting in a 10 amino acid deletion at the N-terminal side of exon 46, and isoform HTT-41b which incorporates an unreported exon located between exons 41 and 42 (i.e., 41b), adding an additional 30 amino acids to the canonical HTT protein. WO / 2019 / 204457 reports that exclusion of exon 12 from mature HTT transcripts created a HTT isoform resistant to proteolysis by caspase-6 that is not cytotoxic.

[0279] As another example, Hughes, A.C., et al. (Identification of Novel Alternative Splicing Events in the Huntingtin Gene and Assessment of the Functional Consequences Using Structural Protein Homology Modelling, Journal of Molecular Biology, 426:1428-1438, 2014) describes splice variants of Htt found in mouse brain that lacked the 111-bp exon 29 (Htt∆ex29) or retained a 57-bp portion of intron 28 (Htt+ 57in28) via use of a cryptic splice site. Similar splice variants lacking exons 28 and 29 were found in human brain.

[0280] In embodiments, HD can be treated using a phosphorodiamidate morpholino oligomer that includes from one to 50 CTG repeats. In embodiments, the CTG repeats are contiguous. In embodiments, the CTG repeats are not contiguous. In embodiments, the ASO includes a nucleotide sequence that includes incomplete CTG repeats on either the 5´ or 3´ end. For example, in embodiments, the ASO may include a sequence such as CT(CTG)n, T(CTG)n, (CTG)nTG, or (CTG)nT where n is an integer from 1 to 50. In embodiments, the ASO comprises a sequence Xp(CTG)nYq, wherein: X and Y are, independently C, T or G; p and q are, independently, an integer from 0 to 2; and n is an integer from 1 to 10. For example, the PMO can comprise a sequence of (CTG)n, G(CTG)n, TG(CTG)n, (CTG)nC, (CTG)nCT, G(CTG)nC, G(CTG)nCT, TG(CTG)nC, TG(CTG)nCT, or combinations thereof, wherein n is an integer from 1 to 10. In embodiments, n is an integer from 5 to 10. In embodiments, n is 1. In embodiments, n is 2. In embodiments, n is 3. In embodiments, n is 4. In embodiments, n is 5. In embodiments, n is 6. In embodiments, n is 7. In embodiments, n is 8. In embodiments, n is 9. In embodiments, n is 10. Myotonic dystrophy

[0281] Myotonic dystrophies (MD) are the most frequent muscle dystrophies in the European population and are caused by repeat expansion mutations. MD are characterized by various symptoms including skeletal muscle wasting, myotonia (muscle hyperexcitability), neurocognitive deficits, cardiac electrical impulse problems, insulin resistance, cataracts, and gastrointestinal problems. Two forms of MD exist which are myotonic dystrophy type I (DM1) and myotonic dystrophy type II (DM2) with an incidence of about 1 in 20,000 people, and 1 in 8000 people, respectively. MD is associated with premature death, often caused by cardiovascular and / or respiratory problems. Both DM1 and DM2 show inheritance in an autosomal dominant manner (e.g., see, Day, J.W., and Ranum, L.P.W. (2005) Genetics and molecular pathogenesis of the myotonic dystrophies, Curr. Neurol. Neurosci. Rep. 5:55–56; Faustino, N.A., and Cooper, T.A. (2003) Pre-mRNA splicing and human disease, Genes Dev.17:419–437; and Liquori, C.L., et al. (2001) Myotonic dystrophy type 2 caused by a CCTG expansion in intron 1 of ZNF9, Science, 293:864–867).

[0282] Both DM1 and DM2 have been shown to be associated with nucleotide repeat expansion abnormalities, but with different genes. Primary RNA transcripts from DM1 and DM2 from thegene produces gain of toxic function, resulting in RNA transcripts with secondary structure and accumulation in the nucleus.

[0283] DM1 (also known as Steiner’s disease) is associated with defects in the myotonic dystrophy protein kinase (DMPK) gene. A common abnormality causative of DM1 is where there is a (CTG)n triplet repeat expansion of in the 3′ untranslated region of the DMPK gene, causing sequestration of transcriptional factors, and reduction in DMPK translation (e.g., see, Brook, J.D., et al. (1992) Molecular basis of myotonic dystrophy: Expansion of a trinucleotide (CTG) repeat at the 3′ end of a transcript encoding a protein kinase family member. Cell 69: 385; Mahadevan, M., et al. (1992) Myotonic dystrophy mutation: An unstable CTG repeat in the 3′ untranslated region of the gene. Science 255:1253–1255). In DM1 patients, the CTG repeats in DMPK typically are in the range from 50 to multiples of thousands, whereas in non-DM1 individuals there are generally less than 35 repeats. The number of nucleotide repeats have been correlated with the disease severity and age of onset.

[0284] DM2 results from an unstable tetranucleotide repeat expansion (CCTG)n in intron 1 of the nucleic acid-binding protein gene (CNBP; also known as ZNF9, for zinc finger 9 gene). CNBP is a conserved protein that includes 6-7 (Cys-Cys-His-Cys)-type 14-amino acids zinc knuckle motifs, and an Arg / Gly (RG)-rich motif for CNBP for protein activity. CNBP binds single-stranded DNA and RNA molecules, has also been shown to be a negative and positive transcriptional regulator of various genes, and functions as a nucleic acid chaperone and remodels G-quadruplex structures See, for example, Armas, P., et al. (2021) What's new about CNBP? Divergent functions and activities for a conserved nucleic acid binding protein, BBA - General Subjects, doi.org / 10.1016 / j.bbagen.2021.129996.

[0285] The size of the (CCTG)nrepeat is below 30 in normal individuals. Patients with myotonic dystrophy type 2 can have as little as 75 (CCTG)n repeat expansions, with the largest expansion measured at about 11,000 (CCTG)n repeats. The size of the (CCTG)n repeat expansion appears to increase over time in the same individual. Meola, G. (2020) Myotonic Dystrophy Type 2: the 2020 update. Acta Myol.39(4):222-234 (doi: 10.36185 / 2532-1900-026).

[0286] The predominant cause of MD pathogenesis is the formation of hairpin loops in the expanded CUG and CCUG repeats in the RNA which leads to dysregulation of two important RNA-binding proteins: muscleblind like 1 (MBNL1) and CUG-binding protein 1 (CUGBP1), also known as CUG-binding protein, Elav-like family member 1 (CELF1). Meola, G. (2020) MyotonicDystrophy Type 2: the 2020 update. Acta Myol. 39(4):222-234 (doi: 10.36185 / 2532-1900-026). In particular, DM pathology is associated with the sequestration of MBNL1 and upregulation of CUGBP1 (CELF), which are tissue-specific regulators of developmentally programmed alternative splicing that act as antagonist regulators of several pre-mRNA targets. In turn, these molecular changes result in the RNA processing alterations of a large number of various genes (e.g., muscle-specific chloride channel (CLCN1), insulin receptor (IR), bridging integrator 1 (BIN1), pyruvate kinase M (PKM), troponin T (TNNT2), etc.), which in turn affect the functionality of the associated proteins. In particular, there is evidence that hundreds of aberrant splicing events as caused by MBNL1 sequestration and CELF1 upregulation (e.g., see, Nakamori, M., et al. (2013) Splicing biomarkers of disease severity in myotonic dystrophy. Ann. Neurol.74:862–872. Thomas, J.D., et al. (2017) Disrupted prenatal RNA processing and myogenesis in congenital myotonic dystrophy. Genes Dev. 31:1122–1133). In turn, this results in decreased cell function and viability, which are associated with clinical characteristic of MD-associated diseases. Diagnosing a Subject

[0287] The disclosure also provides methods for treating a subject using a PMO of the disclosure, wherein the PMO can be prepared according to a method as described herein. The method can include obtaining information about the subject, the subject can be diagnosed to facilitate treatment of the subject, or both, and the subject can be treated using a PMO of the disclosure.

[0288] Treatment using the PMO of the disclosure, whether the treatment is performed prophylactically or therapeutically, can be established based on genomic information obtained from a subject or a group of subjects to be treated. The genomic information can be obtained by methods such as gene expression analysis, gene sequencing, and statistical genetics, including information relating to isoforms or SNPs of genes that are expressed by the individual subject or groups. This genetic information, sometimes referred to as “pharmacogenomics” can be used to determine how an individual or group, for example expressing a certain isoform or SNP of the disorder gene, responds to the PMO. As such, the individual or group may have a particular drug- responsive phenotype or drug-responsive genotype. Accordingly, the disclosure provides methods for establishing a treatment method (prophylactic and / or therapeutic) based on genetic information characteristic of the individual or group, with a PMO of the disclosure based on the individual or group’s drug-responsive phenotype or genotype. By knowing the isoform(s) or SNPs of disordergenes that are expressed by the individual subject or groups, a health care provider can select the most appropriate treatment using a PMO of the disclosure that would best benefit the individual subject or groups. Detection of isoforms in subject

[0289] In embodiments, the information obtained about the subject, or the diagnosis of the subject, relates to the subject expressing one or more isoforms of a gene. The one or more isoforms may or may not correlate with a disease state of a subject for which the PMO is administered to treat the subject. For example, information can be obtained, or diagnosis can be performed, to provide information about a gene isoform, wherein the gene is associated or implicated in Huntington disease (HD) , Fragile X Syndrome, Friedreich’s Ataxia, oculopharyngeal muscular dystrophy (OPMD), dentatorubral-pallidoluysian atrophy (DRPLA), Spinocerebellar ataxia (SCA), Spinal and bulbar muscular atrophy (SBMA), myotonic dystrophy 1 (DM1), or myotonic dystrophy 2 (DM2).

[0290] In embodiments, the PMO includes a nucleotide sequence that is complementary to a (CUG)n expanded repeat in a target RNA sequence. In embodiments, the PMO includes a nucleotide sequence that is complementary to a (CUG)xexpanded repeat in an mRNA transcript. In embodiments, the PMO includes a nucleotide sequence that is complementary to a (CUG)xexpanded repeat in a pre-mRNA. In embodiments, the PMO includes a nucleotide sequence that is complementary to a (CUG)xexpanded repeat in a mature mRNA. In embodiments, the PMO comprises (CAG)xnucleotide repeat (from 5’ to 3’). In embodiments, the PMO comprises a sequence such as Xp(CAG)nYq, wherein: X and Y are, independently C, A or G; p and q are, independently, an integer from 0 to 2; and n is an integer from 1 to 10. For example, the PMO can comprise a sequence of (CAG)x, G(CAG)x, AG(CAG)x, (CAG)xC, (CAG)xCA, G(CAG)xC, G(CAG)xCA, AG(CAG)xC, AG(CAG)xCA, or combinations thereof, wherein x is an integer from 1 to 10. In embodiments, x is 1. In embodiments, x is 2. In embodiments, x is 3. In embodiments, x is 4. In embodiments, x is 5. In embodiments, x is 6. In embodiments, x is 7. In embodiments, x is 8. In embodiments, x is 9. In embodiments, x is 10.

[0291] In embodiments, the PMO includes a nucleotide sequence that is complementary to a (CAG)xexpanded repeat in a target RNA sequence. In embodiments, the PMO includes a nucleotide sequence that is complementary to a (CAG)xexpanded repeat in an mRNA transcript.In embodiments, the PMO includes a nucleotide sequence that is complementary to a (CAG)x expanded repeat in a pre-mRNA. In embodiments, the PMO includes a nucleotide sequence that is complementary to a (CAG)xexpanded repeat in a mature mRNA. In embodiments, the PMO comprises a (CUG)x trinucleotide repeat (from 5’ to 3’). In embodiments, the PMO comprises a sequence Xp(CUG)xYq, wherein: X and Y are, independently C, U or G; p and q are, independently, an integer from 0 to 2; and x is an integer from 1 to 10. For example, the PMO can comprise a sequence of (CUG)x, G(CUG)x, UG(CUG)x, (CUG)xC, (CUG)xCU, G(CUG)xC, G(CUG)xCU, UG(CUG)xC, UG(CUG)xCU, or combinations thereof, wherein x is an integer from 1 to 10. In embodiments, x is 1. In embodiments, x is 2. In embodiments, x is 3. In embodiments, x is 4. In embodiments, x is 5. In embodiments, x is 6. In embodiments, x is 7. In embodiments, x is 8. In embodiments, x is 9. In embodiments, x is 10.

[0292] In embodiments, the PMO includes a nucleotide sequence that is complementary to a (GAA)xexpanded repeat in a target RNA sequence. In embodiments, the PMO includes a nucleotide sequence that is complementary to a (GAA)x expanded repeat in an mRNA transcript. In embodiments, the PMO includes a nucleotide sequence that is complementary to a (GAA)x expanded repeat in a pre-mRNA. In embodiments, the PMO includes a nucleotide sequence that is complementary to a (GAA)xexpanded repeat in a mature mRNA. In embodiments, the PMO comprises a (UUC)x trinucleotide repeat (from 5’ to 3’). In embodiments, the PMO comprises a sequence Xp(UUC)xYq, wherein: X and Y are, independently U or C; p and q are, independently, an integer from 0 to 2; and x is an integer from 1 to 10. For example, the PMO can comprise a sequence of (UUC)x, C(UUC)x, UC(UUC)x, (UUC)xU, (UUC)xUU, C(UUC)xU, C(UUC)xUU, UC(UUC)xU, UC(UUC)xUU, or combinations thereof, wherein x is an integer from 1 to 10. In embodiments, x is 1. In embodiments, x is 2. In embodiments, x is 3. In embodiments, x is 4. In embodiments, x is 5. In embodiments, x is 6. In embodiments, x is 7. In embodiments, x is 8. In embodiments, x is 9. In embodiments, x is 10.

[0293] In embodiments, the PMO includes a nucleotide sequence that is complementary to a (CGG)xexpanded repeat in a target RNA sequence. In embodiments, the PMO includes a nucleotide sequence that is complementary to a (CGG)x expanded repeat in an mRNA transcript. In embodiments, the PMO includes a nucleotide sequence that is complementary to a (CGG)xexpanded repeat in a pre-mRNA. In embodiments, the PMO includes a nucleotide sequence that is complementary to a (CGG)x expanded repeat in a mature mRNA. In embodiments, the PMOcomprises a (CCG)x trinucleotide repeat (from 5’ to 3’). In embodiments, the PMO comprises a sequence Xp(CCG)xYq, wherein: X and Y are, independently C or G; p and q are, independently, an integer from 0 to 2; and x is an integer from 1 to 10. For example, the PMO can comprise a sequence of (CCG)x, G(CCG)x, CG(CCG)x, (CCG)xC, (CCG)xCC, G(CCG)xC, G(CCG)xCC, CG(CCG)xC, CG(CCG)xCC, or combinations thereof, wherein x is an integer from 1 to 10. In embodiments, x is 1. In embodiments, x is 2. In embodiments, x is 3. In embodiments, x is 4. In embodiments, x is 5. In embodiments, x is 6. In embodiments, x is 7. In embodiments, x is 8. In embodiments, x is 9. In embodiments, x is 10.

[0294] In embodiments, the PMO includes a nucleotide sequence that is complementary to a (GCG)xexpanded repeat in a target RNA sequence. In embodiments, the PMO includes a nucleotide sequence that is complementary to a (GCG)x expanded repeat in an mRNA transcript. In embodiments, the PMO includes a nucleotide sequence that is complementary to a (GCG)x expanded repeat in a pre-mRNA sequence. In embodiments, the PMO includes a nucleotide sequence that is complementary to a (GCG)x expanded repeat in a mature mRNA. In embodiments, the PMO comprises a (CGC)x trinucleotide repeat (from 5’ to 3’). In embodiments, the PMO comprises a sequence Xp(CGC)xYq, wherein: X and Y are, independently C or G; p and q are, independently, an integer from 0 to 2; and x is an integer from 1 to 10. For example, the PMO can comprise a sequence of (CGC)x, C(CGC)x, GC(CGC)x, (CGC)xC, (CGC)xCG, C(CGC)xC, C(CGC)xCG, GC(CGC)xC, GC(CGC)xCG, or combinations thereof, wherein x is an integer from 1 to 10. In embodiments, x is 1. In embodiments, x is 2. In embodiments, x is 3. In embodiments, x is 4. In embodiments, x is 5. In embodiments, x is 6. In embodiments, x is 7. In embodiments, x is 8. In embodiments, x is 9. In embodiments, x is 10.

[0295] In embodiments, the PMO includes a nucleotide sequence that is complementary to a (CCUG)x expanded repeat in a target RNA sequence. In embodiments, the PMO includes a nucleotide sequence that is complementary to a (CCUG)x expanded repeat in an mRNA transcript. In embodiments, the PMO includes a nucleotide sequence that is complementary to a (CCUG)xexpanded repeat in a pre-mRNA sequence. In embodiments, the PMO includes a nucleotide sequence that is complementary to a (CCUG)x expanded repeat in a mature mRNA. In embodiments, the PMO comprises a (CAGG)xtrinucleotide repeat (from 5’ to 3’). In embodiments, the PMO comprises a sequence Xp(CAGG)xYq, wherein: X and Y are, independently C, A or G; p and q are, independently, an integer from 0 to 3; and x is an integerfrom 1 to 10. For example, the PMO can comprise a sequence of (CAGG)x, G(CAGG)x, GG(CAGG)x, AGG(CAGG)x, (CAGG)xC, (CAGG)xCA, (CAGG)xCAG, G(CAGG)xC, G(CAGG)xCA, G(CAGG)xCAG, GG(CAGG)xC, GG(CAGG)xCA, GG(CAGG)xCAG, AGG(CAGG)xC, AGG(CAGG)xCA, AGG(CAGG)xCAG, or combinations thereof, wherein x is an integer from 1 to 10. In embodiments, x is 1. In embodiments, x is 2. In embodiments, x is 3. In embodiments, x is 4. In embodiments, x is 5. In embodiments, x is 6. In embodiments, x is 7. In embodiments, x is 8. In embodiments, x is 9. In embodiments, x is 10. Method of Making

[0296] The compounds described herein can be prepared in a variety of ways known to one skilled in the art of organic synthesis or variations thereon as appreciated by those skilled in the art. For example, the compounds can be made as described above.

[0297] The compounds described herein can be prepared from readily available starting materials. Reaction conditions can vary with the particular reactants or solvents used, and such conditions can be determined by one skilled in the art. Reactions can be carried out in solution (use of a solvent or mixture of solvents) or neat (no solvent needed). Reactions can be carried out on solid support.

[0298] Variations on the compounds described herein include the addition, subtraction, or movement of the various constituents as described for each compound. Similarly, when one or more chiral centers are present in a molecule, the chirality of the molecule can be changed. Additionally, compound synthesis can involve the protection and deprotection of various chemical groups. The use of protection and deprotection, and the selection of appropriate protecting groups can be determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in Wuts and Greene, Protective Groups in Organic Synthesis, 4th Ed., Wiley & Sons, 2006, which is incorporated herein by reference in its entirety.

[0299] The starting materials and reagents used in preparing the disclosed compounds and compositions are either available from commercial suppliers such as Aldrich Chemical Co., (Milwaukee, WI), Acros Organics (Morris Plains, NJ), Fisher Scientific (Pittsburgh, PA), Sigma (St. Louis, MO), Pfizer (New York, NY), GlaxoSmithKline (Raleigh, NC), Merck (Whitehouse Station, NJ), Johnson & Johnson (New Brunswick, NJ), Aventis (Bridgewater, NJ), AstraZeneca (Wilmington, DE), Novartis (Basel, Switzerland), Wyeth (Madison, NJ), Bristol-Myers-Squibb(New York, NY), Roche (Basel, Switzerland), Lilly (Indianapolis, IN), Abbott (Abbott Park, IL), Schering Plough (Kenilworth, NJ), or Boehringer Ingelheim (Ingelheim, Germany), or are prepared by methods known to those skilled in the art following procedures set forth in references such as Fieser and Fieser’s Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd’s Chemistry of Carbon Compounds, Volumes 1-5 and Supplementals (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991); March’s Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition); and Larock’s Comprehensive Organic Transformations (VCH Publishers Inc., 1989). Other materials, such as the pharmaceutical carriers disclosed herein can be obtained from commercial sources.

[0300] Reactions to produce the compounds described herein can be carried out in solvents, which can be selected by one of skill in the art of organic synthesis. Solvents can be substantially nonreactive with the starting materials (reactants), the intermediates, or products under the conditions at which the reactions are carried out, i.e., temperature and pressure. Reactions can be carried out in one solvent or a mixture of more than one solvent. Product or intermediate formation can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g.,1H or13C) infrared spectroscopy, spectrophotometry (e.g., UV-visible), or mass spectrometry, or by chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography.

[0301] The disclosure relates to, among other things, methods of synthesizing a phosphorodiamidate morpholino oligomer (PMO) comprising a nucleotide repeat. In embodiments, the PMO comprises a sequence Xp(M1-M2-M3-M4)xYq, wherein: M1and M2are each independently C, A, U, or G; M3and M4are each independently C, A, U, G or are absent; each X and each Y are each independently C, A, U or G; p and q are each independently an integer from 0 to 3; and x is an integer from 1 to 10. In embodiments, the PMO comprises a dinucleotide repeat comprising Xp(M1-M2)xYq, wherein: M1and M2are each independently C, A, U, or G; each X and each Y are each independently C, A, U or G; p and q are each independently an integer from 0 to 1; and x is an integer from 1 to 10. In embodiments, the PMO comprises a trinucleotide repeat comprising Xp(M1-M2-M3)xYq, wherein: M1, M2, and M3are each independently C, A, U, or G; each X and each Y are each independently C, A, U or G; p and q are each independently an integer from 0 to 2; and x is an integer from 1 to 10. In embodiments, the PMO comprises a tetranucleotidesequence comprising Xp(M1-M2-M3-M4)xYq, wherein: M1, M2, M3, and M4are each independently C, A, U, or G; each X and each Y are each independently C, A, U or G; p and q are each independently an integer from 0 to 3; and x is an integer from 1 to 10.

[0302] Phosphorodiamidate morpholino oligomers (PMOs) are synthesized from the 5’ end to the 3’ end on a solid support. The solid support can be polystyrene. The solution phase synthesis for PMO oligomers proceeds in the 3’ to 5’ direction (opposite to the 5’ to 3’ direction for the synthesis of DNA).

[0303] In embodiments, the method comprises coupling a first morpholino nucleotide monomer to a first morpholino nucleotide monomer comprising a phosphoro-terminus to form a morpholino nucleotide multimer comprising a phosphorodiamidate morpholino dimer. In embodiments, the phosphorodiamidate morpholino dimer can be coupled to a second morpholino nucleotide monomer comprising a phosphoro-terminus to form a morpholino nucleotide multimer comprising a phosphorodiamidate morpholino trimer. In embodiments, the phosphorodiamidate morpholino trimer can be coupled to a third nucleotide morpholino monomer comprising a phosphoro-terminus to form a morpholino nucleotide multimer comprising a phosphorodiamidate morpholino tetramer.

[0304] In embodiments, the disclosure relates to a method of synthesizing a phosphorodiamidate morpholino oligomer (PMO) comprising a nucleotide repeat sequence (repeating multimer sequence) comprising: Xp(M1-M2-M3-M4)xYq, wherein: M1and M2are each independently C, A, U, or G; M3and M4are each independently C, A, U, G or are absent; each X and each Y are each independently C, A, U or G; p and q are each independently an integer from 0 to 3; and x is an integer from 1 to 10, wherein the method comprises: (a) synthesizing a nucleotide repeat (multimer repeat) subunit comprising a phosphoro-terminus; and (b) elongating the PMO to a desired chain length by coupling a first nucleotide repeat (multimer repeat) subunit with additional nucleotide repeat (multimer repeat) subunits comprising a phosphoro-terminus.

[0305] In embodiments, the method comprises: (a) synthesizing a nucleotide repeat (multimer repeat) subunit comprising M1-M2-M3- M4, wherein: M1and M2are each independently C, A, U, or G; M3and M4are each independently C, A, U, G or absent; each X and each Y are each independently C,A, U or G; and p and q are each independently an integer from 0 to 3, wherein synthesizing a nucleotide repeat (multimer repeat) subunit comprises: (i) coupling a morpholino monomer to a first morpholino nucleotide monomer comprising a phosphoro-terminus to form a phosphorodiamidate morpholino dimer. (ii) In embodiments, the method includes coupling the phosphorodiamidate morpholino dimer to a second morpholino nucleotide monomer comprising a phosphoro-terminus to form a PMO trimer. (iii) In embodiments, the method includes coupling the PMO trimer to a third morpholino nucleotide monomer comprising a phosphoro-terminus to form a PMO tetramer; and (b) elongating the PMO to a desired chain length by coupling the PMO with one or more additional nucleotide repeat (multimer repeat) subunits.

[0306] In embodiments, the method further comprises elongating the PMO by coupling the PMO with one or more additional morpholino monomers comprising a phosphoro-terminus. In embodiments, the additional morpholino monomers comprising a phosphoro-terminus are coupled to the 3’end, the 5’ end or both the 3’ and 5’ end of a PMO comprising two or more nucleotide repeats.

[0307] In embodiments, the phosphor-terminus can be a phosphorodiamidate, a phosphoric acid, or a phosphonamidite.

[0308] In embodiments, the method comprises coupling a PMO dimer to a PMO dimer. In embodiments, the method comprises coupling a PMO trimer to a PMO trimer. In embodiments, the method comprises coupling a PMO tetramer to a PMO tetramer. In embodiments, the method comprises coupling a PMO pentamer to a PMO pentamer. In embodiments, the method comprises coupling a PMO hexamer to a PMO hexamer.

[0309] In embodiments, the method comprises synthesizing a nucleotide repeat (multimer repeat) PMO oligomer by coupling one or more PMO monomers to the 5’ end of a PMO comprising two or more nucleotide repeats (multimer repeats).

[0310] In embodiments, the method comprises synthesizing a nucleotide repeat PMO oligomer by coupling one or more nucleotide repeat subunits to the 5’ end of a PMO monomer or PMOoligomer. In embodiments, the PMO oligomer comprises from 1 to 4 nucleotides. In embodiments, the PMO oligomer comprises a portion of a nucleotide repeat (multimer repeat).

[0311] In embodiments, the PMO comprises: Xp(M1-M2)xYq, wherein: M1and M2are each independently C, A, U, or G; each X and each Y are each independently C, A, U or G; p and q are each independently an integer from 0 to 1; and x is an integer from 1 to 10. In embodiments, the PMO comprises Xp(M1-M2-M3)xYq, wherein: M1, M2, and M3are each independently C, A, U, or G; each X and each Y are each independently C, A, U or G; p and q are each independently an integer from 0 to 2; and x is an integer from 1 to 10. In embodiments, the PMO comprises: Xp(M1- M2-M3-M4)xYq, wherein: M1, M2, M3, and M4are each independently C, A, U, or G; each X and each Y are each independently C, A, U or G; p and q are each independently an integer from 0 to 3; and x is an integer from 1 to 10.

[0312] The first morpholino nucleotide monomer can be protected at the 5’-OH. The protecting group can be a silyl protecting group.

[0313] The nucleotide repeat (multimer repeat) can be functionalized to be anchored to a resin and then reacted with another nucleotide repeat (multimer repeat) to elongate the PMO. The nucleotide repeat (multimer repeat) can be functionalized to be able to react directly with another nucleotide repeat to elongate the PMO.

[0314] It is understood that the synthesis can be used to make phosphorodiamidate morpholino oligomer (PMO) comprising two or more dimeric, trimeric, or tetrameric repeats. It is also understood that a nucleotide subunit can be coupled with a nucleotide subunit of a different or same sequence and / or length. The synthesis can performed in solution phase or on a solid support or in a combination of solution / solid phases.

[0315] Coupling the morpholino monomer to the PMO can be done in the presence of activating agent.

[0316] The PMO multimer can further be put on solid support at the 5’ end, and successive iterations of coupling to another PMO multimer can be performed and further cleaving the final product from solid support. In embodiments, PMO multimer can be a dimer, trimer, or tetramer.

[0317] In embodiments, the synthesis of the oligonucleotides can be automated. In embodiments, the synthesis of the oligonucleotides can be done manually. In embodiments, the synthesis of the oligonucleotides can be done via a combination of automation and manual synthesis. In embodiments, the synthesis of the oligonucleotides can be on solid support. In embodiments, thesynthesis of the oligonucleotides can be in solution phase. In embodiments, the synthesis of the oligonucleotides can be a combination of synthesis on solid support and in solid phase. Various reagents that can be used in PMO synthesis are listed in Table 3. Table 3. Various reagents that can be used in PMO synthesis Abbreviation IUPAC NameDCM Dichloromethane HFIP 111333-hexafluoro ro an-2-olMethods of Treatment

[0318] In embodiments, an PMO is administered to a patient diagnosed with a disease associated with nucleotide repeat expansion. In embodiments, the patient is diagnosed with a disease associated with a trinucleotide or tetranucleotide repeat expansion.

[0319] A method of treating a disease associated with a nucleotide repeat expansion is provided. In embodiments, a method of treating a disease associated with a trinucleotide or tetranucleotiderepeat expansion in a subject in need thereof is provided, comprising administering a compound disclosed herein.

[0320] The disease can be Huntington disease (HD), Fragile X Syndrome, Friedreich’s Ataxia, oculopharyngeal muscular dystrophy (OPMD), dentatorubral-pallidoluysian atrophy (DRPLA), Spinocerebellar ataxia (SCA), Spinal and bulbar muscular atrophy (SBMA), myotonic dystrophy 1 (DM1) or myotonic dystrophy 2 (DM2). The disease can be myotonic dystrophy 1. The disease can be myotonic dystrophy 2. The disease can be Huntington’s disease.

[0321] In embodiments, treatment refers to partial or complete alleviation, amelioration, relief, inhibition, delaying onset, reducing severity and / or incidence of one or more symptoms in a subject.

[0322] In embodiments, a method is provided for altering the expression of a target gene in a subject in need thereof, comprising administering a compound disclosed herein. In embodiments, the treatment results in the lowered expression of a target protein. In embodiments, the treatment results in the expression of a re-spliced target protein. In embodiments, the treatment results in the preferential expression of a wild-type target protein isomer.

[0323] The terms, “improve,” “increase,” “reduce,” “decrease,” and the like, as used herein, indicate values that are relative to a control. In embodiments, a suitable control is a baseline measurement, such as a measurement in the same individual prior to initiation of the treatment described herein, or a measurement in a control individual (or multiple control individuals) in the absence of the treatment described herein. A “control individual” is an individual afflicted with the same disease, who is about the same age and / or gender as the individual being treated (to ensure that the stages of the disease in the treated individual and the control individual(s) are comparable).

[0324] The individual (also referred to as “patient” or "subject") being treated is an individual (fetus, infant, child, adolescent, or adult human) having a disease or having the potential to develop a disease. The individual may have a disease mediated by aberrant gene expression or aberrant gene splicing.

[0325] In embodiments, the individual is an individual who has been recently diagnosed with the disease. Typically, early treatment (treatment commencing as soon as possible after diagnosis) is important to minimize the effects of the disease and to maximize the benefits of treatment.Methods of Administration

[0326] In vivo application of the disclosed compounds, and compositions containing them, can be accomplished by any suitable method and technique presently or prospectively known to those skilled in the art. For example, the disclosed compounds can be formulated in a physiologically- or pharmaceutically-acceptable form and administered by any suitable route known in the art including, for example, oral and parenteral routes of administration. As used herein, the term parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, intrasternal, and intrathecal administration, such as by injection. Administration of the disclosed compounds or compositions can be a single administration, or at continuous or distinct intervals as can be readily determined by a person skilled in the art.

[0327] The compounds disclosed herein, and compositions comprising them, can also be administered utilizing liposome technology, slow-release capsules, implantable pumps, and biodegradable containers. These delivery methods can, advantageously, provide a uniform dosage over an extended period of time. The compounds can also be administered in their salt derivative forms or crystalline forms. In embodiments, the compounds disclosed herein can be conjugated to a cell penetrating peptide (CPP). In embodiments, the compounds disclosed herein can be conjugated to a cyclic cell penetrating peptide (cCPP). In embodiments, the compounds disclosed herein can be conjugate to an endosomal escape vehicle (EEV), such as those disclosed in WO 2022 / 213118, entitled “CYCLIC CELL PENETRATING PEPTIDES”, or WO 2022 / 241408, entitled “COMPOSITIONS AND METHOD FOR MODULATING TISSUE DISTRIBUTION OF INTRACELLULAR THERAPEUTICS,” the disclosures of which are hereby incorporated by reference in its entirety.

[0328] The compounds disclosed herein can be formulated according to known methods for preparing pharmaceutically acceptable compositions. Formulations are described in detail in a number of sources which are well known and readily available to those skilled in the art. For example, Remington’s Pharmaceutical Science by E.W. Martin (1995) describes formulations that can be used in connection with the disclosed methods. In general, the compounds disclosed herein can be formulated such that an effective amount of the compound is combined with a suitable carrier in order to facilitate effective administration of the compound. The compositions used can also be in a variety of forms. These include, for example, solid, semi-solid, and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspension, suppositories, injectable andinfusible solutions, and sprays. The form depends on the intended mode of administration and therapeutic application. The compositions also include conventional pharmaceutically-acceptable carriers and diluents which are known to those skilled in the art. Examples of carriers or diluents for use with the compounds include ethanol, dimethyl sulfoxide, glycerol, alumina, starch, saline, and equivalent carriers and diluents.

[0329] Formulations suitable for administration include, for example, aqueous sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions, which can include suspending agents and thickening agents. The formulations can be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a freeze dried (lyophilized) condition requiring only the condition of the sterile liquid carrier, for example, water for injections, prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powder, granules, tablets, etc. It should be understood that in addition to the ingredients particularly mentioned above, the compositions can include other agents conventional in the art having regard to the type of formulation in question.

[0330] Compounds and compositions disclosed herein, including pharmaceutically acceptable salts or prodrugs thereof, can be administered intravenously, intramuscularly, or intraperitoneally by infusion or injection. Solutions of the active agent or its salts can be prepared in water. In embodiments, solutions of the active agent or its salts can be mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms.

[0331] The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient, which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions. In embodiments, the pharmaceutical dosage forms can be encapsulated in liposomes. The ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenanceof the required particle size in the case of dispersions or by the use of surfactants. In embodiments, the prevention of the action of microorganisms can be brought about by various other antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, isotonic agents are included, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the inclusion of agents that delay absorption, for example, aluminum monostearate and gelatin.

[0332] Sterile injectable solutions are prepared by incorporating a compound and / or agent in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation include vacuum drying and the freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.

[0333] Useful dosages of the compounds and agents and pharmaceutical compositions can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art.

[0334] The dosage ranges for the administration of the compositions include those large enough to produce the desired effect in which the symptoms or disorder are affected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days.

[0335] Also disclosed are pharmaceutical compositions that comprise a compound in combination with a pharmaceutically acceptable carrier. Pharmaceutical compositions adapted for oral, topical or parenteral administration, comprising an amount of a compound are provided. The dose administered to a patient, particularly a human, should be sufficient to achieve a therapeutic response in the patient over a reasonable time frame, without lethal toxicity, and causing no more than an acceptable level of side effects or morbidity. One skilled in the art will recognize that dosage will depend upon a variety of factors including the condition (health) of the subject, the body weight of the subject, kind of concurrent treatment, if any, frequency of treatment, therapeutic ratio, as well as the severity and stage of the pathological condition.

[0336] Also disclosed are kits that comprise a compound disclosed herein in one or more containers. The disclosed kits can include pharmaceutically acceptable carriers and / or diluents. In embodiments, a kit includes one or more other components, adjuncts, or adjuvants as described herein. In embodiments, a kit includes one or more anti-cancer agents, such as those agents described herein. In embodiments, a kit includes instructions or packaging materials that describe how to administer a compound or composition of the kit. Containers of the kit can be of any suitable material, e.g., glass, plastic, metal, etc., and of any suitable size, shape, or configuration. In embodiments, a compound and / or agent disclosed herein is provided in the kit as a solid, such as a tablet, pill, or powder form. In embodiments, a compound and / or agent disclosed herein is provided in the kit as a liquid or solution. In embodiments, the kit comprises an ampoule or syringe containing a compound and / or agent disclosed herein in liquid or solution form.

[0337] A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims. Certain Definitions

[0338] As used in the description and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” includes mixtures of two or more such compositions, reference to “an agent” includes mixtures of two or more such agents, reference to “the component” includes mixtures of two or more such components, and the like.

[0339] The term “about” when immediately preceding a numerical value means a range (e.g., plus or minus 10% of that value). For example, “about 50” can mean 45 to 55, “about 25,000” can mean 22,500 to 27,500, etc., unless the context of the disclosure indicates otherwise, or is inconsistent with such an interpretation. For example, in a list of numerical values such as “about 49, about 50, about 55, …”, “about 50” means a range extending to less than half the interval(s) within the preceding and subsequent values, e.g., more than 49.5 to less than 52.5. Furthermore, the phrases “less than about” a value or “greater than about” a value should be understood in view of the definition of the term “about” provided herein. Similarly, the term “about” when preceding a series of numerical values or a range of values (e.g., “about 10, 20, 30” or “about 10-30”) refers, respectively, to all values in the series, or the endpoints of the range.

[0340] As used herein, the term “endosomal escape vehicle” (EEV) refers to a cCPP that is conjugated by a chemical linkage (i.e., a covalent bond or non-covalent interaction) to a linker and / or an exocyclic peptide (EP).

[0341] As used herein, the term "oligonucleotide" refers to an oligomeric compound comprising a plurality of linked nucleotides or nucleosides. One or more nucleotides of an oligonucleotide can be modified. An oligonucleotide can comprise ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). Oligonucleotides can be composed of natural and / or modified nucleobases, sugars and covalent internucleoside linkages, and can further include non-nucleic acid conjugates.

[0342] The terms “peptide,” “protein,” and “polypeptide” are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of another. Two or more amino acid residues can be linked by the carboxyl group of one amino acid to the alpha amino group. Two or more amino acids of the polypeptide can be joined by a peptide bond. The polypeptide can include a peptide backbone modification in which two or more amino acids are covalently attached by a bond other than a peptide bond. The polypeptide can include one or more non-natural amino acids, amino acid analogs, or other synthetic molecules that are capable of integrating into a polypeptide. The term polypeptide includes naturally occurring and artificially occurring amino acids. The term polypeptide includes peptides, for example, that include from about 2 to about 100 amino acid residues as well as proteins, that include more than about 100 amino acid residues, or more than about 1000 amino acid residues, including, but not limited to therapeutic proteins such as antibodies, enzymes, receptors, soluble proteins and the like.

[0343] As used herein, by a “subject” is meant an individual. Thus, the “subject” can include domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.), and birds. “Subject” can also include a mammal, such as a primate or a human. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.

[0344] The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. Inaddition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

[0345] The term “isoforms” refers to different mRNA species that are synthesized from a single gene (i.e., “mRNA isoforms” of single gene). If these mRNA isoforms are translated into proteins this can result in “protein isoforms” of a gene that differs in amino acid sequence. mRNA isoforms include, but are not limited to, mRNA isoforms that result from spice variations of a pre-mRNA transcript, for example, having different combinations of exons as compared to wild-type mRNA; mRNA isoforms that include the addition or insertion of nucleotides, resulting in protein products having one or more additional amino acids as compared to the wild-type protein; and mRNA isoforms that remove or delete nucleotides, resulting in protein products having one or more fewer amino acids as compared to the wild-type protein. Resulting protein isoforms can include those lacking various internal amino acid segments or including an additional amino acid segment (e.g., as a result of alternative splicing), having N- and / or C-terminus deletion(s) or truncation(s), or having N- and / or C-terminus addition(s). For example, alternative splicing as caused by post- transcriptional mRNA processing wherein exons are taken together in different combinations and introns are removed, can produce various mature mRNAs. These various mature mRNAs, when translated, produce protein isoforms with different structures and functions.

[0346] The term “isoform of a disorder gene” or “disorder isoform” refers to different mRNA species that are synthesized from a single gene that is associated with a disorder or disease state, such any of those genes listed in Tables 1 and 2. As noted herein, a disorder state can be characterized by a gene that includes nucleotide repeat expansions, which in turn produces a mRNA with the corresponding repeats. Isoforms of mRNAs including these corresponding repeats (e.g., trinucleotide, tetranucleotide, etc.) may also be associated with a subject having a disorder. The subject may therefore express mRNAs of different lengths such as caused alternative splicing of the pre-mRNA, but including the nucleotide repeat. In turn, when translated, this results in the products of various proteins isoforms of different structure and function, but still including amino acids residues encoded by the nucleotide repeat portion of the mRNA, such a polyglutamine tract.The term "polymorphism" refers to one or more variations in a gene sequence from an identified source as compared to the same gene sequence from a different source but from the same organism (e.g., from two different human subjects). The polymorphism can be one or more deletions, insertions, or substitutions in the gene sequence from the identified source. Comparison of the gene sequences from the identified source with the different source allows for identification of the polymorphism. Methods routine in the art can be used for identification of polymorphisms. Polymorphisms can also be identified by the comparison of two alleles of the same gene. Types of polymorphisms include single nucleotide polymorphisms (SNPs).

[0347] The term “therapeutically effective” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.

[0348] The term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0349] The term “carrier” means a compound, composition, substance, or structure that, when in combination with a compound or composition, aids or facilitates preparation, storage, administration, delivery, effectiveness, selectivity, or any other feature of the compound or composition for its intended use or purpose. For example, a carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.

[0350] As used herein, the term “pharmaceutically acceptable carrier” refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms canbe ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use. Suitable inert carriers can include sugars such as lactose.

[0351] As used herein, the term “sequence identity” refers to the percentage of amino acids between two polypeptide sequences that are the same and in the same relative position. As such one polypeptide sequence has a certain percentage of sequence identity compared to another polypeptide sequence. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. Those of ordinary skill in the art will appreciate that two sequences are generally considered to be “substantially identical” if they contain identical residues in corresponding positions. In embodiments, the sequence identity between two amino acid sequences may be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), in the version that exists as of the date of filing. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the −nobrief option) is used as the percent identity and is calculated as follows: (Identical Residues×100) / (Length of Alignment−Total Number of Gaps in Alignment)

[0352] In embodiments, sequence identity may be determined using the Smith-Waterman algorithm, in the version that exists as of the date of filing.

[0353] As is well known in this art, amino acid sequences may be compared using any of a variety of algorithms, including those available in commercial computer programs such as BLASTP, gapped BLAST, and PSI-BLAST, in existence as of the date of filing. Such programs are described in Altschul, et al., Basic local alignment search tool, J. Mol. Biol., 215(3): 403-410, 1990; Altschul, et al., Methods in Enzymology; Altschul, et al., “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”, Nucleic Acids Res.25:3389-3402, 1997; Baxevanis, et al., Bioinformatics A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener, et al., (eds.), Bioinformatics Methods and Protocols (Methods in MolecularBiology, Vol. 132), Humana Press, 1999. In addition to identifying homologous sequences, the programs mentioned above typically provide an indication of the degree of homology.

[0354] As used herein, the terms "antisense compound" and "AC" are used interchangeably to refer to a polymeric nucleic acid structure (which can also be referred to as an oligonucleotide or polynucleotide) which is at least partially complementary to a target nucleic acid molecule to which it (the AC) hybridizes. The AC may be a short (in embodiments, less than 50 base pair) polynucleotide or polynucleotide homologue comprising a sequence complimentary to a target sequence in a target pre-mRNA strand. The AC may be formed of natural nucleic acids, synthetic nucleic acids, nucleic acid homologues, or any combination thereof. In embodiments, the AC comprises oligonucleosides. In embodiments, AC comprises antisense oligonucleotides. In embodiments, the AC comprises conjugate groups. Nonlimiting examples of ACs include, but are not limited to, primers, probes, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, alternate splicers, siRNAs, oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics, and chimeric combinations of these. As such, these compounds can be introduced in the form of single-stranded, double-stranded, circular, branched or hairpins and can contain structural elements such as internal or terminal bulges or loops. Oligomeric double-stranded compounds can be two strands hybridized to form double-stranded compounds or a single strand with sufficient self-complementarity to allow for hybridization and formation of a fully or partially double-stranded compound. In embodiments, an AC modulates (increases, decreases, or changes) expression of a target nucleic acid. Various modifications may be made to the polymeric nucleic acid structure, such as phosphorodiamidate morpholino oligomer (PMO). Therefore, AC as used herein encompasses any modification described herein, such as a PMO. The terms "pre-mRNA" and "primary transcript" as used herein refer to a newly synthesized eukaryotic mRNA molecule directly after DNA transcription. A pre-mRNA must be capped with a 5' cap, modified with a 3' poly-A tail, and spliced to produce a mature mRNA sequence.

[0355] As used herein, the terms “targeting” or “targeted to” refer to the association of an antisense compound (AC) with a target nucleic acid molecule or a region of a target nucleic acid molecule. In embodiments, the AC is capable of hybridizing to a target nucleic acid under physiological conditions. In embodiments, the AC targets a specific portion or site within the target nucleic acid, for example, a portion of the target nucleic acid having at least one identifiable structure, function,or characteristic such as a particular exon or intron, or selected nucleobases or motifs within an exon or intron.

[0356] As used herein, the terms "target nucleic acid" and "target sequence" refer to a nucleic acid molecule having a nucleic acid sequence to which the antisense compound binds or hybridizes. Target nucleic acids include, but are not limited to, RNA (including, but not limited to pre-mRNA and mRNA or portions thereof), cDNA derived from such RNA, as well as non-translated RNA, such as miRNA. For example, in embodiments, a target nucleic acid can be a cellular gene (or mRNA transcribed from such gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In embodiments, the target nucleic acid is a target RNA. In embodiments, the target nucleic acid is a target mRNA. In embodiments, the target nucleic acid is a target pre-mRNA.

[0357] The "target pre-mRNA" is the pre-mRNA comprising the target sequence to which the AC hybridizes.

[0358] The "target mRNA" is the mRNA sequence resulting from splicing of the target pre-mRNA sequence. In embodiments, the target mRNA does not encode a functional protein. In embodiments, the target mRNA retains one or more intron sequences.

[0359] As used herein, the term "gene" refers to a nucleic acid molecule having a nucleic acid sequence that encompasses a 5' promoter region associated with the expression of the gene product, and any intron and exon regions and 3' untranslated regions ("UTR") associated with the expression of the gene product.

[0360] The "target gene" refers to the gene that encodes the target pre-mRNA.

[0361] The "target protein" refers to the amino acid sequence encoded by the target mRNA. In embodiments, the target protein may not be a functional protein.

[0362] "Wild-type target protein" refers to a native, functional protein isomer produced by a wild- type, normal, or unmutated version of the target gene. The wild-type target protein also refers to the protein resulting from a target pre-mRNA that has been properly spliced.

[0363] As used herein, the term “transcript” refers to an RNA molecule transcribed from DNA and includes, but is not limited to mRNA, mature mRNA, pre -mRNA, and partially processed RNA.

[0364] As used herein, the term "nucleoside" means a glycosylamine comprising a nucleobase and a sugar. Nucleosides includes, but are not limited to, natural nucleosides, abasic nucleosides,modified nucleosides, and nucleosides having mimetic bases and / or sugar groups. A "natural nucleoside" or "unmodified nucleoside" is a nucleoside comprising a natural nucleobase and a natural sugar. Natural nucleosides include RNA and DNA nucleosides.

[0365] As used herein, the term "nucleotide" refers to a nucleoside having a phosphate group covalently linked to the sugar. Nucleotides may be modified with any of a variety of substituents.

[0366] As used herein, the term "nucleobase" refers to the base portion of a nucleoside or nucleotide. A nucleobase may comprise any atom or group of atoms capable of hydrogen bonding to a base of another nucleic acid. A natural nucleobase is a nucleobase that is unmodified from its naturally occurring form in RNA or DNA.

[0367] As used herein "oligonucleoside" refers to an oligonucleotide in which the internucleoside linkages do not contain a phosphorus atom.

[0368] As used herein, the term "oligonucleotide" refers to an oligomeric compound comprising a plurality of linked nucleotides or nucleosides. In embodiments, one or more nucleotides of an oligonucleotide is modified. In embodiments, an oligonucleotide comprises ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In embodiments, oligonucleotides are composed of natural and / or modified nucleobases, sugars and covalent internucleoside linkages, and may further include non-nucleic acid conjugates.

[0369] As used herein, “phosphorodiamidate morpholino oligomer (PMO)” refers to a synthetic oligomers comprising a natural nucleobase linked to a methylenemorpholine ring, linked via a phosphorodiamidate group (as compared to a phosphate backbone, found in naturally occurring oligonucleotides). See: Summerton JE (2017). “Invention and Early History of Morpholinos: From Pipe Dream to Practical Products”. Morpholino Oligomers. Methods in Molecular Biology.1565. Humana Press (Springer). pp.1-15. A PMO can have the general structure: , wherein B is a nucleotide base.

[0370] As used herein "internucleoside linkage" refers to a covalent linkage between adjacent nucleosides.

[0371] As used herein "natural internucleotide linkage" refers to a 3' to 5' phosphodiester linkage.

[0372] As used herein, the term "modified internucleoside linkage" refers to any linkage between nucleosides or nucleotides other than a naturally occurring internucleoside linkage.

[0373] As used herein, the term "nucleobase complementarity" refers to a nucleobase that is capable of base pairing with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In embodiments, complementary nucleobase refers to a nucleobase of an antisense compound that is capable of base pairing with a nucleobase of its target nucleic acid. For example, if a nucleobase at a certain position of an antisense compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be complementary at that nucleobase pair.

[0374] As used herein, the term "non-complementary nucleobase" refers to a pair of nucleobases that do not form hydrogen bonds with one another or otherwise support hybridization.

[0375] As used herein, the term "complementary" refers to the capacity of an oligomeric compound to hybridize to another oligomeric compound or nucleic acid through nucleobase complementarity. In embodiments, an antisense compound and its target are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleobases that can bond with each other to allow stable association between the antisense compound and the target. One skilled in the art recognizes that the inclusion of mismatches is possible without eliminating the ability of the oligomeric compounds to remain in association. Therefore, described herein are antisense compounds that may comprise up to about 20% nucleotides that are mismatched (i.e., are not nucleobase complementary to the corresponding nucleotides of the target). In embodiments, the antisense compounds contain no more than about 15%, not more than about 10%, not more than 5% or no mismatches. The remaining nucleotides are nucleobase complementary or otherwise do not disrupt hybridization (e.g., universal bases). One of ordinary skill in the art would recognize the compounds provided herein are at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% nucleobase complementary to a target nucleic acid.

[0376] As used herein, "hybridization" means the pairing of complementary oligomeric compounds (e.g., an antisense compound and its target nucleic acid). While not limited to a particular mechanism, the most common mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases). For example, the natural base adenine is nucleobase complementary to the natural nucleobases thymidine and uracil which pair through the formation of hydrogen bonds. The natural base guanine is nucleobase complementary to the natural bases cytosine and 5-methyl cytosine. Hybridization can occur under varying circumstances.

[0377] As used herein, the term "specifically hybridizes" refers to the ability of an oligomeric compound to hybridize to one nucleic acid site with greater affinity than it hybridizes to another nucleic acid site. In embodiments, an antisense oligonucleotide specifically hybridizes to more than one target site. In embodiments, an oligomeric compound specifically hybridizes with its target under stringent hybridization conditions.

[0378] The terms “modulate”, “modulating” and “modulation” refer to a perturbation of expression, function or activity when compared to the level of expression, function or activity prior to modulation. Modulation can include an increase (stimulation or induction) or a decrease (inhibition or reduction) in expression, function or activity. In embodiments, modulation can include perturbation of splice site selection during pre-mRNA processing.

[0379] The terms “inhibit”, “inhibiting” or “inhibition” refer to a decrease in an activity, expression, function or other biological parameter and can include, but does not require complete ablation of the activity, expression, function or other biological parameter. Inhibition can include, for example, at least about a 10% reduction in the activity, response, condition, or disease as compared to a control. In embodiments, expression, activity or function of a gene or protein is decreased by a statistically significant amount.

[0380] As used herein, the term "expression" refers to all the functions and steps by which a gene's coded information is converted into structures present and operating in a cell. Such structures include, but are not limited to, the products of transcription and translation.

[0381] As used herein, the term "dosage unit" refers to a form in which a pharmaceutical agent is provided. In embodiments, a dosage unit is a vial comprising lyophilized antisenseoligonucleotide. In embodiments, a dosage unit is a vial comprising reconstituted antisense oligonucleotide.

[0382] A number of embodiments have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Other embodiments are within the scope of the following claims.

[0383] All publications, patents and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications, patents and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. EXAMPLES Example 1. Synthesis of 6’-OH CAG Trimer

[0384] FIG.14 shows the synthetic scheme for the preparation of a CAG trimer, with partial views in FIGS.14A-14E. The 6’-OH group of an N-trityl protected morpholino C-monomer is protected with a -TBS protecting group. Next the N-trityl protecting group is removed from the morpholine ring of the C-monomer under acidic conditions to furnish the 6'-OTBS protected-N-deprotected morpholino C-monomer. Next the 6'-OTBS protected-N-deprotected morpholino C-monomer is coupled with a N-trityl protected-6’-chlorophosphoramidate functionalized A-monomer with LiBr as an additive to furnish the CA-dimer. Next the N-trityl protecting group is removed from the morpholine ring of the A-monomer under acidic conditions to furnish the N-deprotected CA-dimer which is coupled with an N-trityl protected-6’-chlorophosphoramidate functionalized G-monomer to form the 6'-OTBS protected-3’-N-trityl protected morpholino CAG trimer. Finally, the -TBS protecting group is removed with triethylamine trihydrofluoride to obtain the 6’-OH CAG trimer. Example 1A: TBS Protection of N-trityl 6'-OH C-monomer

[0385] A clean, dry round-bottom flask was charged with 5 gm of the N-trityl 6'-OH morpholino C-monomer and dry dichloromethane (DCM). Imidazole was added in slight excess, and the solution was stirred until fully dissolved. A slight excess of TBSCl was added slowly in portions. The mixture was stirred at room temperature for 2 hours. After completion, the reaction was quenched by adding a saturated aqueous solution of sodium bicarbonate. The organic layer was separated and washed with brine, then dried over anhydrous magnesium sulfate. The drying agent was removed by filtration, and the solvent evaporated in vacuo. The crude product was purified and then dried under vacuum until a constant weight was achieved, yielding the desired 6’-OTBS protected morpholino C-monomer product in 5.4 gm (90 %) yield. The product's purity was determined to be 99.4% by HPLC. Example 1B: N-trityl deprotection of N-trityl 6'-OTBS protected morpholino C-monomermorpholino C-monomer from Example 1A. Dry dichloromethane (DCM) was added to the flask followed by a catalytic amount of p-toluenesulfonic acid (TsOH), and the mixture stirred at room temperature for 4 hours. After completion, the reaction was quenched by adding a saturated aqueous solution of sodium bicarbonate to neutralize the acid. The organic layer was separated and washed with brine, then dried over anhydrous magnesium sulfate. The drying agent was removed by filtration, and the solvent evaporated in vacuo. The crude product was purified and then dried under vacuum until a constant weight was achieved, yielding the N-deprotected 6’- OTBS protected morpholino C-monomer product in 2.1 gm (64.8 %) yield. The product's purity was determined to be 89.8 % by HPLC.Example 1C: Coupling with N-trityl protected chlorophosphoramidate A-monomerprotected morpholino C-monomer product and dry dichloromethane (DCM). An excess of the N- trityl protected chlorophosphoramidate A-monomer was added to the flask, followed by DBU and the additive lithium bromide (LiBr). The solution was allowed to stir at room temperature for 3 hours. After completion, the reaction was quenched by adding a small volume of water. The organic layer was separated and washed with brine, then dried over anhydrous magnesium sulfate. The drying agent was removed by filtration, and the solvent evaporated in vacuo. The crude product was purified and then dried under vacuum until a constant weight was achieved, yielding the desired phosphorodiamidate C-A-dimer product in 2.8 gm (55.1 %) yield. The product's purity was determined to be 99.3 % by HPLC. Example 1D: N-trityl deprotection of phosphorodiamidate C-A-dimerprocedure and work-up as detailed for the N-trityl deprotection in Example 1B yielded the N-deprotected phosphorodiamidate C-A-dimer product in 2.1 gm (64.8 %) yield. The product's purity was determined to be 89.8 % by HPLC. Example 1E: Coupling with N-trityl protected chlorophosphoramidate G-monomer

[0389] Reaction scheme and conditions are shown in FIG.15.

[0390] Coupling of N-deprotected phosphorodiamidate C-A-dimer with N-trityl protected chlorophosphoramidate G-monomer by following the reaction procedure and work-up as detailed for the coupling reaction of Example 1C yielded the N-deprotected phosphorodiamidate CAG- trimer product in 1.1 gm (64.8 %) yield. The product's purity was determined to be 89.8 % by HPLC. Example 1F: 6’-OTBS deprotection of CAG-trimer

[0391] Reaction scheme and conditions are shown in FIG.16.

[0392] A clean, dry round-bottom flask was charged with 1.1 g of the 6'-TBS protected CAG trimer and dry dichloromethane (DCM). An excess of triethylamine trihydrofluoride (TEA·3HF) was added to the flask. The solution was allowed to stir at room temperature for 12 hours. After completion, the reaction was quenched by adding a small volume of saturated aqueous sodium bicarbonate solution. The organic layer was separated and washed with brine, then dried over anhydrous magnesium sulfate. The drying agent was removed by filtration, and the solvent evaporated in vacuo. The crude product was purified and then dried under vacuum until a constant weight was achieved, yielding the desired deprotected 6’-OH CAG trimer product in 0.6 gm yield. The product's purity was determined to be >97% by HPLC. Example 2. Preparation of Activated 6’-acid-CAG-trimer

[0393] Reaction scheme and conditions are shown in FIG.17.

[0394] A clean, dry round-bottom flask was charged with 500 mg of 6’-OH-CAG-trimer and pyridine as the solvent. To the flask, stoichiometric amount of succinic anhydride and a catalytic amount of 4-dimethylaminopyridine (DMAP) was added and the reaction mixture stirred at room temperature for 16 hours. Upon completion, the reaction mixture was quenched. Wok-up and purification of the crude reaction mixture furnished the product in 293 mg yield after lyophilization.Example 3. Preparation and Determination of Loading of CAG Resin Example 3A: Preparation of CAG Resin

[0395] Reaction scheme and conditions are shown in FIG.18.

[0396] This procedure was performed in a 6 mL syringe-type solid-phase synthesis empty column. the column was connected to the solid phase extractor to allow N2 to through the column or a vacuum extraction.

[0397] The resin treatment / wash steps in the following procedure consist of two basic operations: resin shaken and solvent / solution extraction. For resin shaken, the stopcock was positioned to allow N2flow up through the column and the specified resin treatment / wash was added to the column and allowed to permeate and completely wet the resin. Mixing was then started and the resin slurry mixed for the specified time. For solvent / solution extraction, mixing and N2 flow were stopped and the vacuum pump was started and then the stopcock was positioned to allow evacuation of resin treatment / wash to waste. All resin treatment / wash volumes were 20mL / g of resin unless noted otherwise.

[0398] To aminomethyl polystyrene resin (100-200 mesh; 0.72 mmol / gm N2 substitution; 200 mg, 1 eq, SUNRESIN New Materials Co., Ltd, Xi’an; Catalog No: LXSS5-1-1202) in a 6 mL syringe-type solid-phase synthesis empty column was added 1-methly-2-pyrrolidinone (NMP) and the resin was allowed to swell with mixing for 1 hour. The swell solvent was then removed and the resin washed with dichloromethane (2 x 1-2 min), 5% diisopropylethylamine in 25% isopropanol / dichloromethane (2 x 1-2 min) and dichloromethane (2 x 1-2 min). after evacuation of the final wash, the resin was shaken with a solution of 6’-acid-CAGtrimer (1.5 eq), HBTU (273 mg, 720 μmol, 5 eq), HOBt (97.4 mg, 721 μmol, 5 eq) and NEM (249 mg, 2.16 mmol, 274 μL, 15 eq) in 1-methly-2-pyrrolidinone (2 mL) and the resin / reagent mixture was heated at 35 °C for 16 hrs. On reaction completion, heating was discontinued, and the anchor solution was evacuated and the resin washed with 1-methly-2-pyrrolidinone (4 x 1-2 min) and dichloromethane (6 x 1-2 min). To the resin was added a solution of benzoic anhydride (0.4 M) and NEM (0.4 M). after 25 min, the resin washed with 5% diisopropylethylamine in 25% isopropanol / dichloromethane (3 x 1-2 min) and dichloromethane (5 x 1-2 min). the resin was filtered and dried in a N2 stream for 1 hour and then under high vacuum to constant weight. The product was obtained in 356 mg yield.Example 3B: Determination of the Loading of CAG resin

[0399] The loading of the resin (number of potentially available reactive sites) was determined by a spectrometric assay for the number of triphenylmethyl (trityl) groups per gram of resin. A known weight of dried resin (10±3 mg) was transferred to a silanized 25 mL volumetric flask and ~5mL of 3% (v / v) trifluoracetic acid in dichloromethane is added. The contents were mixed by gentle swirling and then allowed to stand for 30 min. The volume was brought up to 10mL with additional 3% (v / v) trifluoracetic acid in dichloromethane and the contents thoroughly mixed. Using a positive displacement pipette, an aliquot of the trityl-containing solution (500 μL) was transferred to a 10 mL volumetric flask and the volume brought up to 10 mL with methanesulfonic acid. The trityl cation content in the final solution was measured by UV absorbance at 404 nm and the resin loading calculated in trityl groups per gram resin (μmol / gm) using the appropriate volumes, dilutions, extinction coefficient (32.5 umol-1cm-1) and resin weight. The assay was performed in triplicate and an average loading calculated. A loading of 312 μmol / gm was obtained. Example 4. Functionalization of 6’-OH CAG Trimer

[0400] Reaction scheme and conditions are shown in FIG.19.

[0401] General procedure: The reaction was optimized with respect to different variable as presented in the following Table 4. Generally, a clean, dry round-bottom flask was charged with 6'-OH CAG trimer and dissolved in the indicated solvent (dichloromethane (DCM) or trimethyl phosphate (TMP)). The flask was cooled to the indicated temperature (Temp-1), and 2,6-lutidine or proton sponge was added slowly under stirring. Phosphorus oxychloride (POCl₃) was then added dropwise, maintaining the temperature at the indicated temperature (Temp-1). The mixture was stirred at this temperature for the indicated time (Time-1). After completion of the phosphorylation step, dimethylamine was added dropwise to the reaction mixture while maintaining the temperature at the indicated temperature (Temp-2). The solution was stirred for an additional time (Time-2) to allow amination to occur. The product formation was monitored to analyze for crude product formation.Table 4. Reaction Conditions Investigated No. POCl3 in2,6-Lutidine Proton Temp-1 Time-1 DMA Temp-2 Time-2 Crude Solvent(eq) Sponge (oC) (h) (eq) (oC) (h) Product ct ct ct ctExample 5. Solid Phase Synthesis and Purification of CAG Multimer Example 5A: Solid Phase Synthesis of CAG Multimerμmol / gm loading) at 200 mg scale (starting resin weight). Solution used were as follows: Coupling Solution: 0.2 M CAG activated trimer, 0.4 M NEM in DMI; Deprotection Solution: 2.02 gm 4- cyanopyridine, 158 mL DCM, 1.42 mL TFA, 39.0 mL TFE, 2.00 mL EtOH; Neutralization Solution: 35.3 mL isopropyl alcohol, 7.50 mL DIPEA, 107 mL DCM.

[0403] After transfer of the resin to the synthesis reactor and prior to initiating synthesis cycles, N-methly-2-pyrrolidinone (NMP) was added and allowed to sit for 1 hr. after washing 2 times with dichloromethane (10 mL / gm resin), the synthesis cycle in Table 5 was used with addition of the coupling solution of activated morpholino subunit of the desired base and desired linkage type at each cycle to give the proper sequence. Coupling reaction was monitored by chloranil test. Table 5. Synthesis Conditions for CAG Trimer One C cle StepVolume (mL / mmol ofTime FrequencDCM 50 Vol. 1 min 1 30 % TFE / DCM 50 Vol. 1 min 2 t

[0404] , ashed with DCM (3 x 1 min), then drained; washed with 30 % TFE / DCM (2 x 1 min), then drained; treated with CYTFA solution (3 x 10-15 min), then drained; washed with DCM (1 x 1 min) and drained; washed with the neutralization solution (2 x 1 min), then drained; washed with DCM (3 x 1 min) and dried under vacuum at room temperature for 0.5 h to a dried weight of 620 mg. The dried resin was washed 8 times with NMP and then treated with a cleavage solution of 0.1 M 1,4-dithiothreitol (DTT) and 0.73 M DBU in NMP (3 mL / gm starting resin) for 4 hrs. after collection of the protected oligomer solution, the resin (significantly reduced in volume) was washed with two additional portions of the cleavage solution (1 mL / gm starting resin for 15 min each) and the washes were combined with the bulk solution. To the protected oligomer solution in an appropriately sized pressure bottle with a Teflon plug was added concentrated aqueous ammonia (15 mL / gm starting resin), the bottle sealed, and the contents mixed by swirling. The bottle and its contents were heated to a temperature of 45 °C for about 16-20 hrs to remove base and backbone protecting groups. Following ammonolysis, the crude oligomer solution (63.82 % purity by IP-RP-HPLC) was cooled to room temperature and then diafiltered against 0.28 % aqueous ammonia using a 3KD regenerated cellulose membrane to remove solvents and small molecules prior to ion exchange chromatography. Example 5B: Purification of CAGMultimer by Anion Exchange Chromatography

[0405] The crude oligomer solution obtained from diafiltration in Example 4A was adjusted to pH 11-12 and loaded onto a column of SOURCE 15Q anion exchange resin (CYTIVA). The oligomer was eluted with a gradient of 0-48 % B over 18.9 column volume (Buffer A: 10 mM sodium hydroxide in 20% acetonitrile (water:acetonitrile = 80:20); Buffer B: 10 mM sodium hydroxideand 0.6 M sodium chloride in 20 % acetonitrile (water:acetonitrile = 80:20)) and fractions were pooled. The purified drug substance solution was desalted and lyophilized to obtain the purified CAG21-mer product. Example 6: Synthesis of CAG Multimer with purity analysis of each cycle

[0406] Reaction scheme and conditions are shown in FIG.20.

[0407] Following the reaction procedure as detailed in Example 5, a CAG multimer was prepared. Table 6 shows the 6 coupling cycles for the preparation of the 21-mer product. The final purity was determined to be ~20 % by HPLC. After 6 cycles, the coupling efficiency was found to be ~80 %. Table 6. Coupling Cycle Conditions for CAG Multimer Cycle Coupling condition Result y y y y y yExample 7: Optimization of Synthesis of CAG Multimer

[0408] The CAG 21-mer product was prepared under different conditions to optimize the reaction conditions. Table 7 shows various conditions and the purity of the product achieved. Table 7. Reaction Conditions and Purity of CAG Multimer Run CAG NEMSolvent Temp. Time AdditivePurity C)Run CAG NEMSolvent Temp. Time AdPurity No. Trimerconc.ditive(IP-RP-HPLC)Example 8 (Comparative Example): Comparison with conventional monomer synthesis

[0409] The general reaction scheme for conventional monomer synthesis is shown in FIG.21.

[0410] The CAG 21-mer product was also synthesized by the conventional method with 20 sequential coupling cycles. The coupling cycles were performed with each subsequent monomer in 0.1 M (2.5 eq) concentration, with 0.4 M NEM in DMI (~20 V). The reactions were performed at 35oC with each cycle taking about 4 hours of time. The product from Example 8 was compared with the product from Run No.2 of Example 7 by IP-RP-HPLC. The results are presented in Table 8. The comparison of the 21-mer product synthesized by the conventional monomer strategy with the 21-mer product synthesized by the currently disclosed methods showed that the convention method is inferior with respect to the crude purity, the purification yield, and the FLP purity. Table 8. Results of Monomer Method vs. Multimer Method of PMO Synthesis ConventionalMM hMultimer MethodExample 9. Preparation and Determination of Loading of CTG Resin Example 9A: Synthesis of CTG Resin

[0411] The reaction scheme and conditions are shown in FIG.22.

[0412] This procedure was performed in a 6 mL syringe-type solid-phase synthesis empty column. the column was connected to the solid phase extractor to allow N2 to through the column or a vacuum extraction.

[0413] The resin treatment / wash steps in the following procedure consist of two basic operations: resin shaken and solvent / solution extraction. For resin shaken, the stopcock was positioned to allow N2flow up through the column and the specified resin treatment / wash was added to the column and allowed to permeate and completely wet the resin. Mixing was then started, and the resin slurry mixed for the specified time. For solvent / solution extraction, mixing and N2 flow were stopped, and the vacuum pump was started and then the stopcock was positioned to allow evacuation of resin treatment / wash to waste. All resin treatment / wash volumes were 20mL / g of resin unless noted otherwise.

[0414] To aminomethyl polystyrene resin (100-200 mesh; 0.72 mmol / gm N2 substitution; 200 mg, 1 eq, SUNRESIN New Materials Co., Ltd, Xi’an; Catalog No: LXSS5-1-1202) in a 6 mL syringe-type solid-phase synthesis empty column was added 1-methly-2-pyrrolidinone (NMP) and the resin was allowed to swell with mixing for 1 hour. The swell solvent was then removed, and the resin washed with dichloromethane (2 x 1-2 min), 5% diisopropylethylamine in 25% isopropanol / dichloromethane (2 x 1-2 min) and dichloromethane (2 x 1-2 min). after evacuation of the final wash, the resin was shaken with a solution of 6’-acid-CTG trimer (341 mg, 216 μmol, 1.5 eq), HBTU (273 mg, 720 μmol, 5 eq), HOBt (97.4 mg, 721 μmol, 5 eq) and NEM (249 mg, 2.16 mmol, 274 μL, 15 eq) in 1-methyl-2-pyrrolidinone (2 mL) and the resin / reagent mixture was heated at 35 °C for 16 hrs. On reaction completion, heating was discontinued, and the anchor solution was evacuated, and the resin washed with 1-methyl-2-pyrrolidinone (4 x 1-2 min) and dichloromethane (6 x 1-2 min). To the resin was added a solution of benzoic anhydride (0.4 M) and NEM (0.4 M). after 25 min, the resin washed with 5% diisopropylethylamine in 25% isopropanol / dichloromethane (3 x 1-2 min) and dichloromethane (5 x 1-2 min). the resin was filtered and dried under a N2stream for 1 hour and then under high vacuum to constant weight. A yield of 150-200% of the original resin weight was obtained. Example 9B:Determination of the Loading of CTG resin

[0415] The loading of the resin (number of potentially available reactive sites) was determined by a spectrometric assay for the number of triphenylmethyl (trityl) groups per gram of resin. A knownweight of dried resin (10±3 mg) was transferred to a silanized 25 mL volumetric flask and ~5mL of 3% (v / v) trifluoracetic acid in dichloromethane is added. The contents were mixed by gentle swirling and then allowed to stand for 30 min. The volume was brought up to 10mL with additional 3% (v / v) trifluoracetic acid in dichloromethane and the contents thoroughly mixed. Using a positive displacement pipette, an aliquot of the trityl-containing solution (500 μL) was transferred to a 10 mL volumetric flask and the volume brought up to 10 mL with methanesulfonic acid. The trityl cation content in the final solution was measured by UV absorbance at 404 nm and the resin loading calculated in trityl groups per gram resin (μmol / gm) using the appropriate volumes, dilutions, extinction coefficient (32.5 umol-1cm-1) and resin weight. The assay was performed in triplicate and an average loading calculated. The resin loading procedure in this example provided resin with a loading of approximately 700 μmol / gm. A loading of 300-400 μmol / gm was obtained. Example 10: Solid Phase Synthesis and Purification of CTG Multimer Example 10A: Solid Phase Synthesis of CTG Multimer

[0416] Protected oligomers were prepared by solid phase oligomer synthesis on CTG resin (~345 μmol / gm loading) at 200 mg scale (starting resin weight). Solution used were as follows: Coupling Solution: 0.2 M CTG activated trimer, 0.4 M NEM in DMI; Deprotection Solution: 2.02 gm 4- cyanopyridine, 158 mL DCM, 1.42 mL TFA, 39.0 mL TFE, 2.00 mL EtOH; Neutralization Solution: 35.3 mL isopropyl alcohol, 7.50 mL DIPEA, 107 mL DCM.

[0417] After transfer of the resin to the synthesis reactor and prior to initiating synthesis cycles, 1-methly-2-pyrrolidinone (NMP) was added and allowed to sit for 1 hr. after washing 2 times with dichloromethane (10 mL / gm resin), the synthesis cycle in Table 9 was used with addition of the coupling solution of activated morpholino subunit of the desired base and desired linkage type at each cycle to give the proper sequence. Table 9. One cycle stepVolume (mL / mmol ofstarting resin) Time (min) Frequencyt

[0418] After the final coupling step was performed as described above, the resin was washed with DCM (3 x 1 min), then drained; washed with 30 % TFE / DCM (2 x 1 min), then drained; treated with CYTFA solution (3 x 10-15 min), then drained; washed with DCM (1 x 1 min) and drained; washed with the neutralization solution (2 x 1 min), then drained; washed with DCM (3 x 1 min) and dried under vacuum at room temperature for 0.5 h to a dried weight of 620 mg. The dried resin was washed 8 times with NMP and then treated with a cleavage solution of 0.1 M 1,4-dithiothreitol (DTT) and 0.73 M DBU in NMP (3 mL / gm starting resin) for 4 hrs. after collection of the protected oligomer solution, the resin (significantly reduced in volume) was washed with two additional portions of the cleavage solution (1 mL / gm starting resin for 15 min each) and the washes were combined with the bulk solution. To the protected oligomer solution in an appropriately sized pressure bottle with a Teflon plug was added concentrated aqueous ammonia (15 mL / gm startingresin), the bottle sealed, and the contents mixed by swirling. The bottle and its contents were heated to a temperature of 45 °C for about 16-20 hrs. to remove base and backbone protecting groups. Following ammonolysis, the crude oligomer solution (63.82 % purity by IP-RP-HPLC) was cooled to room temperature and then diafiltered against 0.28 % aqueous ammonia using a 3KD regenerated cellulose membrane to remove solvents and small molecules prior to ion exchange chromatography. Example 10B: Purification of CTG Multimer by Anion Exchange Chromatography

[0419] The crude oligomer solution obtained from diafiltration in Example 4A was adjusted to pH 11-12 and loaded onto a column of SOURCE 15Q anion exchange resin (CYTIVA). The oligomer was eluted with a gradient of 0-48 % B over 18.9 column volume (Buffer A: 10 mM sodium hydroxide in 20% acetonitrile (water:acetonitrile = 80:20); Buffer B: 10 mM sodium hydroxide and 0.6 M sodium chloride in 20 % acetonitrile (water:acetonitrile = 80:20)) and fractions were pooled. The purified drug substance solution was desalted and lyophilized to obtain the purified CTG 21mer in 118 mg (22.6 %) yield. The product’s purity was determined to be 97.90 % by IP- RP-HPLC. Example 11: Phosphorylation of 6’-OH morpholino substrate

[0420] The reaction scheme and conditions are shown in FIG.23.

[0421] Phosphorylation of 6’-OH-CAG trimer substrate was performed with the variables as shown in Table 6. A clean, dry round-bottom flask was charged with the indicated amount of the of 6’-OH-CAG trimer and pyridine added to it. Next, diphenylphosphite was added dropwise to the flask while stirring at 0-5 °C. The solution was allowed to stir for 2 hours at 0-5 °C. After completion, triethylamine (TEA) and deionized water were added sequentially, and the reaction mixture was stirred for an additional 2 hours at room temperature. The crude product was analyzed product purity by RP-HPLC as indicated in Table 10. Table 10. Reaction Conditions and Purity of CAG Morpholino Substrate Run 6’-OH-CrudePurification by RP-ts1) Diphenylphosphite, NH4HCO3: ~25 % Pyridine, 0-5oC, 2 h Detritylation 99.2 % ,Example 12. H-Phosphonate Coupling and Oxidative Amination

[0422] The reaction scheme and conditions are shown in FIG.24.

[0423] Coupling of 6’-H-phosphonate substrate with solid supported N-deprotected morpholino substrate to furnish the H-phosphonamidate product followed by oxidative amination of the H- phosphonamidate to prepare the phosphorodiamidate product was performed with the variables as shown in Table 11. A clean, dry round-bottom flask was charged with the indicated amount of the N-deprotected morpholino substrate (Resin CAG trimer) and 20 volume equivalents of a 1:1 mixture of acetonitrile (MeCN) and pyridine (Py) as solvent. 6'-H-phosphonate substrate (5 equivalents) was added to the flask, followed by the indicated equivalents of the coupling agent. The solution was allowed to stir at 25oC temperature for 30 minutes. Samples were taken during the reaction to analyze the progress of the reaction. After 30 minutes, the reaction was quenched. Work-up and purification of the reaction mixture furnished the H-phosphonamidate product as indicated. Next, the indicated amounts of halogenating agent and 20 volume equivalents of a 1:1 mixture of acetonitrile (MeCN) and pyridine (Py) as solvent were added sequentially to the flask at the indicated temperature with stirring. The reaction was allowed to proceed before the addition of the aminating agent (dimethylamine in THF (40 equivalents) and the reaction mixture allowed to stir at the indicated temperature for the indicated time. Samples were taken during the reaction to analyze the progress of the reaction. After completion, the reaction was quenched. Work-up and purification of the reaction mixture furnished the results as indicated. Only coupling product was obtained in all the runs.Table 11. Coupling Reaction Conditions Amination Run Resin Reaction CAG Coupling HalogenatinResult20 V; Temperature 25oC; Time 30 min. Amination Reaction Conditions: Aminating Agent: Dimethylamine in THF (40 equiv). Example 13. Phosphorylation of 6’-OH morpholino substrate

[0425] The reaction scheme and conditions are shown in FIG.25.

[0426] Phosphorylation of 6’-OH-CAG trimer substrate was performed with the variables as shown in Table 12. A clean, dry round-bottom flask was charged with the indicated amount of the of 6’-OH-CAG trimer and the solvent added to it. Next, the activator was added to the flask while stirring at 10-20 °C. The solution was allowed to stir for 1 hour at 0-20 °C. The crude product was analyzed by31P-NMR and UPLC.Table 12. Phosphorylation Conditions and Crude Purity Run 6’-OH- No. CAG Conditions Crude purityExample 14. Phosphoramidite Coupling and Oxidative Amination

[0427] The reaction scheme and conditions are shown in FIG.26.

[0428] Coupling of the 6’-phosphoramidite substrate with solid supported N-deprotected morpholino substrate to furnish the phosphoramidite product followed by oxidative amination of the phosphoramidite to prepare the phosphorodiamidate product was performed with the variables as shown in Table 9. A clean, dry round-bottom flask was charged with the indicated amount of the N-deprotected morpholino substrate (Resin CAG trimer), and the solvent as indicated in Table 13.6'-Phosphoramidite substrate (5 equivalents) was added to the flask, followed by the indicated equivalents of the coupling agent. The solution was allowed to stir at 35oC temperature for 1 hour minutes. Samples were taken during the reaction to analyze the progress of the reaction. After 1 hour, the reaction was quenched. Work-up and purification of the reaction mixture furnished the phosphoramidite product. Next, the indicated amounts of amination reaction reagents were added sequentially to the flask at the indicated temperature and the reaction mixture was allowed to stir at the 35oC for the indicated time. Samples were taken during the reaction to analyze the progress of the reaction. After completion, the reaction was quenched. Work-up and purification of the reaction mixture furnished the results as indicated. Only coupling product was obtained in all the runs.Table 13. Coupling Conditions for Phosphoramidite Coupling Run Resin C Amination Reaction Conditions CAG oupling No.AgentSolventReagents TimeResultng ng ng ngngngngngngngngng ng50 mg BTT0.5 M I2 in THF:Py = 7:2, Only coupling (30 eq)MeCN (10 V)v:v, 18 V + 2M NMe2in 10 min product

Claims

CLAIMS 1. A method for synthesizing a phosphorodiamidate morpholino oligomer (PMO), the method comprising: (a) synthesizing a first PMO multimer, wherein synthesizing the first PMO multimer comprises: (i) coupling a morpholino monomer to a first morpholino nucleotide monomer comprising a phosphor-terminus to form a morpholino nucleotide multimer comprising a phosphorodiamidate morpholino dimer; (b) coupling the first PMO multimer in (a) with one or more additional PMO multimers to form an extended PMO.

2. The method of claim 1, wherein (a) further comprises coupling the phosphorodiamidate morpholino dimer to a second morpholino nucleotide monomer comprising a phosphor- terminus to form a morpholino nucleotide multimer comprising a phosphorodiamidate morpholino trimer.

3. The method of claim 1, 2, wherein (a) further comprises coupling the phosphorodiamidate morpholino trimer to a third morpholino nucleotide monomer comprising a phosphor-terminus to form a morpholino nucleotide multimer comprising a phosphorodiamidate morpholino tetramer.

4. The method of claim 1, wherein each of the one or more additional PMO multimers are formed as in (a).

5. The method of any of claims 1 to 4, wherein the first PMO multimer and each of the additional PMO multimers consist of the same nucleotide sequence.

6. The method of any one of claims 1 to 5, wherein the first morpholino nucleotide monomer is protected at the 5’-OH.

7. The method of any one of claims 1 to 6, further comprising functionalizing the first PMOmultimer of step (a) or the second PMO multimer of step (b).

8. The method of claim 7, wherein the second PMO multimer of step (b) is functionalized and wherein the method further comprises coupling the functionalized second phosphorodiamidate morpholino multimer in (b) to the first phosphorodiamidate morpholino multimer of (a) to from the extended PMO.

9. The method of claim 7, wherein the first phosphorodiamidate morpholino multimer of step (a) is functionalized and wherein the method further comprises attaching the functionalized first phosphorodiamidate morpholino multimer of (a) to a solid support.

10. The method of claim 9, wherein the solid support comprises a polystyrene resin.

11. The method of claim 9 or 10, wherein first phosphorodiamidate morpholino multimer of (a) is conjugated to the solid support at the 5’ end.

12. The method of any one of claims 9 to 11, further comprising cleaving the PMO comprising the extended PMO from the solid support.

13. The method of any one of claims 1 to 12, further comprising coupling the extended PMO with one or more additional phosphorodiamidate morpholino (PMO) monomers at the 3’ end, the 5’ end, or both the 3’ end and the 5’ end of the extended PMO.

14. The method of claim 13, wherein the additional PMO monomer is functionalized and attached to a solid support.

15. The method of any one of claims 1 to 14, wherein the phosphorodiamidate morpholino oligomer (PMO) comprises the first phosphorodiamidate morpholino multimer and at least one additional phosphorodiamidate morpholino multimer and comprises the following sequence: Xp(M1-M2-M3-M4)xYq, wherein: M1and M2are each independently C, A, U, or G; M3and M4are each independently C, A, U, G or are absent; each X andeach Y are each independently C, A, U or G; p and q are each independently an integer from 0 to 3; and x is an integer from 1 to 10.

16. The method of any one of claims 1 to 14, wherein the phosphorodiamidate morpholino oligomer (PMO) comprises a dinucleotide repeat and comprises the following sequence: Xp(M1-M2)xYq, wherein: M1and M2are each independently C, A, U, or G; each X and each Y are each independently C, A, U or G; p and q are each independently an integer from 0 to 1; and x is an integer from 1 to 10.

17. The method of any one of claims 1 to14, wherein the phosphorodiamidate morpholino oligomer (PMO) comprises a trinucleotide repeat and comprises the following sequence: Xp(M1-M2-M3)xYq, wherein: M1, M2, and M3are each independently C, A, U, or G; each X and each Y are each independently C, A, U or G; p and q are each independently an integer from 0 to 2; and x is an integer from 1 to 10.

18. The method of any one of claims 1 to 14, wherein the phosphorodiamidate morpholino oligomer (PMO) comprises a tetranucleotide sequence and comprises the following sequence: Xp(M1-M2-M3-M4)xYq, wherein: M1, M2, M3, and M4are each independently C, A, U, or G; each X and each Y are each independently C, A, U or G; p and q are each independently an integer from 0 to 3; and x is an integer from 1 to 10.

19. The method of any one of claims 1 to 18, wherein the phosphorodiamidate morpholino oligomer (PMO) comprises from 3 to 20, 3 to 15, 3 to 10, 3 to 8 or 4 to 6 nucleotide repeats.

20. The method of any one of claims 1 to 18, wherein the phosphorodiamidate morpholino oligomer (PMO) comprises 3, 4, 5, 6, 7, 8, 9 or 10 nucleotide repeats.

21. The method of any one of claims 1 to 15, 17, 19 or 20, wherein the phosphorodiamidate morpholino oligomer (PMO) comprises a nucleotide sequence that is complementary to a (CUG) expanded repeat in a target RNA sequence.

22. The method of any one of claims 1 to 15, 17, 19 or 20, wherein the phosphorodiamidate morpholino oligomer (PMO) comprises a nucleotide sequence that is complementary to a (CAG) expanded repeat in a target RNA sequence.

23. The method of claim 15, wherein the phosphorodiamidate morpholino oligomer (PMO) comprises the following sequence: Xp(CAG)xYq, wherein: each X and each Y are each independently C, A or G; p and q are each independently an integer from 0 to 2; and x is an integer from 1 to 10.

24. The method of claim 16, wherein the phosphorodiamidate morpholino oligomer (PMO) comprises a sequence of (CAG)x, G(CAG)x, AG(CAG)x, (CAG)xC, (CAG)xCA, G(CAG)xC, G(CAG)xCA, AG(CAG)xC, AG(CAG)xCA, or combinations thereof, wherein x is an integer from 1 to 10.

25. The method of claim 22, wherein the phosphorodiamidate morpholino oligomer (PMO) comprises Xp(CUG)xYq, wherein: each X and each Y are each independently C, U or G; p and q are each independently an integer from 0 to 2; and x is an integer from 1 to 10.

26. The method of claim 25, wherein the phosphorodiamidate morpholino oligomer (PMO) comprises a sequence of (CUG)x, G(CUG)x, UG(CUG)x, (CUG)xC, (CUG)xCU, G(CUG)xC, G(CUG)xCU, UG(CUG)xC, UG(CUG)xCU, or combinations thereof, wherein x is an integer from 1 to 10.

27. The method of any one of claims 1 to 20, wherein the phosphorodiamidate morpholino oligomer (PMO) comprises a nucleotide sequence that is complementary to a (GAA) expanded repeat in a target RNA sequence.

28. The method of claim 27, wherein the phosphorodiamidate morpholino oligomer (PMO) comprises the following sequence: Xp(UUC)xYq, wherein: each X and each Y are each independently U or C; p and q are each independently an integer from 0 to 2; and x is aninteger from 1 to 10.

29. The method of claim 28, wherein the phosphorodiamidate morpholino oligomer (PMO) comprises a sequence of (UUC)x, C(UUC)x, UC(UUC)x, (UUC)xU, (UUC)xUU, C(UUC)xU, C(UUC)xUU, UC(UUC)xU, UC(UUC)xUU, or combinations thereof, wherein x is an integer from 1 to 10.

30. The method of any one of claims 1 to 20, wherein the phosphorodiamidate morpholino oligomer (PMO) comprises a nucleotide sequence that is complementary to a (CGG) expanded repeat in a target RNA sequence.

31. The method of claim 30, wherein the phosphorodiamidate morpholino oligomer (PMO) comprises a sequence of Xp(CCG)xYq, wherein: each X and each Y are each independently C or G; p and q are each independently an integer from 0 to 2; and x is an integer from 1 to 10.

32. The method of claim 31, wherein the phosphorodiamidate morpholino oligomer (PMO) comprises a sequence of (CCG)x, G(CCG)x, CG(CCG)x, (CCG)xC, (CCG)xCC, G(CCG)xC, G(CCG)xCC, CG(CCG)xC, CG(CCG)xCC, or combinations thereof, wherein x is an integer from 1 to 10.

33. The method of any one of claims 1 to 20, wherein the phosphorodiamidate morpholino oligomer (PMO) comprises a nucleotide sequence that is complementary to a (GCG) expanded repeat in a target RNA sequence.

34. The method of claim 33, wherein the phosphorodiamidate morpholino oligomer (PMO) comprises a sequence of Xp(CGC)xYq, wherein: each X and each Y are each independently C or G; p and q are each independently an integer from 0 to 2; and x is an integer from 1 to 10.

35. The method of claim 34, wherein the phosphorodiamidate morpholino oligomer (PMO)comprises a sequence of (CGC)x, C(CGC)x, GC(CGC)x, (CGC)xC, (CGC)xCG, C(CGC)xC, C(CGC)xCG, GC(CGC)xC, GC(CGC)xCG, or combinations thereof, wherein x is an integer from 1 to 10.

36. The method of any one of claims 1 to 18, wherein the phosphorodiamidate morpholino oligomer (PMO) comprises a nucleotide sequence that is complementary to a (CCUG) expanded repeat in a target RNA sequence.

37. The method of claim 36, wherein the phosphorodiamidate morpholino oligomer (PMO) comprises a nucleotide sequence of Xp(CAGG)xYq, wherein: each X and each Y are each independently C, A or G; p and q are each independently an integer from 0 to 3; and x is an integer from 1 to 10.

38. The method of claim 37, wherein the phosphorodiamidate morpholino oligomer (PMO) comprises a sequence of (CAGG)x, G(CAGG)x, GG(CAGG)x, AGG(CAGG)x, (CAGG)xC, (CAGG)xCA, (CAGG)xCAG, G(CAGG)xC, G(CAGG)xCA, G(CAGG)xCAG, GG(CAGG)xC, GG(CAGG)xCA, GG(CAGG)xCAG, AGG(CAGG)xC, AGG(CAGG)xCA, AGG(CAGG)xCAG, or combinations thereof, wherein x is an integer from 1 to 10.

39. The method of any one of claims 1 to 38, wherein the PMO comprises a phosphoro- terminus, and wherein the phosphoro-terminus is a phosphorodiamidate.

40. The method of any one of claims 1 to 38, wherein the PMO comprises a phosphoro- terminus, and wherein the phosphoro-terminus is a phosphoric acid.

41. The method of any one of claims 1 to 38, wherein the PMO comprises a phosphoro- terminus, and wherein the phosphoro-terminus is a phosphonamidite.

42. A method for elongating a growing chain during synthesis of a PMO, the method comprising:reacting a deprotected morpholino oligonucleotide unit attached to a solid support a morpholino multimer comprising the following structure: , whereinLG is a leaving group; R1and R2are each independently H or substituted or unsubstituted alkyl, aryl, alkynyl, alkenyl, cycloalkyl, heterocyclyl, or heteroaryl; Mnis a PMO monomer; (Mn)m-1 is a PMO multimer having m-1 PMO nucleotides; position of the Mnmonomer in the (Mn)m-1multimer sequence; and m is the number of Mnmonomers in the (Mn)mmultimer.

43. The method of claim 42, wherein LG is chloro group.

44. The method of claim 42 or 43, wherein R1and R2are methyl.

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