Polynucleotide compositions and methods for the treatment of neurodegenerative diseases

Abstract

The present disclosure relates to an engineered polynucleotide that interacts with pre-mRNA and spliceosome to regulate gene expression.The engineered polynucleotide can have a stem-loop structure that recruits spliceosome and targeting sequence that is complementary to the target sequence at the exon-intron splice junction, and can include nucleotides with 2' modification and phosphorothioate linkage.The engineered polynucleotide can be administered to a subject to treat neurodegenerative diseases.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 63 / 480,464, filed January 18, 2023, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Abnormal splicing and premature polyadenylation are involved in many disease states. Proper splicing of pre-messenger RNA (pre-mRNA) and suppression of premature polyadenylation are important processes for proper protein translation. Splicing efficiency and suppression of premature polyadenylation must be regulated to ensure proper splicing of pre-mRNA and avoid the production of misfolded and possibly disease-causing proteins. The efficiency of gene regulation at the ribonucleic acid (RNA) level remains limited. Therefore, there is a need to develop polynucleotide compositions and methods for regulating gene expression and activity at therapeutically effective and safe levels, for example. Summary of the Invention

[0003] Described herein, in some embodiments, are methods of treating a neurodegenerative disease in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an engineered polynucleotide, the engineered polynucleotide comprising: (1) one or more targeting moieties configured to specifically bind to a pre-messenger ribonucleic acid (pre-mRNA) at a target sequence therein; and (2) a recruitment moiety configured to recruit a spliceosome moiety, wherein upon association with the pre-mRNA and the engineered polynucleotide, the spliceosome moiety alters the pre-mRNA at or near the target sequence.

[0004] In some embodiments, the present disclosure provides a method for treating a neurodegenerative disease in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an engineered polynucleotide, the engineered polynucleotide comprising one or more targeting moieties configured to specifically bind to a pre-messenger ribonucleic acid (pre-mRNA) at a target sequence therein and a recruitment moiety configured to recruit a spliceosome moiety, wherein upon association with the pre-mRNA and the engineered polynucleotide, the spliceosome moiety alters the pre-mRNA at or near the target sequence. In some embodiments, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Pick's disease, chronic traumatic encephalopathy, progressive supranuclear palsy, argyrophilic grain disease, amyotrophic lateral sclerosis (ALS), and frontotemporal dementia. In some embodiments, the neurodegenerative disease is a neurodegenerative disease associated with the presence of tau in the subject's brain. In some embodiments, tau expression is altered. In some embodiments, tau expression is decreased. In some embodiments, the total amount of tau in the subject's brain is decreased. In some embodiments, the neurodegenerative disease is a neurodegenerative disease associated with the presence of amyloid beta in the subject's brain. In some embodiments, the method alters amyloid beta expression. In some embodiments, the method reduces amyloid beta expression. In some embodiments, the method reduces the total amount of amyloid beta in the subject's brain. In some embodiments, the method reduces the total amount of amyloid plaques in the subject's brain. In some embodiments, the engineered polynucleotide is administered at a concentration of at least 5 nM. In some embodiments, the engineered polynucleotide is administered at a concentration of at least 10 nM. In some embodiments, the engineered polynucleotide is administered at a concentration of about 5 nM to about 20 nM.

[0005] Described herein, in some embodiments, are methods for reducing Tau expression by neurons, the methods comprising administering to the neuron an engineered polynucleotide, wherein the engineered polynucleotide comprises one or more targeting moieties configured to specifically bind to a pre-messenger ribonucleic acid (pre-mRNA) at a target sequence therein and a recruitment moiety configured to recruit a spliceosome moiety, wherein upon association with the pre-mRNA and the engineered polynucleotide, the spliceosome moiety alters the pre-mRNA at or near the target sequence. In some embodiments, the neuron is derived from an individual suffering from a neurodegenerative disorder. In some embodiments, the neuron is Alzheimer's disease. In some embodiments, the neuron is a healthy neuron. In some embodiments, the neuron is derived from a healthy subject. In some embodiments, the engineered polynucleotide is administered at a concentration of at least 5 nM. In some embodiments, the engineered polynucleotide is administered at a concentration of at least 10 nM. In some embodiments, the engineered polynucleotide is administered at a concentration of about 5 nM to about 20 nM.

[0006] In some embodiments, described herein are methods for increasing fiber width in neurons, the methods comprising administering to the neuron an engineered polynucleotide, the engineered polynucleotide comprising one or more targeting moieties configured to specifically bind to a pre-messenger ribonucleic acid (pre-mRNA) at an internal target sequence and a recruitment moiety configured to recruit a spliceosome moiety, wherein upon association with the pre-mRNA and the engineered polynucleotide, the spliceosome moiety alters the pre-mRNA at or near the target sequence. In some embodiments, the neuron is derived from an individual suffering from a neurodegenerative disorder. In some embodiments, the neurodegenerative disease is Alzheimer's disease. In some embodiments, the neuron is a healthy neuron. In some embodiments, the neuron is derived from a healthy subject. In some embodiments, the engineered polynucleotide is administered at a concentration of at least 5 nM. In some embodiments, the engineered polynucleotide is administered at a concentration of at least 10 nM. In some embodiments, the engineered polynucleotide is administered at a concentration of about 5 nM to about 20 nM.

[0007] In some embodiments, described herein are methods for increasing the number of branches in neurons, the methods comprising administering to the neurons an engineered polynucleotide, the engineered polynucleotide comprising one or more targeting moieties configured to specifically bind to a pre-messenger ribonucleic acid (pre-mRNA) at a target sequence therein and a recruitment moiety configured to recruit a spliceosome moiety, wherein upon association with the pre-mRNA and the engineered polynucleotide, the spliceosome moiety alters the pre-mRNA at or near the target sequence. In some embodiments, the neuron is derived from an individual suffering from a neurodegenerative disorder. In some embodiments, the neuron is Alzheimer's disease. In some embodiments, the neuron is a healthy neuron. In some embodiments, the neuron is derived from a healthy subject.

[0008] Described herein, in some embodiments, are methods of increasing electrical activity in neurons, the methods comprising administering to a neuron an engineered polynucleotide, the engineered polynucleotide comprising one or more targeting moieties configured to specifically bind to a pre-messenger ribonucleic acid (pre-mRNA) at a target sequence therein, and a recruitment moiety configured to recruit a spliceosome moiety, wherein upon association with the pre-mRNA and the engineered polynucleotide, the spliceosome moiety alters the pre-mRNA at or near the target sequence.

[0009] In some embodiments, the neuron is derived from an individual suffering from a neurodegenerative disorder. In some embodiments, the neurodegenerative disorder is selected from the group consisting of Alzheimer's disease, Pick's disease, chronic traumatic encephalopathy, progressive supranuclear palsy, argyrophilic grain disease, amyotrophic lateral sclerosis (ALS), and frontotemporal dementia. In some embodiments, the neuron is a healthy neuron. In some embodiments, the neuron is derived from a healthy subject.

[0010] In some embodiments, the method alters expression of a gene in a neuron. In some embodiments, the method alters expression of a gene that is part of the MAPK signaling pathway. In some embodiments, the method alters expression of a gene that is part of the cholesterol metabolic pathway. In some embodiments, the method alters expression of a gene that is part of the steroid biosynthesis pathway. In some embodiments, the method alters expression of a gene that is part of the PI3K-Akt signaling pathway. In some embodiments, the method alters expression of a gene that is part of a cell cycle regulation or cellular senescence pathway. In some embodiments, the method alters expression of a gene that is part of the pyruvate metabolic pathway. In some embodiments, the method alters expression of a gene that is part of the RAS signaling pathway. In some embodiments, the method alters expression of a gene that is part of the AMPK signaling pathway. In some embodiments, the method alters expression of a gene that is part of the fatty acid metabolic pathway. In some embodiments, the method alters expression of a gene that is part of the fatty acid synthesis pathway. In some embodiments, the method alters expression of a gene that is part of the PPAR signaling pathway. In some embodiments, the method alters expression of a gene associated with mRNA transport. In some embodiments, the method increases mRNA transport in a neuron. In some embodiments, the method reduces excitotoxicity in a neuron. In some embodiments, the method increases synaptic integrity in a neuron. In some embodiments, the method reduces premature polyadenylation of one or more transcripts in a neuron.

[0011] In some embodiments, the present specification describes a method for treating a subject with Alzheimer's disease, comprising administering to the subject a pharmaceutical composition comprising an engineered polynucleotide having the nucleotide sequence of SEQ ID NO: 3, thereby modulating pre-mRNA affected by U1 snRNP dysfunction in the subject's brain. In some embodiments, one or more, e.g., all, internucleotide linkages of the engineered polynucleotide comprise phosphorothioate linkages. In some embodiments, nucleotides 1-5 and 20-24 of the engineered polynucleotide comprise 2'-O-methyl moieties. In some embodiments, the pre-mRNA comprises tau, and the method reduces tau protein levels in the brain.

[0012] Described herein, in some embodiments, are methods of reducing amyloid-beta expression and / or aggregation in neurons, the methods comprising administering to neurons an engineered polynucleotide, the engineered polynucleotide comprising (i) one or more targeting moieties configured to specifically bind to a pre-messenger ribonucleic acid (pre-mRNA) at an internal target sequence, which may be a conserved splice site, and (ii) a recruitment moiety configured to recruit a spliceosome moiety, wherein upon association with the pre-mRNA and the engineered polynucleotide, the spliceosome moiety alters the pre-mRNA at or near the target sequence.

[0013] Described herein, in some embodiments, are methods for reducing U1-70K aggregation and / or mislocalization in neurons, the methods comprising administering to neurons an engineered polynucleotide comprising: (i) one or more targeting moieties configured to specifically bind to pre-messenger ribonucleic acid (pre-mRNA) at an internal target sequence; and (ii) a recruitment moiety configured to recruit a spliceosome moiety, wherein upon association with the pre-mRNA and the engineered polynucleotide, the spliceosome moiety alters the pre-mRNA at or near the target sequence.

[0014] Described herein, in some embodiments, are methods of modulating astrogliosis, the methods comprising administering to a neuron an engineered polynucleotide comprising: (i) one or more targeting moieties configured to specifically bind to a pre-messenger ribonucleic acid (pre-mRNA) at a target sequence therein; and (ii) a recruitment moiety configured to recruit a spliceosome moiety, wherein upon association with the pre-mRNA and the engineered polynucleotide, the spliceosome moiety alters the pre-mRNA at or near the target sequence.

[0015] In some embodiments, the engineered polynucleotide is administered intratumorally. In some embodiments, the engineered polynucleotide is administered intravenously. In some embodiments, the engineered polynucleotide is administered intrathecally. In some embodiments, the engineered polynucleotide is administered via subcutaneous injection, intramuscular injection, intradermal injection, transdermal administration, intranasal administration, intralymphatic injection, intrathecal administration, pulmonary administration, rectal administration, intragastric administration, or any other suitable parenteral administration.

[0016] In some aspects, the present specification describes engineered polynucleotides for use in the methods disclosed throughout this disclosure. In some embodiments, the engineered polynucleotide comprises a nucleotide sequence that is at least 70%, 80%, 85%, or 90% identical to or complementary to any one of SEQ ID NOs: 1-4. In some embodiments, the engineered polynucleotide comprises a nucleotide sequence that is identical to or complementary to any one of SEQ ID NOs: 3 or 4.

[0017] Described herein, in some embodiments, are engineered polynucleotides that include one or more targeting moieties configured to specifically bind to a pre-messenger ribonucleic acid (pre-mRNA) at an internal target sequence, and a recruitment moiety configured to recruit a post-transcriptional regulatory moiety (e.g., a spliceosome moiety), wherein, upon association with the pre-mRNA and the engineered polynucleotide, the post-transcriptional regulatory moiety alters the pre-mRNA at or near the target sequence.

[0018] In some embodiments, one targeting moiety of the one or more targeting moieties is sufficiently identical to or complementary to a consensus sequence in the target sequence of the target gene. In some embodiments, the targeting moiety is complementary to and / or hybridizes with the target sequence. In some embodiments, the targeting moiety is complementary to and / or hybridizes with the consensus sequence of the target sequence. In some embodiments, the target sequence comprises a splice site. In some embodiments, the splice site is a conserved splice site. In some embodiments, the splice site comprises 5'-GU-3'. In some embodiments, the pre-mRNA is encoded by the target gene. In some embodiments, the method alters the expression or activity of the target gene.

[0019] In some embodiments, the one or more targeting moieties include a first targeting moiety configured to specifically bind to a first targeting sequence in the target sequence of the pre-mRNA and a second targeting moiety configured to specifically bind to a second targeting sequence in the target sequence of the pre-mRNA. In some embodiments, the first targeting sequence includes a consensus sequence in the target sequence. In some embodiments, the second targeting sequence includes a consensus sequence in the target sequence. In some embodiments, the first targeting sequence and the second targeting sequence are separated in the target sequence by a spacing sequence of 5 nucleotides or less (e.g., 1 or 2 nucleotides). In some embodiments, the target sequence includes an exon-intron boundary in the pre-mRNA. In some embodiments, both the first targeting sequence and the second targeting sequence are 5' or 3' to the exon-intron boundary. In some embodiments, one of the first targeting sequence and the second targeting sequence is 5' to the exon-intron boundary, and the other of the first targeting sequence and the second targeting sequence is 3' to the exon-intron boundary. In some embodiments, the target sequence comprises a splice site in the pre-mRNA. In some embodiments, the first targeting sequence or the second targeting sequence comprises a splice site (e.g., 5'ss) in the pre-mRNA. In some embodiments, one of the first targeting moiety and the second targeting moiety is 5' to the recruitment moiety, and the other of the first targeting moiety and the second targeting moiety is 3' to the recruitment moiety. In some embodiments, the targeting moiety comprises a sequence that is at least 80%, 90%, or identical to the ribosome binding site of a spliceosomal snRNA, e.g., U1 snRNA. In some embodiments, the first targeting moiety comprises a sequence that is at least 80%, 90%, or identical to the ribosome binding site of a spliceosomal snRNA, e.g., U1 snRNA. In some embodiments, the second targeting moiety comprises a sequence that is at least 80%, 90%, or identical to the ribosome binding site of a spliceosomal snRNA, e.g., U1 snRNA.The sequence at least 80%, 90%, or identical to the ribosome binding site can be from about 2 nucleotides to about 10 nucleotides. In some embodiments, the recruitment nucleotide sequence comprises (i) a nucleotide sequence complementary to at least 4 nucleotides of stem-loop II (SL2) of the U1 snRNA.

[0020] In some embodiments, the first targeting moiety or the second targeting moiety comprises a sequence identical to or complementary to a sequence shown in Table 1. In some embodiments, the first targeting moiety comprises a sequence identical to or complementary to a sequence selected from the 5'-targeting moiety sequence column of Table 1, and the second targeting moiety comprises a sequence identical to or complementary to a sequence shown in the 3'-targeting moiety sequence column of Table 1. In some embodiments, the first targeting moiety comprises a sequence identical to or complementary to a sequence shown in the 3'-targeting moiety sequence column of Table 1, and the second targeting moiety comprises a sequence identical to or complementary to a sequence shown in the 5'-targeting moiety sequence column of Table 1. In some embodiments, the first targeting moiety or the second targeting moiety comprises a sequence identical to or complementary to a consensus sequence for an intron donor site (e.g., selected from GU, GT, GC, and CA). In some embodiments, the first targeting moiety or the second targeting moiety comprises a sequence identical to or complementary to a consensus sequence of an exon donor site (e.g., G). In some embodiments, the first targeting moiety or the second targeting moiety comprises a sequence identical to or complementary to a consensus sequence selected from GU, GC, G, and CA. In some embodiments, the spliceosome moiety is selected from a spliceosome ribonucleoprotein complex, a spliceosome small nuclear ribonucleic acid (snRNA), a spliceosome protein, a functional variant thereof, or a functional fragment thereof. In some embodiments, the spliceosome moiety comprises a U1 snRNA and a spliceosome protein. In some embodiments, the spliceosome snRNA is selected from U1, U2, U4, U5, U6, U11, U12, U14atac, U6atac, and a combination thereof. In some embodiments, the spliceosome protein is selected from Sm, U1-70k, U1A, U1C, and combinations thereof. In some embodiments, the recruitment portion comprises a nucleotide sequence that is at least 70%, 80%, 85%, or 90% identical to or complementary to a sequence set forth in Tables 2-3.In some embodiments, the recruitment portion is complementary to the stem-loop-II region of a snRNA, e.g., U1 snRNA. In some embodiments, the recruitment portion hybridizes to the stem-loop-II region of a snRNA, e.g., U1 snRNA. In some embodiments, the recruitment portion comprises AGGCC. In some embodiments, the recruitment portion comprises a nucleotide sequence that is at least 80%, 90%, or identical to at least 5 contiguous nucleotides of a sequence set forth in Tables 2-3. In some embodiments, the recruitment portion comprises a nucleotide sequence that is at least 80%, 90%, or identical to about 5 to about 10 contiguous nucleotides of a sequence set forth in Tables 2-3. In some embodiments, the recruitment portion comprises a nucleotide sequence that is identical to or complementary to a sequence set forth in Tables 2-3. In some embodiments, the engineered polynucleotide comprises a (e.g., secondary) structural feature. In some embodiments, the engineered polynucleotide comprises an apical loop, an upper stem, an internal loop, a lower stem, or a combination thereof. In some embodiments, the engineered polynucleotide comprises a loop (e.g., an internal loop) adjacent to a stem (e.g., a lower stem or an upper stem) comprising two complementary stem sequences. In some embodiments, the stem sequence of the stem (e.g., a lower stem or an upper stem) comprises about 5 or fewer, about 4 or fewer, or about 3 or fewer nucleotides. In some embodiments, the loop is an internal loop adjacent to a stem (e.g., a lower stem) and an additional stem (e.g., an upper stem) comprising two complementary stem sequences. In some embodiments, the internal loop comprises a nucleic acid sequence of 10 or fewer, 9 or fewer, or 8 or fewer nucleotides. In some embodiments, the stem sequence of the additional stem (e.g., an upper stem) comprises about 5 or fewer, 4 or fewer, or 3 or fewer nucleotides. In some embodiments, the engineered polynucleotide further comprises an apical loop. In some embodiments, the apical loop comprises a nucleic acid sequence of 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, or 5 or fewer nucleotides.In some embodiments, the engineered polynucleotide does not contain any intramolecular disulfide bonds. In some embodiments, when the engineered polynucleotide is associated with a spliceosome portion, the pre-mRNA exhibits substantially no base pairing with the RNA binding domain (RBD) of U1 snRNA. In some embodiments, when the engineered polynucleotide is associated with a spliceosome portion, the pre-mRNA exhibits substantially no base-specific interaction with the U1-C protein. In some embodiments, the engineered polynucleotide is configured to specifically interact with a zinc finger of the U1-C protein. In some embodiments, the 5' targeting portion of the engineered polynucleotide is configured to specifically interact with a zinc finger of the U1-C protein. In some embodiments, the engineered polynucleotide is configured to covalently interact with a zinc finger of the U1-C protein (e.g., via a disulfide bond). In some embodiments, the engineered polynucleotide is configured to non-covalently interact with a zinc finger of the U1-C protein (e.g., via a hydrogen bond). In some embodiments, the engineered polynucleotide comprises a nucleotide sequence complementary to a subsequence of stem-loop II (SL2) of U1 snRNA. In some embodiments, the side of the stem-loop structure of the engineered polynucleotide comprises a nucleotide sequence complementary to a subsequence of stem-loop II (SL2) of U1 snRNA. In some embodiments, the subsequence comprises a sequence corresponding to 5'-GGCCU-3' of SL2 of U1 snRNA. In some embodiments, the subsequence does not comprise a sequence corresponding to 5'-CACGUUA-3' of SL2 of U1 snRNA. In some embodiments, the engineered polynucleotide exhibits substantially no base pairing with the anchor sequence of SL2 of U1 snRNA. In some embodiments, the internal loop of the engineered polynucleotide exhibits substantially no base pairing with the anchor sequence of SL2 of U1 snRNA.In some embodiments, the lower stem of the engineered polynucleotide exhibits substantially no base pairing with the anchor sequence of SL2 of U1 snRNA. In some embodiments, the anchor sequence comprises a sequence corresponding to 5'-CACGUUA-3'. In some embodiments, the engineered polynucleotide exhibits substantially no base pairing with the H helix of U1 snRNA. In some embodiments, the engineered polynucleotide comprises at least one chemical modification. In some embodiments, the engineered polynucleotide comprises at least one 2'-modified (e.g., 2'-methoxy, 2'-methoxymethyl, or 2'-methoxyethyl) nucleotide. In some embodiments, at least about 50%, 60%, 70%, 80%, or 90% of the nucleotides of the engineered polynucleotide are chemically modified nucleotides. In some embodiments, at least about 50%, 60%, 70%, 80%, or 90% of the nucleotides of the engineered polynucleotide comprise 2'-modified (e.g., 2'-methoxy, 2'-methoxymethyl, or 2'-methoxyethyl) nucleotides. In some embodiments, the 2'-modified nucleotides comprise 2'-methoxy, 2'-methoxymethyl, 2'-methoxyethyl, 2'-fluoro, or 2'-aminoethyl nucleotides. In some embodiments, the engineered polynucleotide comprises nucleotides connected by internucleotide linkages, wherein at least one of the internucleotide linkages does not comprise a phosphate. In some embodiments, the engineered polynucleotide comprises nucleotides connected by internucleotide linkages, wherein at least one of the internucleotide linkages comprises sulfur (S), selenium (Se), BR3 (where each R is independently selected from the group consisting of hydrogen, alkyl, and aryl), carbon (C), or NR2 (where each R is independently selected from the group consisting of hydrogen, alkyl, and aryl).

[0021] In some embodiments, the engineered polynucleotide comprises at least one phosphorothioate internucleotide linkage. In some embodiments, at least about 50%, 60%, 70%, 80%, or 90% of the internucleotide linkages of the engineered polynucleotide are chemically modified. In some embodiments, at least about 50%, 60%, 70%, 80%, or 90% of the internucleotide linkages are phosphorathioate. In some embodiments, the internucleotide linkages comprise methylphosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo, or methyleneoxymethylimino. In some embodiments, the engineered polynucleotide comprises about 10 to about 40 nucleotides, about 10 to about 35 nucleotides, about 10 to about 30 nucleotides, or about 10 to about 25 nucleotides. In some embodiments, the recruitment moiety comprises about 10 to about 30 nucleotides, or about 10 to about 20 nucleotides. In some embodiments, one or more targeting moieties each independently comprise about 2 to about 15 nucleotides, about 2 to about 10 nucleotides, or about 2 to about 8 nucleotides. In some embodiments, one of the first targeting moiety and the second targeting moiety comprises about 2 nucleotides, and the other of the first targeting moiety and the second targeting moiety comprises about 5 or 6 nucleotides. In some embodiments, upon association with the engineered polynucleotide and the pre-mRNA, the spliceosome moiety cleaves or splices the pre-mRNA in the target sequence. In some embodiments, the spliceosome moiety further facilitates modification of the cleaved pre-mRNA.

[0022] Described herein, in some embodiments, are engineered polynucleotides comprising nucleotide sequences at least 70%, 80%, 85%, or 90% identical to or complementary to a sequence set forth in Tables 2-3, where the engineered polynucleotides are characterized by (e.g., secondary) structural features. In some embodiments, the nucleotide sequences are identical to or complementary to a sequence set forth in Tables 2-3. In some embodiments, the structural features comprise one or more stem-loop structures. In some embodiments, the structural features comprise an apical loop, an upper stem, an internal loop, a lower stem, or a combination thereof. In some embodiments, the engineered polynucleotide comprises a loop (e.g., an internal loop) adjacent to a stem (e.g., a lower stem or an upper stem) comprising two complementary stem sequences. In some embodiments, the stem sequence of the stem (e.g., a lower stem or an upper stem) comprises no more than about 5, no more than about 4, or no more than about 3 nucleotides. In some embodiments, the loop is an internal loop flanked by a stem (e.g., a lower stem) and an additional stem (e.g., an upper stem) comprising two complementary stem sequences. In some embodiments, the internal loop comprises a nucleic acid sequence of 10 or fewer, 9 or fewer, or 8 or fewer nucleotides. In some embodiments, the stem sequence of the additional stem (e.g., the upper stem) comprises about 5 or fewer, 4 or fewer, or 3 or fewer nucleotides. In some embodiments, the engineered polynucleotide further comprises an apical loop. In some embodiments, the apical loop comprises a nucleic acid sequence of 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, or 5 or fewer nucleotides. In some embodiments, the engineered polynucleotide further comprises one or more targeting moieties that are sufficiently identical to or complementary to a target sequence of the target gene. In some embodiments, one targeting moiety of the one or more targeting moieties is sufficiently identical to or complementary to a consensus sequence in the target sequence of the target gene. In some embodiments, the target gene is microtubule-associated protein tau (MAPT).In some embodiments, the engineered polynucleotide comprises at least one chemical modification. In some embodiments, the engineered polynucleotide comprises at least one phosphorothioate internucleotide linkage. In some embodiments, at least about 50%, 60%, 70%, 80%, or 90% of the internucleotide linkages of the engineered polynucleotide are chemically modified. In some embodiments, at least about 50%, 60%, 70%, 80%, or 90% of the internucleotide linkages are phosphorathioate. In some embodiments, the engineered polynucleotide comprises at least one 2'-modified (e.g., 2'-methoxy, 2'-methoxymethyl, or 2'-methoxyethyl) nucleotide. In some embodiments, at least about 50%, 60%, 70%, 80%, or 90% of the nucleotides of the engineered polynucleotide are chemically modified nucleotides. In some embodiments, at least about 50%, 60%, 70%, 80%, or 90% of the nucleotides of the engineered polynucleotide comprise 2'-modified (e.g., 2'-methoxy, 2'-methoxymethyl, or 2'-methoxyethyl) nucleotides. In some embodiments, the engineered polynucleotide is about 10 to about 40 nucleotides, about 10 to about 35 nucleotides, about 10 to about 30 nucleotides, or about 10 to about 25 nucleotides.

[0023] In some embodiments, the present specification describes a method for altering pre-messenger ribonucleic acid (pre-mRNA) in a cell, the method comprising contacting the cell with an engineered polynucleotide comprising one or more targeting moieties and a recruitment moiety, wherein the one or more targeting moieties bind to the pre-mRNA at an internal target sequence, and the recruitment moiety recruits a post-transcriptional regulatory moiety (e.g., a spliceosome moiety) near the target sequence of the pre-mRNA, thereby altering the pre-mRNA in the cell, resulting in one or more altered pre-mRNAs. In some embodiments, one targeting moiety of the one or more targeting moieties is sufficiently identical to or complementary to a consensus sequence in the target sequence of the target gene. In some embodiments, the pre-mRNA corresponds to a target gene. In some embodiments, the target gene is microtubule-associated protein tau (MAPT). In some embodiments, the method alters the expression or activity of the target gene. In some embodiments, prior to contacting, the cell exhibits aberrant messenger ribonucleic acid (mRNA) or protein corresponding to the target gene.

[0024] In some embodiments, the engineered polynucleotide comprises: (i) a first targeting moiety configured to specifically bind to a pre-messenger ribonucleic acid (pre-mRNA) at an internal first targeting sequence, the first targeting moiety comprising a sequence identical to or complementary to 5'-GTCCA-3'; (ii) a recruitment moiety comprising a sequence at least 90% similar to or complementary to SEQ ID NO: 1 and configured to recruit spliceosome components including U1 snRNA and U1-C protein, the recruitment moiety comprising an apical loop, an upper stem adjacent to the apical loop, a lower stem, and an internal loop located between the upper and lower stems; and (iii) a second targeting moiety configured to specifically bind to a pre-mRNA at an internal second targeting sequence, the second targeting moiety comprising a sequence identical to or complementary to 5'-CG-3'.

[0025] Described herein, in some embodiments, is a set of engineered polynucleotides each independently comprising one or more targeting moieties configured to bind to pre-messenger ribonucleic acid (pre-mRNA) at a target sequence and a recruitment moiety configured to recruit a post-transcriptional regulatory moiety (e.g., a spliceosome moiety), wherein the set of engineered polynucleotides is configured to specifically bind to pre-mRNA at a plurality of target sequences, including the target sequence.

[0026] Another aspect described herein is an engineered polynucleotide comprising a first targeting portion configured to specifically bind to a pre-messenger ribonucleic acid (pre-mRNA) at an internal first targeting sequence, a recruitment portion configured to recruit a spliceosome portion, and a second targeting portion configured to specifically bind to the pre-mRNA at an internal second targeting sequence, wherein the recruitment portion comprises an apical loop, an upper stem adjacent to the apical loop, a lower stem, and an internal loop located between the upper and lower stems, and the spliceosome portion, upon association with the pre-mRNA and the engineered polynucleotide, alters the pre-mRNA in a target sequence comprising the first and second targeting sequences. In some embodiments, the first targeting portion is complementary to and / or hybridizes with the first target sequence. In some embodiments, the second targeting portion is complementary to and / or hybridizes with the second target sequence. In some embodiments, the first targeting sequence and the second targeting sequence are separated by a spacing sequence of 5 or fewer nucleotides in the target sequence. In some embodiments, the target sequence comprises an exon-intron boundary in the pre-mRNA. In some embodiments, the first targeting sequence is 5' of the exon-intron boundary and the second targeting sequence is 3' of the exon-intron boundary. In some embodiments, the first targeting moiety comprises a sequence identical to or complementary to a sequence shown in the exon sequence column of Table 1, and the second targeting moiety comprises a sequence identical to or complementary to a sequence shown in the intron sequence column of Table 1. In some embodiments, the first targeting moiety comprises a sequence at least 80%, 90%, or identical to a ribosome binding site of a spliceosomal snRNA, e.g., U1 snRNA. In some embodiments, the second targeting moiety comprises a sequence that is at least 80%, 90%, or identical to a ribosome binding site of a spliceosomal snRNA, e.g., U1 snRNA. In some embodiments, the sequence that is at least 80%, 90%, or identical to the ribosome binding site can be from about 2 nucleotides to about 10 nucleotides.In some embodiments, the recruitment portion is complementary to the stem-loop-II region of a snRNA, e.g., U1 snRNA. In some embodiments, the recruitment portion hybridizes to the stem-loop-II region of a snRNA, e.g., U1 snRNA. In some embodiments, the recruitment portion comprises AGGCC. In some embodiments, the recruitment portion comprises a nucleotide sequence that is at least 80%, 90%, or identical to at least 5 contiguous nucleotides of a sequence set forth in Tables 2-3. In some embodiments, the recruitment portion comprises a nucleotide sequence that is at least 80%, 90%, or identical to about 5 to about 10 contiguous nucleotides of a sequence set forth in Tables 2-3. In some embodiments, the spliceosome portion comprises U1 snRNA and U1-C protein. In some embodiments, the upper stem or the lower stem comprises two complementary sequences, each of which comprises five or fewer nucleotides; the internal loop comprises two nucleic acid sequences, each of which comprises five or fewer nucleotides; and the apical loop comprises a nucleic acid sequence of eight or fewer nucleotides. In other embodiments, the pre-mRNA, when associated with the engineered polynucleotide, exhibits substantially no base-pairing with the RNA-binding domain (RBD) of the U1 snRNA, and the spliceosome portion, when associated with the engineered polynucleotide and the spliceosome portion, exhibits substantially no base-specific interaction with the U1-C protein. In another embodiment, the 5' targeting portion of the engineered polynucleotide is configured to specifically interact with the zinc finger of the U1-C protein. In another embodiment, the recruitment portion comprises a nucleotide sequence complementary to at least four nucleotides of the sequence of stem-loop II (SL2) of the U1 snRNA. In another embodiment, the sequence of SL2 of the U1 snRNA comprises 5'-GGCCU-3'. The engineered polynucleotide may have 2'-modified nucleotides. At least 50% of the nucleotides of the engineered polynucleotide may be 2'-modified nucleotides. The 2'-modified nucleotides may be 2'-methoxy nucleotides.In another embodiment, the engineered polynucleotide comprises nucleotides connected by internucleotide linkages, wherein at least one of the internucleotide linkages does not contain a phosphate. At least one of the internucleotide linkages, or 50%, 60%, 70%, 80%, or 90% thereof, can be phosphorothioate. [Brief explanation of the drawings]

[0027] The patent or patent application file will contain at least one drawing executed in color. Copies of this patent or patent application including the color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Figure 1]A schematic diagram for identifying splice donor and splice acceptor sites is shown. An exemplary consensus sequence for messenger ribonucleic acid (mRNA) splicing in animals and plants is "GU_AG," where "GU" is an exemplary splice donor sequence and "AG" is an exemplary splice acceptor sequence. The longer splice donor consensus sequence in mammals can be "GUrAGU," where "r" represents either "G" or "A." Typically, the expression "GU_AG" means that only the two nucleotides at the 5' and 3' ends of the sequence are constant, "GU" and "AG," respectively, and that the underlined sequence can be any sequence. However, as described herein, this expression indicates that the underlined sequence can be any sequence except for sequences that do not match any of the other consensus sequences. The splice acceptor consensus sequence is preceded by a branch point sequence containing an adenine that ligates to the ribonucleotide of the 5' splice site to form an intron lariat, and a polypyrimidine tract (C or U) between the branch point and the splice acceptor sequence. Although the short GU_AG consensus sequence of an intron is clearly not sufficient to distinguish between multiple alternative splicing events, surprisingly, little is known about the need for other sequence information to regulate alternative RNA splicing. One or two nucleotides flanking both sides of the intron are also often conserved; these are included in our supplementary table but are not discussed in detail in the text, allowing us to focus our analysis on the consensus sequence at the end of the intron. In this sense, rational design of engineered polynucleotides logically identifies the splice intron consensus sequence (GU_AG). It is then possible to determine the conserved regions of the donor site (5' exon and downstream intron) and the acceptor site (3' exon and downstream intron). It is important to note that the conserved and consensus regions are located within the same site of the constitutive splice donor or acceptor.Recognition of consensus regions determines the location of 5' splice sites, i.e., junctions between exons and introns, while recognition of conserved regions identifies the identity of transcripts selected for regulation. [Figure 2A] 1 shows an exemplary engineered polynucleotide described herein, comprising: (1) 3'-targeting portion: 3'-GC-5', (2) lower stem: 3'-GA-5' / 5'-CT3', (3) internal loop: 3'-CC-5' / 5'-AA-3', (4) upper stem: 3'-GGA-5' / 5'-CCT-3', (5) apical loop: 3'-CTT-5', and (6) 5'-targeting portion: 5'-GTCCA-3'. [Figure 2B] 1 shows an exemplary engineered polynucleotide described herein, comprising: (1) 3'-targeting portion: 3'-GC-5', (2) lower stem: 3'-GA-5' / 5'-CT3', (3) internal loop: 3'-CC-5' / 5'-AA-3', (4) upper stem: 3'-GGA-5' / 5'-CCT-3', (5) apical loop: 3'-CTT-5', and (6) 5'-targeting portion: 5'-GTCCA-3'. [Figure 2C] 1 shows the interaction of an exemplary engineered polynucleotide with a target pre-mRNA sequence. [Figure 2D] 1 shows the interactions of an exemplary engineered polynucleotide with various components of the U1 RNP complex. [Figure 3A] Anchoring is shown to occur via an engineered polynucleotide "stem 5' / 3'" (also known as a 5'-targeting portion or / and a 3'-targeting portion) designed to interact with conserved regions present in the constitutive donor site. In Figure 3A, substitution of the stem 5' / 3' (GTCCA and CG), for example, with phosphorothioate internucleotide linkages and 2'O-methyl (2'O-ME) molecular sugars, increases resistance to endonucleases and increases the molecular strength of the interaction between the stem 5' / 3' bases and the conserved region from the constitutive donor. [Figure 3B]Anchoring is shown to occur via an engineered polynucleotide "stem 5' / 3'' (also known as a 5'-targeting portion or / and a 3'-targeting portion) designed to interact with a conserved portion present in the constitutive donor site. Figure 3B shows the interaction of the engineered polynucleotides described herein with constitutive donor splicing and the silencing of the RNA-binding domain (RBD) of U1 snRNA by the constitutive donor splicing exon. [Figure 4] A diagram of the human U1 snRNP is shown. The U1 snRNP is composed of one U1 snRNA, seven common Sm proteins, and three U1 snRNP-specific proteins (U1-70K, U1A, and U1C). The secondary structure of the U1 snRNA consists of four stem-loops (SLs) and a highlighted H helix. The nucleotides that form the H helix are indicated. Additionally, the U1 snRNA sequence relevant to RNA:protein or RNA:5'ss interactions is also given. The loop portion of SL1 is depicted according to the crystal structure. This portion is closed by a trans WC / Hoogsteen base pair formed between A29 and A36. The protein components of the U1 snRNP, their sizes, and their approximate locations are also indicated. The Sm ring formed by the Sm proteins, indicated by the green circle, binds to the boxed Sm site. U1-70K, indicated in red, recognizes SL1. U1A, indicated in yellow, binds to SL2. U1C, shown in blue, is recruited to the U1 snRNP through protein-protein interactions with U1-70K and Sm proteins. Note the interaction between the U1C ring and the Sm ring. [Figure 5A]This work demonstrates the U1 snRNP, which binds to the 5' exon-intron junction of pre-mRNA and thereby plays a key role in the early stages of pre-mRNA splicing. Two crystal structures of engineered U1 substructures are presented, which together reveal at atomic resolution a nearly complete network of protein-protein and RNA-protein interactions within the U1 snRNP and demonstrate how the 5' splice site of pre-mRNA is recognized by the U1 snRNP. The zinc finger of U1-C interacts with the duplex between the pre-mRNA and the 5' end of the U1 snRNA. RNA duplex binding is stabilized by hydrogen bonds and electrostatic interactions between U1-C and the RNA backbone surrounding the splice junction, but U1-C does not make base-specific contacts with the pre-mRNA. This structure, along with RNA-binding assays, demonstrates that 5'-splice site nucleotide selection by U1 snRNP is primarily achieved by base pairing with U1 snRNA, but U1-C fine-tunes the relative affinity of mismatched 5'-splice sites. In Figure 5A, U1-70K forms a complex with the U1 snRNA stem-loop, and the U1-A RRM forms a complex with stem-loop 2. [Figure 5B]This work demonstrates the U1 snRNP, which binds to the 5' exon-intron junction of pre-mRNA and thereby plays a key role in the early stages of pre-mRNA splicing. Two crystal structures of engineered U1 substructures are presented, which together reveal at atomic resolution a nearly complete network of protein-protein and RNA-protein interactions within the U1 snRNP and demonstrate how the 5' splice site of pre-mRNA is recognized by the U1 snRNP. The zinc finger of U1-C interacts with the duplex between the pre-mRNA and the 5' end of the U1 snRNA. RNA duplex binding is stabilized by hydrogen bonds and electrostatic interactions between U1-C and the RNA backbone surrounding the splice junction, but U1-C does not make base-specific contacts with the pre-mRNA. This structure, along with RNA-binding assays, demonstrates that 5'-splice site nucleotide selection by U1 snRNP is primarily achieved by base pairing with U1 snRNA, but U1-C fine-tunes the relative affinity of mismatched 5'-splice sites. In Figure 5B, U1 snRNA stem-loops 1 and 2 (55-MER). [Figure 5C]This work demonstrates the U1 snRNP, which binds to the 5' exon-intron junction of pre-mRNA and thereby plays a key role in the early stages of pre-mRNA splicing. Two crystal structures of engineered U1 substructures are presented, which together reveal at atomic resolution a nearly complete network of protein-protein and RNA-protein interactions within the U1 snRNP and demonstrate how the 5' splice site of pre-mRNA is recognized by the U1 snRNP. The zinc finger of U1-C interacts with the duplex between the pre-mRNA and the 5' end of the U1 snRNA. RNA duplex binding is stabilized by hydrogen bonds and electrostatic interactions between U1-C and the RNA backbone surrounding the splice junction, but U1-C does not make base-specific contacts with the pre-mRNA. This structure, along with RNA-binding assays, demonstrates that 5'-splice site nucleotide selection by U1 snRNP is primarily achieved by base pairing with U1 snRNA, but U1-C fine-tunes the relative affinity of mismatched 5'-splice sites. In Figure 5C, U1 small nuclear ribonucleoprotein A and 70 kDa. [Figure 6] Shown are U1-70K in complex with the U1 snRNA stem-loop and the U1-A RRM in complex with stem-loop 2, stabilized via the U1-C zinc finger. [Figure 7]A schematic diagram of the modulation of the spliceosome machinery by the engineered polynucleotide described herein (ASMO1, also known as APT20TTMG) is shown. Anchoring of the targeting moiety ("stem 5' / 3'") (5'-GTCCA-3' and 5'-CG-3') allows interaction with a conserved site in the constitutive donor via silencing of the RNA-binding domain (RBD) of U1 snRNA. Stabilization of the U1 snRNP complex can be observed through the strong ionic attraction of the zinc fingers of U1-C, induced by disulfide bridges with thiols in the ASMO1 stem 5' / 3'. The pre-mRNA / ASMO1 duplex is stabilized by hydrogen bonding and electrostatic interactions between U1-C and the backbone of the pre-mRNA around the seam joint, but U1-C does not make base-specific contacts with the pre-mRNA. This structure demonstrates that nucleotide selection for 5'-splice by U1 snRNP is primarily achieved through interactions between the 5' / 3' stem and pre-mRNA. Meanwhile, U1-C adjusts the relative affinity of the 5'-splice mismatch site and stabilizes the central core of the spliceosome machinery by bridging interactions between U1-70K and the Sm ring. Electrostatic interactions and hydrogen bridges of stem-loop II with specific bases (5'-AGGCC-3') in the upper stem (also known as "hairpin-2," see Figure 2B) (3'-GGA-5' / 5'-CCT-3') and the internal loop (3'-mCC-5' / 5'-AA-3') can be observed in association with the regulation of polyadenylation signals and acetylation by U1-A. Note that the anchor portion of U1-A in stem-loop II (5'-CAACGUUA-3') is not silenced by the upper stem and induces regulation of gene expression and acetylation levels. In addition, the presence of the 2'-OME group induces a conformational change in U1-snRNA and a change in molecular dynamics that encourages stem-loop II to approach ASMO1. The ASMO1 targeting or recruitment portion, unlike the U1-A protein, reduces the possibility of premature interruption of the reading frame due to deregulation of the polyadenylation signal. [Figure 8]The U1-70K complexed with the U1 snRNA stem-loop and the U1-A RRM complexed with stem-loop 2 are shown, stabilized via the U1-C zinc finger. Electrostatic interactions and hydrogen bridges of stem-loop II with a specific base (3'-CCGGA-5') in the upper stem (3'-GGA-5' / 5'-CCT-3') and the internal loop (3'-mCC-5' / 5'-AA-3') can be observed in association with the regulation of polyadenylation and acetylation by U1-A. Note that the anchor portion of U1-A in stem-loop II (5'-CAACGUUA-3') is not silenced by the upper stem, inducing regulation of gene expression and acetylation levels. In addition, the presence of the 2'-OME group induces a conformational change in U1-snRNA and a change in the molecular dynamics of the medium that allows stem-loop II to approach the engineered polynucleotide (ASMO1) described herein. [Figure 9A] We show that U1-C is located on SmD3 and that its binding can be stabilized by the N-terminus of U1-70K. [Figure 9B] It shows that U1-C forms hydrogen bonds with the sugar-phosphate backbone atoms but does not contact the RNA bases. On the 5'SS strand, nucleotides are colored teal for exon sequences and light tan for intron sequences. [Figure 9C] A schematic diagram of 5'-splice site recognition is shown. The red dotted lines are hydrogen bonds created by the amino acid side chains of the U1-C zinc finger. The blue dotted lines are hydrogen bonds created by the main chain atoms of the U1-C zinc finger. The green dotted lines are disulfide bonds created by the amino acid side chains of the U1-C zinc finger. The orange dotted lines are disulfide bonds created by the atoms in the main chain of the U1-C zinc finger. The 5'SS nucleotide is encoded by the nucleotide as in Figure 9B. [Figure 10A] The fingerprint Z1 U1-C snRNP is shown in blue, represented by 36 amino acid residues. [Figure 10B]The Z1 finger portion of the U1-C snRNP is shown, showing the key residues that interact with the pre-mRNA / ASMO1 duplex in the 5′ constitutive donor region. [Figure 10C] A representative sequence of the U1-C snRNP containing 145 aa is shown, with the 36 aa highlighted in green indicating the zinc finger portion. [Figure 11] A shows mitochondrial activity in neurons derived from induced pluripotent stem cells (iPSCs) from healthy patients (HDCs) after incubation with ASMO1. B shows mitochondrial activity in neurons derived from induced pluripotent stem cells (iPSCs) from patients diagnosed with Alzheimer's disease (ADCs) after incubation with ASMO1. [Figure 12] Figure 1 shows mitochondrial activity in neurons from healthy donors (HDCs) after incubation with ASMO1. [Figure 13] A shows mitochondrial activity in microglia derived from iPSCs from healthy patients (HDC) after incubation with ASMO1. B shows mitochondrial activity in microglia derived from iPSCs from patients diagnosed with AD (ADC) after incubation with ASMO1. [Figure 14] 1 shows mitochondrial activity in astrocytes after incubation with ASMO1. [Figure 15] A shows extracellular glutamate levels in neurons derived from iPSCs from healthy patients (HDCs) after incubation with ASMO1. B shows extracellular glutamate levels in neurons derived from iPSCs from patients diagnosed with AD (ADCs) after incubation with ASMO1. [Figure 16]This figure shows a representation of a well containing iPSC-derived neurons in a NeuroHTS™ technology plate, and the parameters that can be evaluated in three distinct regions of the neuron using this system. In the upper section of the well, cell number and nuclear aggregates are evaluated in the soma and dendrites. In the central section of the channel (axon), the thickness of the axonal fibers is examined. In the lower section, where the axon and dendrites are located, four parameters are evaluated: the number of branches, the number of branch junctions, the straightness of the neurites, and the axonal material. [Figure 17] A shows the number of cultured neurons derived from iPSCs from healthy patients (HDCs) after incubation with ASMO1. B shows the number of cultured neurons derived from iPSCs from patients diagnosed with AD (ADCs) after incubation with ASMO1. [Figure 18] A shows the formation of cell aggregates in neurons derived from iPSCs from a healthy patient (HDC) after incubation with ASMO1. B shows the formation of cell aggregates in neurons derived from a patient diagnosed with AD (ADC) after incubation with ASMO1. [Figure 19] A shows fiber width in neurons derived from iPSCs from healthy patients (HDCs) after incubation with ASMO1. B shows fiber width in neurons derived from patients diagnosed with AD (ADCs) after incubation with ASMO1. [Figure 20] A shows the number of branches in neurons derived from iPSCs from healthy patients (HDC) after incubation with ASMO1. B shows the number of branches in neurons derived from iPSCs from patients diagnosed with AD (ADC) after incubation with ASMO1. C shows the number of branches per cell in neurons derived from iPSCs from healthy patients (HDC) after incubation with ASMO1. D shows the number of branches per cell in neurons derived from iPSCs from patients diagnosed with AD (ADC) after incubation with ASMO1. [Figure 21]A shows branch junctions in neurons derived from iPSCs from a healthy patient (HDC) after incubation with ASMO1. B shows branch junctions in neurons derived from iPSCs from a patient diagnosed with AD (ADC) after incubation with ASMO1. C shows branch junctions per cell in neurons derived from iPSCs from a healthy patient (HDC) after incubation with ASMO1. D shows branch junctions per cell in neurons derived from iPSCs from a patient diagnosed with AD (ADC) after incubation with ASMO1. [Figure 22] A shows the linearity of neurites in neurons derived from iPSCs from healthy patients (HDC) after incubation with ASMO1. B shows the linearity of neurites in neurons derived from iPSCs from patients diagnosed with AD (ADC) after incubation with ASMO1. [Figure 23] A shows total axonal material in neurons derived from iPSCs from a healthy patient (HDC) after incubation with ASMO1. B shows total axonal material in neurons derived from iPSCs from a patient diagnosed with AD (ADC) after incubation with ASMO1. C shows axonal material per cell in neurons derived from iPSCs from a healthy patient (HDC) after incubation with ASMO1. D shows axonal material per cell in neurons derived from iPSCs from a patient diagnosed with AD (ADC) after incubation with ASMO1. [Figure 24] Representative images of morphological changes in neurons derived from iPSCs of healthy patients (HDCs) after incubation with ASMO1 are shown. [Figure 25] Representative images of morphological changes in neurons derived from iPSCs from patients diagnosed with AD (ADC) after incubation with ASMO1 are shown. [Figure 26]Western blotting analysis of ASMO1 binding to U1-70K protein in neuroblastoma cell lines (A), U1-C protein in neuroblastoma cell lines (B), U1-A protein in neuroblastoma cell lines (C), and GAPDH protein in neuroblastoma cell lines (D). [Figure 27] Binding of ASMO1 to U1 snRNA and pre-mRNA is shown. A: U1 snRNA, B: tau pre-mRNA, and C: GAPDH. [Figure 28] A shows the difference in tau levels normalized to the negative control in neurons derived from iPSCs of healthy patients (HDC) after incubation with ASMO1. B shows the difference in tau levels normalized to the negative control in neurons derived from iPSCs from patients diagnosed with AD (ADC) after incubation with ASMO1. [Figure 29] Figure 1 shows a plot of the mapping profile (percentage) of reads against the human reference genome using the STAR RNA-seq aligner for RNA-seq differential gene expression analysis. [Figure 30] Use featureCounts to indicate the percentage of reads that mapped to genomic features such as genes, exons, promoters, gene bodies, genome bins, and chromosomal locations. [Figure 31] A shows a principal component analysis (PCA) plot of an individual sample. B shows a PCA plot with gray circles to indicate the first three clusters identified in the PCA results. [Figure 32] A shows a volcano plot of differentially expressed (DEG) genes in ADC cells treated with 18.52 nM ASMO1 relative to untreated ADC neurons. B shows a volcano plot of differentially expressed genes in ADC cells treated with 500 nM ASMO1 relative to untreated ADC neurons. [Figure 33] A shows a volcano plot of differentially expressed genes in HDC cells treated with 18.52 nM ASMO1 relative to untreated HDC neurons. B shows a volcano plot of differentially expressed genes in HDC cells treated with 500 nM ASMO1 relative to untreated HDC neurons. [Figure 34] Volcano plot of expressed genes in HDC untreated neurons relative to untreated ADC neurons. [Figure 35] Figure 1 shows the top-ranked Gene Ontology biological process entries obtained from enrichment analysis of DEGs from ADC neurons treated with 18.52 nM ASMO1 relative to untreated ADC (media only). [Figure 36] Figure 1 shows the top-ranked Gene Ontology biological process entries obtained in the enrichment analysis of DEGs from HDC neurons treated with 18.52 nM ASMO1 relative to untreated HDCs (medium only). [Figure 37] Figure 1 shows the top-ranked Gene Ontology biological processes obtained in the enrichment analysis of DEGs from HDC neurons treated with 500 nM ASMO1 relative to untreated HDCs (medium only). [Figure 38] Shown are all items related to gene ontology biological process (GO:BP) obtained for DEGs in ADC neurons treated with 500 nM ASMO1 in relation to untreated ADC (medium only). [Figure 39]A shows all Kyoto Encyclopedia of Genes and Genomes (KEGG) entries obtained by enrichment analysis of DEGs in ADC neurons treated with 18.52 nM ASMO1 relative to untreated ADC (medium only). B shows all Kyoto Encyclopedia of Genes and Genomes (KEGG) entries obtained by enrichment analysis of DEGs in ADC neurons treated with 500 nM ASMO1 relative to untreated ADC (medium only). [Figure 40] A shows all KEGG items obtained by enrichment analysis of DEGs in HDC neurons treated with 18.52 nM ASMO1 relative to untreated HDCs (medium only). B shows all KEGG items obtained by enrichment analysis of DEGs in HDC neurons treated with 500 nM ASMO1 relative to untreated HDCs (medium only). C shows all KEGG items obtained by enrichment analysis of DEGs in HDC untreated neurons relative to untreated ADCs (medium only). [Figure 41] Figure 1 shows the protein-protein interaction network and clustering of DEGs in ADCs treated with 18.52 nM ASMO1. [Figure 42] 3'UTR / CDS expression ratios of genes associated with senescence are shown, indicating potential early polyadenylation profiles and the resulting sizes of expressed transcripts. Senescence. [Figure 43] DEGs in ADC neurons treated with 18.52 nM ASMO1 and their proposed primary roles in the mechanism of action of ASMO1 are shown. [Figure 44] A-B show spontaneous electrophysiological activity in neurons derived from HDC-derived iPSCs after incubation with APT20TTMG (ASMO1). [Figure 45]A-B show spontaneous electrophysiological activity in neurons derived from ADC-derived iPSCs after incubation with APT20TTMG (ASMO1). [Figure 46] A-D show the levels of soluble Aβ40 and Aβ42 in the cortex in an aging animal model (SAMP-8) measured by ELISA. Graphs depict the levels of (A) soluble Aβ40, (B) soluble Aβ42, (C) insoluble Aβ40, and (D) insoluble Aβ42 in cortical samples from n = 7 animals per treatment. Biological outliers identified by histology were excluded from the analysis. Data are presented as pg / mg protein (Aβ40 or Aβ42). [Figure 47] A-D show the levels of soluble Aβ40 and Aβ42 in the hippocampus in an aging animal model (SAMP-8) measured by ELISA. Graphs depict the levels of (A) soluble Aβ40, (B) soluble Aβ42, (C) insoluble Aβ40, and (D) insoluble Aβ42 in cortical samples from n = 7 animals per treatment. Biological outliers identified by histology were excluded from the analysis. Data are shown as pg / mg protein (Aβ40 or Aβ42). [Figure 48] A to D show quantification of amyloid immunofluorescence in the cerebral cortex in an aging animal model (SAMP-8). [Figure 49] A to D show quantification of amyloid immunofluorescence in the hippocampus in an aging animal model (SAMP-8). [Figure 50] A to D show the levels of soluble and insoluble tau and ptau (ptau)(T231) in the cortex in an aging animal model (SAMP-8) measured by ELISA. [Figure 51] A to D show the levels of soluble and insoluble tau and ptau (ptau)(T231) in the hippocampus in an aging animal model (SAMP-8) measured by ELISA. [Figure 52] A to D show quantification of pSer202 / pThr205-tau immunofluorescence in the cerebral cortex in an aging animal model (SAMP-8). [Figure 53] A to D show quantification of pSer202 / pThr205-tau immunofluorescence in the hippocampus in an aging animal model (SAMP-8). [Figure 54] A to D show quantification of U1-70K immunofluorescence in the cerebral cortex in an aging animal model (SAMP-8). [Figure 55] A to D show quantification of U1-70K immunofluorescence in the hippocampus in an aging animal model (SAMP-8). [Figure 56] A-C show quantification of GFAP in the cerebral cortex in an aging animal model (SAMP-8) by ELISA (58A) and immunofluorescence (58B and 58C). [Figure 57] A-C show quantification of GFAP in the hippocampus in an aging animal model (SAMP-8) by ELISA (58A) and immunofluorescence (58B and 58C). [Figure 58] A to H show quantification of proinflammatory cytokines in plasma. [Figure 59] Panels A to B show immunofluorescence signals in an aging animal model (SAMP-8). [Figure 60] The 3' untranslated region to coding sequence (3'UTR / CDS) ratios of TARDBP and STMN2 for HDC and ADC cells are shown.

[0028] The novel features of the present disclosure are set forth with particularity in the appended claims. The features and advantages of the present disclosure will be better understood by reference to the following detailed description that sets forth illustrative embodiments. DETAILED DESCRIPTION OF THE INVENTION

[0029] Described herein are (e.g., engineered) polynucleotides and (e.g., pharmaceutical) compositions, as well as methods for utilizing such polynucleotides and compositions, e.g., to modulate gene expression or activity.

[0030] This specification describes a method for treating neurodegenerative diseases and Alzheimer's disease using the engineered polynucleotides described in this disclosure.The engineered polynucleotides described herein can be effective in regulating pre-mRNA splicing by recruiting spliceosome components, or in suppressing premature polyadenylation by recruiting spliceosome components involved in the telescripting process.Without being bound by a specific mechanism, improving the recruitment of spliceosome components to pre-mRNA can be effective in regulating the cellular mechanisms responsible for neurodegenerative diseases.The engineered polynucleotides can be administered to subjects suffering from neurodegenerative diseases, and can treat neurodegenerative diseases, for example, by reducing the number of protein aggregates, fibroids, or plaques in the subject's brain, or by preventing the growth of fibroids, protein aggregates, plaques, or other structures associated with neurodegenerative diseases.

[0031] Neurodegenerative diseases affect the brain and nervous system and often result in the progressive deterioration of neurons and their supporting cells. Progressive deterioration of cells in the nervous system, typically exacerbated by the aging process, can impair an individual's ability to interact with the environment and function independently. Alzheimer's disease (AD) is a neurodegenerative disorder characterized by memory loss and progressive loss of the ability to perform daily activities. Millions of people worldwide suffer from various manifestations of neurodegenerative diseases. While many environmental and genetic causes of neurodegenerative diseases have been identified, no cures exist for these disorders.

[0032] AD is one of many diseases classified as a tauopathy. It is characterized by the presence of amyloid-β plaques and hyperphosphorylated tau aggregates in neurofibrillary tangles, neuropil threads, and neuritic plaques in the brain. Progressive loss of white matter has been observed in areas showing tau pathology. Furthermore, AD may also be characterized by the depletion, accumulation, and aggregation of U1 small nuclear ribonucleoprotein (snRNP) nuclei in the cytoplasm along with impaired splicing. Additionally, the basic-acidic dipeptide domain of U1-70K has been demonstrated to interact with tau from AD brains, and both U1-70K and tau colocalize in neurofibrillary tangles in late-onset sporadic and familial cases of AD.

[0033] Studies integrating data from human postmortem brain tissue and Drosophila melanogaster models have shown that AD tau neurofibrillary tangle pathology disrupts spliceosome activity, leading to transcriptome dysfunction and ultimately CNS dysfunction and neurodegeneration. Tau may be involved in spliceosome cytoplasmic sequestration and disruption of snRNP assembly and / or stability. For example, several spliceosome components (including U1-70K) can physically associate with tau in human brains with AD pathology, and genetic manipulation of these factors in Drosophila can enhance tau neurotoxicity. Furthermore, increased cryptic splicing load has been observed in postmortem brains of humans with tau pathology.

[0034] Additionally, the molecular events involved in the transformation from the initiation of AD associated with Aβ accumulation to mild cognitive impairment (MCI) and symptomatic AD primarily associated with tau aggregation remain poorly understood (Hales et al., 2016a). U1 small nuclear ribonucleoprotein particles (snRNPs), particularly U1-70K and U1-A proteins, are enriched in the AD insoluble proteome of asymptomatic AD, MCI, and postmortem brain tissue. Furthermore, insoluble U1-A and U1-70K strongly correlate with the quantification of both insoluble Aβ and tau (Hales et al., 2016b). This evidence not only suggests the potential involvement of U1 snRNP aggregation in the mechanism linking plaque development to NFT formation and, consequently, to the pathogenesis of Alzheimer's disease, but also proposes that snRNP dysfunction, characterized by aggregation, hyperactivation, and mislocalization, may represent an upstream event that could significantly contribute to the early development of Alzheimer's disease.

[0035] The U1 snRNP complex is one of five complexes (designated U1, U2, U4, U5, and U6) that comprise the human spliceosome. It is involved in processing pre-messenger RNA (pre-mRNA) by removing intronic regions and producing mature mRNA in the nucleus. The U1 snRNP complex specifically plays a role in the recognition of pre-mRNA splicing sites during the early stages of spliceosome assembly. Furthermore, U1 snRNP plays an essential global role in 3'-end pre-mRNA processing by actively suppressing premature 3'-end cleavage and polyadenylation, particularly within intronic GU-rich regions. Due to this important physiological role, U1 snRNP dysfunction in Alzheimer's disease (AD) can result in RNA splicing defects and suppression of premature cleavage and polyadenylation in RNA transcripts and the autophagy-lysosomal system. Furthermore, mislocalization and depletion of U1 from the nucleus and translocation to the cytoplasm can trigger cell cycle reentry (CCR) in neurons, potentially leading to mitotic cell death and potential neuronal death. Furthermore, as previously described, U1 snRNP aggregation may be involved in the mechanism linking plaque development to neurofibrillary tangle (NFT) formation and, therefore, to the development of AD. The disclosed engineered polynucleotides (e.g., ASMO1) can generally target pre-mRNAs, such as tau pre-mRNA, recruit the U1 complex, and help the U1 complex stabilize early spliceosome assembly at splicing sites, enhancing the accuracy of splice site selection. Furthermore, interaction with key domains of the U1 complex, such as the U1-C zinc finger, which has previously been proposed to be involved in binding of the U1-C snRNP to the U1 complex, may occur, although this is not essential. These features may enable engineered polynucleotides (e.g., ASMO1) to promote proper recognition of the 5' end and initiation of the splicing process even under pathological conditions, while also preventing cytoplasmic aggregation associated with U1 nuclear depletion, reduced 3' processing / premature polyadenylation (in other words, reduced U1 telescript function).

[0036] Thus, the present disclosure provides engineered polynucleotides that function as neurodegenerative disease treatments, as well as corresponding methods for using engineered polynucleotides. Engineered polynucleotides can function through homeostatic regulation of U1 snRNP function, regulation of tau expression, or a combination (e.g., synergistic) of both, enabling broad application for the treatment of neurodegenerative diseases. To enable this U1 regulation, engineered polynucleotides can be designed to have a size, conformation, and strategic chemical modifications that enable direct attraction and interaction with U1C and indirect attraction and interaction with U1-70K. Engineered polynucleotides (e.g., ASMO1) can have sequences complementary to highly conserved regions that present pre-mRNAs at the exon-intron junction at the 5' end of introns, commonly referred to as donor splice sites. U1 regulation can ensure the precise assembly (with the correct distance, position, and behavior) of the snRNPs that make up the U1 complex, which is critically related to the splicing process. Because splicing can control expression patterns at the post-transcriptional level in cells with aberrant gene expression caused by U1 dysfunction, U1 regulation induced by engineered polynucleotides (e.g., ASMO1) can help normalize protein expression. Regulation of tau expression upon treatment with engineered polynucleotides (e.g., ASMO1) can be a response to this splicing regulation in dysregulated cells and may be useful for treating neurodegenerative diseases. Taken together, the important roles of U1 snRNP and tau protein in the development of neurodegenerative disease treatments, as well as our in vitro and in vivo findings, confirm that the engineered polynucleotides contemplated in this disclosure can function as therapeutic agents for the treatment of neurodegenerative diseases.

[0037] Engineered Polynucleotides The present disclosure provides engineered polynucleotides and methods for using the engineered polynucleotides. Engineered polynucleotides generally refer to polynucleotides that do not occur in nature. These engineered polynucleotides are not limited to any synthetic form and can be generated by any synthetic method (e.g., recombinant technology or solid-phase synthesis). In some embodiments described herein, the engineered polynucleotides (i) include one or more targeting moieties configured to bind (e.g., specifically) to ribonucleic acid (RNA) (e.g., messenger ribonucleic acid (mRNA) such as pre-messenger ribonucleic acid (pre-mRNA)) at an internal target sequence. The engineered polynucleotides may further include (ii) a recruitment moiety configured, upon association with the RNA (e.g., mRNA such as pre-mRNA) and the engineered polynucleotide, to recruit a post-transcriptional regulatory moiety (e.g., spliceosome moiety) such that the post-transcriptional regulatory moiety alters the RNA (e.g., mRNA such as pre-mRNA) at or near the target sequence. In some embodiments, "configured to specifically bind to" refers to hybridizing. In some embodiments, a portion configured to specifically bind to a pre-mRNA refers to a portion that is at least 80%, 90%, or 100% complementary to the pre-mRNA sequence to which it specifically binds. In some embodiments, the RNA (e.g., mRNA, such as pre-mRNA) encodes a target gene. In some embodiments, a pre-mRNA associated with an engineered polynucleotide refers to a pre-mRNA that hybridizes with at least one nucleotide (and up to about 100%) of the engineered polynucleotide. In some embodiments, "altering the RNA" refers to modifying a nucleic acid molecule by cleaving the nucleic acid, reacting nucleotides of the nucleic acid, or splicing the nucleic acid. In some embodiments, "proximity" refers to a distance of 5 or fewer nucleotides apart.

[0038] An engineered polynucleotide or the engineered polynucleotides described herein can include various moieties. A "moiety" can refer to a region of an engineered polynucleotide. In some cases, a moiety can be described in terms of the function of that moiety. For example, a "targeting moiety" can refer to a region of an engineered polynucleotide, at least a portion of which can be complementary to a target RNA, a "recruitment moiety" can refer to a moiety that can recruit any one of the regulatory moieties described herein, and a "spacing sequence" can refer to a moiety that provides spacing between other moieties. In some cases, the recitation of the name of a moiety does not limit that moiety to a particular function. For example, a "targeting moiety," at least a portion of which can be complementary to a target RNA, can in some cases recruit a regulatory moiety. The various moieties described throughout this disclosure can be combined to generate engineered polynucleotides that can perform a particular function. For example, an engineered polynucleotide can include a targeting moiety as disclosed herein along with a recruitment moiety as disclosed herein.

[0039] targeting part In various aspects, the engineered polynucleotide comprises one or more targeting moieties. The targeting moiety may enable the engineered polynucleotide to interact with a ribonucleic acid (e.g., a pre-mRNA). The targeting moiety may comprise a sequence complementary to or identical to a sequence of a ribonucleic acid (e.g., a pre-mRNA) so that the engineered polynucleotide and the ribonucleic acid can interact or hybridize. The targeting moiety may be complementary to or identical to a target sequence. In some embodiments of the engineered polynucleotides described herein, one targeting moiety of the one or more targeting moieties is sufficiently identical to or complementary to a consensus sequence in the target sequence. The target sequence may be in a target gene. The target sequence may be a conserved or consensus sequence present in multiple different pre-mRNAs. For example, the target sequence may allow the engineered polynucleotide to interact with a conserved region present in a first pre-mRNA encoding a gene and a second pre-mRNA encoding another gene. Targeting moieties can be found at the 5' and 3' ends of the engineered polynucleotide and are sometimes referred to as the lower stem or foot. Without wishing to be bound by theory, stable binding of one or two targeting moieties to the constitutive donor 5' of the target pre-mRNA allows interaction with a conserved site in the constitutive donor, silencing the U1 snRNA RNA-binding domain (RBD). The targeting moiety can contain a sequence nearly identical to that of the U1 snRNA RBD. For example, the RBD can contain the following sequence: 3'-GUCCAUUCAUA-5', and the targeting moiety can contain GTCCA (or GUCCA). Based on the sequence similarity between the U1 snRNA RBD and the targeting moiety, the targeting moiety can effectively displace or prevent the U1 snRNA RBD from binding to the pre-mRNA.

[0040] Consensus sequences can be determined based on the identification of genetic variants of unknown significance (VUS). Any exon or intron VUS can be spliceogenic by disrupting cis-DNA sequences that define exons, introns, and regulatory sequences required for accurate RNA splicing. Cis-DNA elements can include consensus nucleotides at the exon-intron boundary core (e.g., GT at +1 and +2 of the 5' donor site and AG at -1 and -2 of the 3' acceptor site) or intronic and exon nucleotides adjacent to these invariant nucleotides that are highly conserved and known to be involved in splice site selection (e.g., CAG / GUAAGU at the donor site and NYAG / G at the acceptor site). Nucleotide changes in any of these elements can lead to incorrect splice site recognition, creating new splice sites or activating cryptic splice sites, resulting in aberrant transcripts or nonfunctional proteins associated with disease or disorders. In some embodiments, at least one targeting moiety of the one or more targeting moieties is sufficiently identical to or complementary to a consensus sequence in the target sequence of the target ribonucleic acid or target gene. In some embodiments, the consensus sequence comprises about 2 to about 15 nucleotides, about 2 to about 10 nucleotides, or about 2 to about 8 nucleotides. In some embodiments, at least two targeting moieties of the one or more targeting moieties are sufficiently identical to or complementary to at least two consensus sequences in the target sequence of the target gene. In some embodiments, each of the one or more targeting moieties is independently sufficiently identical to or complementary to a consensus sequence in the target sequence of the target gene.

[0041] In some embodiments of the engineered polynucleotides described herein, one or more targeting moieties each independently comprise from about 2 to about 15 nucleotides, from about 2 to about 10 nucleotides, or from about 2 to about 8 nucleotides. In some embodiments of the engineered polynucleotides described herein, one or more targeting moieties each independently comprise 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

[0042] In some embodiments of the engineered polynucleotides described herein, the one or more targeting moieties comprise: (1) a first targeting moiety configured to specifically bind to a first targeting sequence in a target sequence of RNA (e.g., mRNA, such as pre-mRNA); and (2) a second targeting moiety configured to specifically bind to a second targeting sequence in a target sequence of RNA (e.g., mRNA, such as pre-mRNA). In some embodiments, the first targeting sequence comprises a consensus sequence in the target sequence. In some embodiments, the consensus sequence of the first targeting sequence comprises about 2 to about 15 nucleotides, about 2 to about 10 nucleotides, or about 2 to about 8 nucleotides. In some embodiments, the consensus sequence of the first targeting sequence comprises 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, or 8 to 10 nucleotides. In some embodiments, the second targeting sequence comprises a consensus sequence in the target sequence. In some embodiments, the consensus sequence of the second targeting sequence comprises about 2 to about 15 nucleotides, about 2 to about 10 nucleotides, or about 2 to about 8 nucleotides. In some embodiments, the consensus sequence of the second targeting sequence comprises 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, or 8 to 10 nucleotides. In some embodiments, the consensus sequence of the first targeting sequence and the consensus sequence of the second targeting sequence are sequences of different nucleotide lengths. In some embodiments, one of the consensus sequences of the first and second targeting sequences comprises about 1 to about 5 nucleotides, and the other of the consensus sequences of the first and second targeting sequences comprises about 4 to about 8 nucleotides. In some embodiments, one of the consensus sequences of the first and second targeting sequences comprises at least about 2 nucleotides, and the other of the consensus sequences of the first and second targeting sequences comprises at least about 5 or 6 nucleotides. In some embodiments, one of the consensus sequences of the first and second targeting sequences comprises about 2 nucleotides, and the other of the consensus sequences of the first and second targeting sequences comprises about 5 or 6 nucleotides.

[0043] In some embodiments of the engineered polynucleotides described herein, the first and second targeting moieties are moieties of different nucleotide lengths. In some embodiments, one of the first and second targeting moieties comprises about 1 to about 5 nucleotides, and the other of the first and second targeting moieties comprises about 4 to about 8 nucleotides. In some embodiments, one of the first and second targeting moieties comprises at least about 2 nucleotides, and the other of the first and second targeting moieties comprises at least about 5 or 6 nucleotides. In some embodiments, one of the first and second targeting moieties comprises about 2 nucleotides, and the other of the first and second targeting moieties comprises about 5 or 6 nucleotides.

[0044] In some embodiments, the (e.g., first or second) targeting moiety comprises a nucleic acid sequence having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides. In some embodiments, the (e.g., first or second) targeting moiety comprises a nucleic acid sequence having up to 10, 9, 8, 7, 6, 5, 4, 3, or 2 nucleotides. In some embodiments, the (e.g., first or second) targeting moiety comprises a nucleic acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, or a range between any two of the foregoing values.

[0045] In some embodiments, the (e.g., first or second) targeting moiety comprises a nucleic acid sequence that targets a nucleotide in an exon. In some embodiments, the (e.g., first or second) targeting moiety comprises a nucleic acid sequence that targets a nucleotide in an exon immediately adjacent to an intron. In some embodiments, the (e.g., first or second) targeting moiety comprises a nucleic acid sequence that targets a nucleotide in an exon adjacent to or immediately adjacent to an intron at the 3' end of the exon. In some embodiments, the (e.g., first or second) targeting moiety comprises a nucleic acid sequence that targets a nucleotide in an exon adjacent to or immediately adjacent to an intron at the 5' end of the exon. In some embodiments, the (e.g., first or second) targeting moiety comprises a nucleic acid sequence comprising 5'-AA-3', 5'-AT-3', 5'-AC-3', 5'-AG-3', 5'-TA-3', 5'-TT-3', 5'-TC-3', 5'-TG-3', 5'-CA-3', 5'-CT-3', 5'-CC-3', 5'-CG-3', 5'-GA-3', 5'-GT-3', 5'-GC-3', or 5'-GG-3' that targets a nucleotide in an exon. In some embodiments, the (e.g., first or second) targeting moiety comprises a nucleic acid sequence that targets a nucleotide in an exon immediately adjacent to an intron at the 5' end of the exon. In some embodiments, the (e.g., first or second) targeting moiety comprises a nucleic acid sequence comprising 5'-AA-3', 5'-AT-3', 5'-AC-3', 5'-AG-3', 5'-TA-3', 5'-TT-3', 5'-TC-3', 5'-TG-3', 5'-CA-3', 5'-CT-3', 5'-CC-3', 5'-CG-3', 5'-GA-3', 5'-GT-3', 5'-GC-3', or 5'-GG-3' that targets a nucleotide in an exon adjacent to an intron. In some embodiments, the (e.g., first or second) targeting moiety comprises a nucleic acid sequence that targets a nucleotide in an exon immediately adjacent to an intron at the 5' end of the exon.In some embodiments, the (e.g., first or second) targeting moiety comprises a nucleic acid sequence comprising 5'-AA-3', 5'-AT-3', 5'-AC-3', 5'-AG-3', 5'-TA-3', 5'-TT-3', 5'-TC-3', 5'-TG-3', 5'-CA-3', 5'-CT-3', 5'-CC-3', 5'-CG-3', 5'-GA-3', 5'-GT-3', 5'-GC-3', or 5'-GG-3' that targets a nucleotide in an exon 5' to an intron. In some embodiments, the (e.g., first or second) targeting moiety comprises a nucleic acid sequence comprising 5'-AA-3', 5'-AT-3', 5'-AC-3', 5'-AG-3', 5'-TA-3', 5'-TT-3', 5'-TC-3', 5'-TG-3', 5'-CA-3', 5'-CT-3', 5'-CC-3', 5'-CG-3', 5'-GA-3', 5'-GT-3', 5'-GC-3', or 5'-GG-3' that targets a nucleotide in an exon 3' to an intron. In some embodiments, the (e.g., first or second) targeting moiety comprises a nucleic acid sequence comprising 5'-AA-3', 5'-AT-3', 5'-AC-3', 5'-AG-3', 5'-TA-3', 5'-TT-3', 5'-TC-3', 5'-TG-3', 5'-CA-3', 5'-CT-3', 5'-CC-3', 5'-CG-3', 5'-GA-3', 5'-GT-3', 5'-GC-3', or 5'-GG-3' that targets a nucleotide in an exon that is not immediately adjacent to an intron.

[0046] In some embodiments, the (e.g., first or second) targeting moiety comprises a nucleic acid sequence that targets a nucleotide in an exon. In some embodiments, the (e.g., first or second) targeting moiety comprises a nucleic acid sequence that targets a nucleotide in an intron adjacent to or immediately adjacent to an exon. In some embodiments, the (e.g., first or second) targeting moiety comprises a nucleic acid sequence that targets a nucleotide in an intron adjacent to or immediately adjacent to an exon at the 5' end of the intron. In some embodiments, the (e.g., first or second) targeting moiety comprises a nucleic acid sequence that targets a nucleotide in an intron adjacent to or immediately adjacent to an exon at the 5' end of the intron. In some embodiments, the (e.g., first or second) targeting moiety comprises a nucleic acid sequence that targets a nucleotide located entirely within the intron. In some embodiments, the (e.g., first or second) targeting moiety comprises a nucleic acid sequence comprising 5'-AA-3', 5'-AT-3', 5'-AC-3', 5'-AG-3', 5'-TA-3', 5'-TT-3', 5'-TC-3', 5'-TG-3', 5'-CA-3', 5'-CT-3', 5'-CC-3', 5'-CG-3', 5'-GA-3', 5'-GT-3', 5'-GC-3', or 5'-GG-3' that targets a nucleotide in an intron adjacent to the exon. In some embodiments, the (e.g., first or second) targeting moiety comprises a nucleic acid sequence comprising 5'-AA-3', 5'-AT-3', 5'-AC-3', 5'-AG-3', 5'-TA-3', 5'-TT-3', 5'-TC-3', 5'-TG-3', 5'-CA-3', 5'-CT-3', 5'-CC-3', 5'-CG-3', 5'-GA-3', 5'-GT-3', 5'-GC-3', or 5'-GG-3' that targets a nucleotide in an intron and immediately adjacent to an exon at the 5' end of the intron.In some embodiments, the (e.g., first or second) targeting moiety comprises a nucleic acid sequence comprising 5'-AA-3', 5'-AT-3', 5'-AC-3', 5'-AG-3', 5'-TA-3', 5'-TT-3', 5'-TC-3', 5'-TG-3', 5'-CA-3', 5'-CT-3', 5'-CC-3', 5'-CG-3', 5'-GA-3', 5'-GT-3', 5'-GC-3', or 5'-GG-3' that targets a nucleotide in an intron and immediately adjacent to an exon at the 3' end of the intron. In some embodiments, the (e.g., first or second) targeting moiety comprises a nucleic acid sequence comprising 5'-AA-3', 5'-AT-3', 5'-AC-3', 5'-AG-3', 5'-TA-3', 5'-TT-3', 5'-TC-3', 5'-TG-3', 5'-CA-3', 5'-CT-3', 5'-CC-3', 5'-CG-3', 5'-GA-3', 5'-GT-3', 5'-GC-3', or 5'-GG-3' that targets a nucleotide in an intron that is not adjacent to an exon.

[0047] As described, the targeting moiety (e.g., the first or second) may be gene-agnostic, so that it can target a conserved region shared (or substantially similar) in the pre-mRNA of multiple different genes. Furthermore, the targeting moiety (e.g., the first or second) may target a specific gene or specific exons and introns of a gene. As an example, Table 1 provides MAPT gene exon-intron junction sequences for multiple exon-intron junctions. For example, using Table 1 (or other sequences from the gene or exon-intron junctions of the gene of interest), engineered polynucleotides can be designed to target specific exon-intron junctions, or can be designed to target more than one exon-intron junction. In some embodiments of the engineered polynucleotides described herein, the (e.g., first or second) targeting moiety comprises a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to or complementary to a sequence shown in Table 1. In some embodiments of the engineered polynucleotides described herein, the (e.g., first or second) targeting moiety comprises a sequence that is identical to or complementary to a sequence shown in Table 1. In some embodiments, the (e.g., first or second) targeting moiety comprises a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to or complementary to a sequence selected from the "Exon Sequence" column of Table 1 and the "Intron Sequence" column of Table 1. In some embodiments, the (e.g., first or second) targeting moiety comprises a sequence that is identical to or complementary to a sequence selected from the "Exon Sequence" column of Table 1 and the "Intron Sequence" column of Table 1.

[0048] In some embodiments, the first targeting moiety is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to or complementary to a sequence selected from the "Exon Sequence" column of Table 1. In some embodiments, the first targeting moiety comprises a sequence identical to or complementary to a sequence selected from the "Exon Sequence" column of Table 1, and the second targeting moiety comprises a sequence at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to or complementary to a sequence selected from the "Exon Sequence" column of Table 1, and the second targeting moiety comprises a sequence identical to or complementary to a sequence shown in the "Intron Sequence" column of Table 1. In some embodiments, the first targeting moiety is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to or complementary to a sequence shown in the "Intron Sequence" column of Table 1. In some embodiments, the first targeting moiety comprises a sequence identical to or complementary to a sequence shown in the "Intron Sequence" column of Table 1, and the second targeting moiety comprises a sequence at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to or complementary to a sequence shown in the "Exon Sequence" column of Table 1. In some embodiments, the first targeting moiety comprises a sequence identical to or complementary to a sequence shown in the "Intron Sequence" column of Table 1, and the second targeting moiety comprises a sequence identical to or complementary to a sequence shown in the "Exon Sequence" column of Table 1.In some embodiments, the (e.g., first or second) targeting moiety comprises a sequence identical to or complementary to a consensus sequence of an intron donor site (e.g., selected from GU, GT, GC, and CA). In some embodiments, the (e.g., first or second) targeting moiety comprises a sequence identical to or complementary to a consensus sequence of an exon donor site (e.g., G). In some embodiments, the (e.g., first or second) targeting moiety comprises a sequence identical to or complementary to a consensus sequence selected from GU, GC, G, and CA.

[0049] [Table 1-1]

[0050] [Table 1-2]

[0051] Exemplary consensus sequences include (e.g., 5'-) intron donor site #1: GU, (e.g., 5'-) intron donor site #2: GC, (e.g., 5'-) exon donor site #1: G, and (e.g., 5'-) intron donor site #3: CA.

[0052] The targeting moiety (e.g., first or second) can be specific to a gene or to a particular exon or intron of a gene. For example, the targeting moiety can be specific to an exon-intron junction and a portion of an exon or intron. In some embodiments, the targeting moiety is specific to a portion of a gene outside of a splice site. In some embodiments, the targeting moiety targets a specific gene. For example, the targeting moiety can include a splice site-specific and gene-specific section. As described elsewhere herein, the targeting moiety can include a consensus splice site sequence. By having a targeting moiety specific to a consensus splice site, the targeting moiety can be gene-independent and target multiple different mRNAs of different genes. Alternatively, the targeting moiety can include a gene-specific portion. The engineered polynucleotide may then be able to regulate the splicing of a specific target gene (as opposed to any gene / mRNA containing a consensus splice site). For example, the targeting moiety can include a sequence complementary to a sequence unique to MAPT.

[0053] Target sequence As described in the present disclosure, an engineered polynucleotide can include one or more targeting moieties. The targeting moiety can target a target sequence (e.g., configured to bind, hybridize, or otherwise interact with the target sequence), for example, the targeting moiety targets an adjacent or nearby region in the target sequence. The targeting moiety can be complementary to the targeting sequence. In some embodiments of the engineered polynucleotide described herein, the first targeting sequence and the second targeting sequence are separated in the target sequence by a spacing sequence of 5, 4, or 3 nucleotides or less (e.g., 1 or 2 nucleotides).

[0054] In some embodiments of the engineered polynucleotides described herein, the first targeting sequence and the second targeting sequence are contiguous or adjacent to one another.

[0055] In some embodiments, when a spacing sequence in a target sequence is adjacent to the 5' or 3' end of a targeting sequence of the target sequence, the spacing sequence may not be complementary to the targeting portion of an engineered polynucleotide. In some embodiments, when a spacing sequence in a target sequence is adjacent to the 5' or 3' end of a targeting sequence of the target sequence, the spacing sequence may not be complementary to any targeting portion of an engineered polynucleotide.

[0056] In some embodiments, the spacing sequence separates the first targeting sequence and the second targeting sequence described herein.In some embodiments, the spacing sequence is not complementary to the targeting portion of the engineered polynucleotide and does not bind to it.In some embodiments, the spacing sequence is not complementary to all of the targeting portions of the engineered polynucleotide and does not bind to them.

[0057] In some embodiments of the engineered polynucleotides described herein, the target sequence may comprise an exon-intron boundary in an RNA (e.g., an mRNA, such as a pre-mRNA). In some embodiments, both the first targeting sequence and the second targeting sequence are 5' or 3' to the exon-intron boundary. In some embodiments, one of the first targeting sequence and the second targeting sequence is 5' to the exon-intron boundary, and the other of the first targeting sequence and the second targeting sequence is 3' to the exon-intron boundary.

[0058] In some embodiments of the engineered polynucleotides described herein, the target sequence comprises a splice site in an RNA (e.g., an mRNA, such as a pre-mRNA). In some embodiments, the (e.g., first or second) targeting sequence comprises a splice site (e.g., a 5'ss) in an RNA (e.g., an mRNA, such as a pre-mRNA).

[0059] In some embodiments, the two targeting sequences (e.g., the first targeting sequence and the second targeting sequence) are part of a single nucleic acid molecule (i.e., RNA, e.g., mRNA, such as pre-mRNA). In some embodiments, the first and second targeting sequences are spaced apart on the single nucleic acid molecule. In some embodiments, the first and second targeting sequences span an exon-intron boundary of the single nucleic acid molecule. In some embodiments, the first and second targeting sequences do not span an exon-intron boundary of the single nucleic acid molecule. In some embodiments, the first and second targeting sequences are adjacent to an exon-intron boundary of the single nucleic acid molecule. In some embodiments, the first and second targeting sequences both target an intron of the single nucleic acid molecule. In some embodiments, the first and second targeting sequences span a splice site of the single nucleic acid molecule. In some embodiments, the first and second targeting nucleic acid sequences do not span a splice site of the single nucleic acid molecule.

[0060] In some embodiments of the engineered polynucleotides described herein, the consensus sequence in the target sequence comprises from about 2 to about 15 nucleotides, from about 2 nucleotides to about 10 nucleotides, or from about 2 nucleotides to about 8 nucleotides.

[0061] In some embodiments, the engineered polynucleotide is complementary to and binds to a target sequence. In some embodiments, at least a portion of the engineered polynucleotide binds to the target sequence. In some embodiments, the target sequence encodes a gene (e.g., a target gene). A non-limiting example of a gene can include microtubule-associated protein tau (MAPT).

[0062] In some embodiments, the target sequence comprises an RNA sequence. In some embodiments, the RNA is nuclear RNA, cytoplasmic RNA, or mitochondrial RNA. In some embodiments, the target RNA sequence comprises messenger RNA (mRNA), pre-messenger RNA (pre-mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), ribozyme, recombinant polynucleotide, branched polynucleotide, isolated RNA, guide RNA, oligonucleotide, nucleic acid probe, primer, snRNA, long non-coding RNA, small RNA, snoRNA, siRNA, miRNA, tRNA-derived small RNA (tsRNA), antisense RNA, shRNA, or small rDNA-derived RNA (srRNA). In some embodiments, the target RNA sequence is pre-mRNA. In some embodiments, the engineered polynucleotide is not an antisense oligonucleotide.

[0063] In some embodiments, the target RNA sequence comprises at least one exon. In some embodiments, the target RNA sequence comprises at least one intron. In some embodiments, the target RNA sequence comprises at least one exon or at least one intron. In some embodiments, the target RNA sequence comprises at least one exon-intron boundary. The target RNA sequence may comprise a gene-specific sequence. For example, the target RNA sequence may comprise a sequence encoding a polypeptide. For example, the target RNA sequence may comprise a sequence encoding tau. The target sequence may comprise a sequence corresponding to an exon-intron boundary and a sequence corresponding to a specific gene or a consensus / conserved sequence present in many genes.

[0064] In some embodiments, the target sequence is an endogenous nucleic acid molecule. In some embodiments, binding of the engineered polynucleotide to the target sequence is by base pairing, such as Watson-Crick base pairing.

[0065] Mobilization part As described herein, the engineered polynucleotide can include a recruitment moiety. The recruitment moiety can recruit one or more components of the spliceosome. In some embodiments, a recruitment moiety configured to recruit a spliceosome moiety refers to a recruitment moiety that hybridizes to the spliceosome moiety. In some embodiments, a recruitment moiety configured to recruit a spliceosome moiety refers to a recruitment moiety that is at least 80%, 90%, or 100% complementary to the spliceosome moiety or the sequence of the spliceosome moiety. In some embodiments, "recruitment" refers to the formation of at least one hydrogen bond between the recruitment moiety and the spliceosome moiety. In some embodiments, "recruitment" refers to hybridization between at least one nucleotide of the recruitment moiety and at least one nucleotide of the spliceosome moiety. The recruitment moiety comprises a hairpin structure. The hairpin may be a complete hairpin or may have an internal loop intervening. The hairpin structure may consist of 13 to 17 nucleotides. The recruitment moiety may interact with stem-loop II of the U1 snRNA. The recruitment moiety can contain a sequence complementary to the sequence or portion of stem-loop II. For example, stem-loop II of U1 snRNA contains the sequence 5'-GUAGGCCUCACGUUACCUAU-3', and the recruitment moiety can contain 5'-CCGGA-3'. Ul-A can also bind to stem-loop II of U1 snRNA. Hydrogen bridges in stem-loop II with the hairpin / internal loop region can indirectly regulate polyadenylation and acetylation signaling by U1-A. The recruitment moiety may not silence the anchor domain of U1-A in stem-loop II. This interaction can regulate gene expression and acetylation.

[0066] In some embodiments of the engineered polynucleotides described herein, the recruitment portion comprises a nucleotide sequence that is at least 70%, 80%, 85%, or 90% identical to or complementary to a sequence shown in Table 2. In some embodiments, the recruitment portion comprises a nucleotide sequence that is identical to or complementary to a sequence shown in Table 2. In some embodiments, the engineered polynucleotide comprises a nucleotide sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to or complementary to any sequence selected from SEQ ID NOs: 1-2. In some embodiments, the engineered polynucleotide comprises a nucleotide sequence that is at least 70%, 80%, 85%, or 90% identical to or complementary to any sequence selected from SEQ ID NOs: 1-2. In some embodiments, the engineered polynucleotide comprises a nucleotide sequence that is identical to or complementary to any sequence selected from SEQ ID NOs: 1-2.

[0067] [Table 2]

[0068] In some embodiments described herein, the engineered polynucleotide (e.g., recruitment portion) comprises a (e.g., secondary) structural feature (see Figures 2A-2D). In some embodiments, the engineered polynucleotide (e.g., recruitment portion) comprises an apical loop, an upper stem, an internal loop, a lower stem, or a combination thereof. In some embodiments, the engineered polynucleotide (e.g., recruitment portion) comprises a loop (e.g., an internal loop) adjacent to a stem (e.g., a lower stem or an upper stem) comprising two complementary stem sequences. In some embodiments, the stem sequence of the stem (e.g., a lower stem or an upper stem) comprises about 5 or fewer, about 4 or fewer, or about 3 or fewer nucleotides. In some embodiments, the loop is an internal loop adjacent to a stem (e.g., a lower stem) and an additional stem (e.g., an upper stem) comprising two complementary stem sequences. In some embodiments, the internal loop comprises a nucleic acid sequence of 10 or fewer, 9 or fewer, or 8 or fewer nucleotides. In some embodiments, the stem sequence of the additional stem (e.g., upper stem) comprises about 5 or fewer, 4 or fewer, or 3 or fewer nucleotides. In some embodiments, the engineered polynucleotide (e.g., recruitment moiety) further comprises an apical loop. In some embodiments, the apical loop comprises a nucleic acid sequence of 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, or 5 or fewer nucleotides.

[0069] In some embodiments of the engineered polynucleotides described herein, the recruitment portion comprises about 10 to about 30 nucleotides, about 10 to about 25 nucleotides, or about 10 to about 20 nucleotides.

[0070] In some embodiments, the recruitment moiety is partially complementary to a post-transcriptional regulatory moiety (i.e., regulatory moiety) comprising a ribonucleoprotein complex (e.g., a spliceosome moiety). For example, the recruitment moiety can be partially complementary to a regulatory moiety comprising a spliceosome ribonucleoprotein complex, which comprises a small nuclear ribonucleic acid (snRNA). In some embodiments, the recruitment moiety is not complementary to and does not bind to a target sequence described herein. For example, the recruitment moiety is not complementary to and does not bind to a pre-mRNA described herein.

[0071] Structural composition In various aspects, an engineered polynucleotide comprises one or more moieties capable of performing a function. These one or more moieties can be present in the engineered polypeptide in various structural configurations that allow the engineered polynucleotide to perform a given function (e.g., recruiting regulatory moieties or components of the spliceosome, or binding to pre-mRNA). In some embodiments of the engineered polynucleotides described herein, one of the first targeting moiety and the second targeting moiety is 5' to the recruitment moiety, and the other of the first targeting moiety and the second targeting moiety is 3' to the recruitment moiety.

[0072] In some embodiments, the engineered polynucleotide has the following structural arrangement from the 5' to the 3' end: a first targeting moiety, a recruitment moiety, and a second targeting moiety. In some embodiments, the engineered polynucleotide has the following structural arrangement from the 5' to the 3' end: a second targeting moiety, a recruitment moiety, and a first targeting moiety.

[0073] Examples of Polynucleotides In some embodiments of the engineered polynucleotides described herein, the engineered polynucleotide comprises about 10 to about 40 nucleotides, about 10 to about 35 nucleotides, about 10 to about 30 nucleotides, or about 10 to about 25 nucleotides. In some embodiments, the engineered polynucleotide comprises at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, or more nucleotides in length. In some embodiments, an engineered polynucleotide comprises at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, or fewer nucleotides in length. In some embodiments, an engineered polynucleotide comprises at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50 nucleotides in length, or a range between any two of the foregoing values.

[0074] In some embodiments of the engineered polynucleotides described herein, the recruitment portion comprises a nucleotide sequence that is at least 70%, 80%, 85%, or 90% identical to or complementary to a sequence shown in Table 3. In some embodiments, the recruitment portion comprises a nucleotide sequence that is identical to or complementary to a sequence shown in Table 3.

[0075] [Table 3]

[0076] In some embodiments, an engineered polynucleotide can be generated from a precursor of the engineered polynucleotide. Optionally, the precursor of the engineered polynucleotide can be linear. For example, the precursor of the engineered polynucleotide can be a linear polynucleotide transcribed from a plasmid. In another example, the precursor of the engineered polynucleotide can be constructed to be a linear polynucleotide having moieties such as a ribozyme moiety and a ligation moiety that allow circularization of the engineered polynucleotide in a cell. The linear engineered polynucleotide having the ligation and ribozyme moiety can be transfected into a cell, where it can be circularized. Optionally, the engineered polynucleotide can be circular. Optionally, the engineered polynucleotide comprises DNA, RNA, or both. Optionally, the precursor of the engineered polynucleotide comprises a precursor of the engineered polynucleotide. Optionally, the precursor of the engineered polynucleotide can be used to generate the engineered polynucleotide.

[0077] In some embodiments, the engineered polynucleotide comprises at least one secondary structure (e.g., as described elsewhere herein). For example, the engineered polynucleotide comprises at least one, two, three, four, or more secondary structures, which may be any one or any combination of an apical loop, a stem, a stem-loop, or an internal loop. The one or more secondary structures may serve a variety of different functions. For example, the secondary structure may provide stability or otherwise help stabilize the engineered polynucleotide. The secondary structure may bind to or interact with a polypeptide or other polynucleotide. For example, the secondary structure may interact with a component of the spliceosome. In some embodiments, the recruitment portion of the polynucleotide comprises at least one, two, three, four, or more secondary structures. In some embodiments, the targeting portion does not have a secondary structure. In some embodiments, the secondary structure is an apical loop comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides. In some embodiments, the apical loop is complementary to and binds to the regulatory portion. In some embodiments, the apical loop is not complementary to and does not bind to the regulatory portion. In some embodiments, the secondary structure is at least one stem. In some embodiments, the engineered polynucleotide comprises two stems, one an upper stem near the apical loop and the other a lower stem near the targeting portion. In some embodiments, the upper stem comprises at least 2, 4, 6, 8, 10, or more nucleotides, where the nucleotides pair to form the upper stem. In some embodiments, the upper stem is complementary to and binds to the regulatory portion. In some embodiments, the upper stem is not complementary to and does not bind to the regulatory portion. In some embodiments, the secondary structure is a lower stem, where the lower stem comprises at least 2, 4, 6, 8, 10, or more nucleotides, where the nucleotides pair to form the lower stem. In some embodiments, the lower stem is complementary to and binds to the regulatory portion.In some embodiments, the lower stem is not complementary to and does not bind to the regulatory portion. In some embodiments, the engineered polynucleotide comprises an internal loop between the upper stem and the lower stem. In some embodiments, the internal loop comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides. In some embodiments, the internal loop is complementary to and binds to the regulatory portion. In some embodiments, the internal loop is not complementary to and does not bind to the regulatory portion. In some embodiments, the engineered polynucleotide comprises a secondary structure comprising an apical loop, an upper stem, an internal loop, and a lower stem, wherein the upper stem and internal loop are at least partially complementary to and bind to the regulatory portion. In some embodiments, the upper stem and internal loop are complementary to and bind to a regulatory portion comprising a snRNA. In some embodiments, the snRNA is a U1 snRNA, such as a US-A snRNA. In some embodiments, the snRNA is a U2 snRNA.

[0078] In some embodiments, the nucleic acid sequence of at least one secondary structure is partially complementary to a regulatory portion comprising a ribonucleoprotein complex. In some embodiments, the nucleic acid sequence of at least one secondary structure is not complementary to a targeting nucleic acid sequence. In some embodiments, the engineered polynucleotide comprises at least one nucleic acid secondary structure. In some embodiments, the engineered polynucleotide comprises at least one, two, three, four, or more nucleic acid secondary structures. In some embodiments, at least one secondary structure improves binding between a recruitment portion and a regulatory portion. In some embodiments, at least one secondary structure stabilizes assembly of a regulatory portion. In some embodiments, at least one secondary structure stabilizes assembly of a regulatory portion with other additional portions. In some embodiments, at least one secondary structure increases the efficiency of regulating expression or activity of a gene encoded by a target sequence. In some embodiments, at least one secondary structure increases the specificity of regulating expression or activity of a gene encoded by a target sequence. In some embodiments, at least one secondary structure increases the resistance of the engineered polynucleotide to hydrolytic degradation. In some embodiments, the at least one secondary structure increases the resistance of the engineered polynucleotide to degradation by nuclease digestion. In some embodiments, the at least one secondary structure increases the half-life of the engineered polynucleotide. In some embodiments, the at least one secondary structure reduces the immunogenicity induced by the engineered polynucleotide.

[0079] In some embodiments, the engineered polynucleotide is characterized by a secondary structure. In some embodiments, the secondary structure comprises one or more stem-loop structures. In some embodiments, the secondary structure comprises an apical loop, an upper stem, an internal loop, and a lower stem. In some embodiments, the engineered polynucleotide comprises at least one secondary structure. In some embodiments, the first or second targeting moiety is not part of the secondary structure. In some embodiments, the engineered polynucleotide comprises at least one, two, three, four, or more secondary structures. In some embodiments, the engineered polynucleotide comprises a secondary structure comprising a stem-loop, a cruciform, a toe hold, a mismatched bulge, or any combination thereof. In some embodiments, the engineered polynucleotide comprises a secondary structure comprising an apical loop, an upper stem, an internal loop, or a lower stem. In some embodiments, the engineered polynucleotide comprises a secondary structure comprising an apical loop, an upper stem, an internal loop, and a lower stem. In some cases, the secondary structure can comprise a stem, a hairpin loop, a pseudoknot, a bulge, an internal loop, a multi-loop, a G-quadruplex, or any combination thereof. In some embodiments, the engineered polynucleotide can adopt an A-form, a B-form, a Z-form, or any combination thereof. In some embodiments, the secondary structure is formed at least in part based on the nucleotide sequence of the engineered polynucleotide. In some embodiments, the secondary structure is formed within the nucleotide sequence of the engineered polynucleotide.

[0080] In some embodiments, at least one secondary structure improves binding between the recruitment moiety and the regulatory moiety, ie, at least one secondary structure improves binding between the recruitment moiety and the regulatory moiety by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more compared to binding between the recruitment moiety and the regulatory moiety without the secondary structure.

[0081] In some embodiments, at least one chemical modification improves binding between the recruitment moiety and the regulatory moiety, ie, at least one chemical modification improves binding between the recruitment moiety and the regulatory moiety by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more compared to binding between the recruitment moiety and the regulatory moiety without the chemical modification.

[0082] In some embodiments, at least one chemical modification of the engineered polynucleotide stabilizes the assembly of a spliceosome containing the regulatory moiety when the regulatory moiety is associated with a target sequence. In some embodiments, the assembly of a spliceosome containing the regulatory moiety is stabilized by an engineered polynucleotide containing a chemical modification by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more compared to a comparable polynucleotide without the chemical modification. In some embodiments, the regulatory moiety is U1 (U1 SNP), U2 (U2 SNP), U4, U5, U6, U11, U12, U14, or U16 of the spliceosome. In some embodiments, the regulatory moiety is U1 SNP of the spliceosome. In some embodiments, the regulatory moiety is U1-A of the spliceosome. In some embodiments, the regulatory moiety is U2 SNP of the spliceosome. In some embodiments, at least one chemical modification of the engineered polynucleotide stabilizes the assembly of a spliceosome comprising a regulatory portion and at least one additional portion. For example, at least one chemical modification of the engineered polynucleotide stabilizes the assembly of a spliceosome comprising a regulatory portion comprising U1-A and at least one additional portion comprising U1-70K, UC-1, SmD1, SmD2, SmD3, SmE, SmF, or SmG. In some embodiments, the at least one additional portion is U4, U5, U6, U11, U12, U14, or U16 of the spliceosome.

[0083] In some embodiments, the at least one chemical modification increases the efficiency of the engineered polynucleotide to modulate expression or activity of a gene encoded by the target sequence compared to an equivalent polynucleotide without the chemical modification, hi some embodiments, the efficiency of the engineered polynucleotide to modulate expression or activity of a gene encoded by the target sequence is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more compared to the efficiency of an equivalent polynucleotide without the chemical modification to modulate expression or activity of a gene encoded by the target sequence.

[0084] In some embodiments, the at least one chemical modification increases the specificity of the engineered polynucleotide to modulate the expression or activity of a gene encoded by the target sequence compared to an equivalent polynucleotide without the chemical modification, hi some embodiments, the specificity of the engineered polynucleotide to modulate the expression or activity of a gene encoded by the target sequence is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more compared to the efficiency of an equivalent polynucleotide without the chemical modification to modulate the expression or activity of a gene encoded by the target sequence.

[0085] In some embodiments, at least one chemical modification increases the resistance of the engineered polynucleotide to hydrolytic degradation (e.g., endonuclease-mediated degradation). In some embodiments, at least one chemical modification increases the resistance of the engineered polynucleotide to hydrolytic degradation compared to the resistance of an equivalent engineered polynucleotide without the chemical modification. In some embodiments, the resistance of an engineered polynucleotide comprising at least one chemical modification to hydrolytic degradation is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more compared to the resistance of an equivalent engineered polynucleotide without the chemical modification.

[0086] In some embodiments, at least one chemical modification increases the resistance of the engineered polynucleotide to degradation by nuclease digestion. In some embodiments, at least one chemical modification increases the resistance of the engineered polynucleotide to degradation by nuclease digestion compared to the resistance of an equivalent engineered polynucleotide without the chemical modification. In some embodiments, the resistance of an engineered polynucleotide comprising at least one chemical modification to degradation by nuclease digestion is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more compared to the resistance of an equivalent engineered polynucleotide without the chemical modification.

[0087] In some embodiments, at least one chemical modification increases the half-life of the engineered polynucleotide compared to the half-life of an equivalent engineered polynucleotide without the chemical modification. In some embodiments, the half-life of an engineered polynucleotide comprising at least one chemical modification is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more compared to the half-life of an equivalent engineered polynucleotide without the chemical modification. The chemical modification increases the half-life of the engineered polynucleotide.

[0088] In some embodiments, at least one chemical modification reduces the immunogenicity induced by the engineered polynucleotide. In some embodiments, at least one chemical modification reduces the immunogenicity induced by the engineered polynucleotide compared to the immunogenicity of an equivalent engineered polynucleotide without the chemical modification. In some embodiments, the immunogenicity of an engineered polynucleotide comprising at least one chemical modification is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more compared to the immunogenicity of an equivalent engineered polynucleotide without the chemical modification.

[0089] chemical modification In some embodiments described herein, the engineered polynucleotide comprises at least one chemical modification. As described in this disclosure, chemical modifications may confer structural or functional advantages on the engineered polynucleotide (e.g., increased half-life), reduced immunogenicity, increased resistance to hydrolysis or enzymatic degradation, or improved reactivity or binding to polypeptides (e.g., components of the spliceosome) or polynucleotides (e.g., pre-mRNA, or components of the spliceosome).

[0090] In some embodiments, all nucleotides of the targeting moiety are linked by phosphorothioate bonds. In some embodiments, all nucleotides of the targeting moiety contain 2' O-methyl modifications. The 2' modifications can prevent nuclease degradation and / or increase the affinity of the targeting moiety for the pre-mRNA target.

[0091] In some embodiments, all nucleotides of the recruitment moiety are linked by phosphorothioate bonds. In some embodiments, three nucleotides of the recruitment moiety contain 2' O-methyl modifications. The 2' modifications can induce changes in the molecular dynamics of the recruitment moiety, thereby promoting a conformational change in stem-loop II of the U1-snRNA and binding of the recruitment moiety to the U1-snRNA.

[0092] In some embodiments, an engineered polynucleotide comprises at least one 2'-modified (e.g., 2'-methoxy, 2'-methoxymethyl, or 2'-methoxyethyl) nucleotide. In some embodiments, at least about 50%, 60%, 70%, 80%, or 90% of the nucleotides of an engineered polynucleotide are chemically modified nucleotides. In some embodiments, at least about 50%, 60%, 70%, 80%, or 90% of the nucleotides of an engineered polynucleotide comprise 2'-modified (e.g., 2'-methoxy, 2'-methoxymethyl, or 2'-methoxyethyl) nucleotides. In some embodiments, an engineered polynucleotide comprises at least one phosphorothioate internucleotide linkage. In some embodiments, at least about 50%, 60%, 70%, 80%, or 90% of the internucleotide linkages of an engineered polynucleotide are chemically modified. In some embodiments, at least about 50%, 60%, 70%, 80%, or 90% of the internucleotide linkages are phosphorathioate.

[0093] In some embodiments, the engineered polynucleotide comprises at least one chemical modification of the nucleic acid. In some embodiments, the engineered polynucleotide comprises at least one, two, three, four, or more chemical modifications of the nucleic acid. In some embodiments, at least one chemical modification improves the bond between the recruitment moiety and the regulatory moiety. In some embodiments, at least one chemical modification stabilizes the assembly of the regulatory moiety. In some embodiments, at least one chemical modification stabilizes the assembly of the regulatory moiety and other additional moieties. In some embodiments, at least one chemical modification increases the efficiency of regulating the expression or activity of a gene encoded by a target sequence. In some embodiments, at least one chemical modification increases the specificity of regulating the expression or activity of a gene encoded by a target sequence. In some embodiments, at least one chemical modification increases the resistance of the engineered polynucleotide to degradation by hydrolysis. In some embodiments, at least one chemical modification increases the resistance of the engineered polynucleotide to degradation by nuclease digestion. In some embodiments, at least one chemical modification increases the half-life of the engineered polynucleotide. In some embodiments, at least one chemical modification reduces immunogenicity induced by the engineered polynucleotide.

[0094] In some embodiments, the chemical modification of the engineered polynucleotide comprises at least one substitution for one or both of the non-linking phosphate oxygen atoms in the phosphodiester backbone linkages of the engineered polynucleotide. In some embodiments, the at least one chemical modification of the engineered polynucleotide comprises one or more substitutions of the linking phosphate oxygen atoms in the phosphodiester backbone linkages of the engineered polynucleotide. A non-limiting example of a chemical modification of a phosphate oxygen atom is a sulfur atom. Further non-limiting examples are included in Table 3. In some embodiments, the chemical modification of the engineered polynucleotide comprises at least one chemical modification to the sugar of a nucleotide of the engineered polynucleotide. In some embodiments, the chemical modification of the engineered polynucleotide comprises at least one chemical modification to the sugar of a nucleotide of the engineered polynucleotide, wherein the chemical modification comprises at least one locked nucleic acid (LNA). In some embodiments, the chemical modification of the engineered polynucleotide comprises at least one chemical modification to the sugar of a nucleotide of the engineered polynucleotide comprising at least one unlocked nucleic acid (UNA). In some embodiments, the chemical modification of the engineered polynucleotide comprises at least one chemical modification to the sugar, wherein the sugar is a ribose sugar, wherein the sugar is a ribose sugar. In some embodiments, the chemical modification of the engineered polynucleotide comprises at least one chemical modification to a ribose sugar component of a nucleotide of the engineered polynucleotide, which comprises a 2'-O-methyl group. In some embodiments, the chemical modification comprises 2'-F-RNA instead of a 2'-O-methyl group modification. In such cases, the 2'-F-RNA and pre-mRNA duplex does not activate RNase H (degradation by nuclease digestion) and is more stable, as determined by a higher melting temperature (Tm), than the 2'-O-methyl-RNA and pre-mRNA duplex.

[0095] In some embodiments, the chemical modification of the engineered polynucleotide comprises at least one chemical modification comprising a substitution of a phosphate moiety of the engineered polynucleotide with a dephosphoryl linker. In some embodiments, the chemical modification of the engineered polynucleotide comprises at least one chemical modification of the phosphate backbone of the engineered polynucleotide. In some embodiments, the engineered polynucleotide comprises a phosphothioate group. In some embodiments, the chemical modification of the engineered polynucleotide comprises at least one chemical modification comprising a modification to a base of a nucleotide of the engineered polynucleotide. In some embodiments, the chemical modification of the engineered polynucleotide comprises at least one chemical modification comprising a non-natural base of a nucleotide. In some embodiments, the chemical modification of the engineered polynucleotide comprises at least one chemical modification comprising a morpholino group, a cyclobutyl group, a pyrrolidine group, or a peptide nucleic acid (PNA) nucleoside surrogate. In some embodiments, the chemical modification of the engineered polynucleotide comprises at least one chemical modification comprising at least one stereopure nucleic acid. In some embodiments, at least one chemical modification can be located proximal to the 5' end of the engineered polynucleotide. In some embodiments, at least one chemical modification can be located proximal to the 3' end of the engineered polynucleotide, hi some embodiments, at least one chemical modification can be located proximal to both the 5' and 3' ends of the engineered polynucleotide.

[0096] In some embodiments, at least one chemical modification of an engineered polynucleotide comprises modification of one or both of the non-linking phosphate oxygens in a phosphodiester backbone linkage, modification of one or more of the linking phosphate oxygens in a phosphodiester backbone linkage, modification of a component of the ribose sugar, replacement of a phosphate moiety with a "dephospho" linker, modification or replacement of a naturally occurring nucleobase, modification of the ribose-phosphate backbone, modification of the 5' end of a polynucleotide, modification of the 3' end of a polynucleotide, modification of the deoxyribose phosphate backbone, replacement of a phosphate group, modification of the ribophosphate backbone, modification to the sugar of a nucleotide, modification to the base of a nucleotide, or stereopurity of a nucleotide, or any combination thereof. Examples of chemical modifications for engineered polynucleotides can be found in Table 4.

[0097] [Table 4-1]

[0098] [Table 4-2]

[0099] Modification of the phosphate backbone

[0100] In some embodiments, chemical modifications include modification of one or both of the non-linking phosphate oxygens in the phosphodiester backbone linkages or modification of one or more of the linking phosphate oxygens in the phosphodiester backbone linkages. As used herein, "alkyl" is intended to refer to a saturated hydrocarbon group that is linear or branched. Examples of alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl or isopropyl), butyl (e.g., n-butyl, isobutyl, or t-butyl), or pentyl (e.g., n-pentyl, isopentyl, or neopentyl). Alkyl groups can contain 1 to about 20, 2 to about 20, 1 to about 12, 1 to about 8, 1 to about 6, 1 to about 4, or 1 to about 3 carbon atoms. As used herein, "aryl" refers to a monocyclic or polycyclic (e.g., having 2, 3, or 4 fused rings) aromatic hydrocarbon, such as phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, or indenyl. In some embodiments, an aryl group has 6 to about 20 carbon atoms. As used herein, "alkenyl" refers to an aliphatic group containing at least one double bond. As used herein, "alkynyl" refers to a straight or branched hydrocarbon chain containing 2 to 12 carbon atoms and characterized by one or more triple bonds. Examples of alkynyl groups include ethynyl, propargyl, and 3-hexynyl. "Arylalkyl" or "aralkyl" refers to an alkyl moiety in which an alkyl hydrogen atom is replaced by an aryl group. Aralkyl includes groups in which one or more hydrogen atoms are replaced by an aryl group. Examples of "arylalkyl" or "aralkyl" include benzyl, 2-phenylethyl, 3-phenylpropyl, 9-fluorenyl, benzhydryl, and trityl. "Cycloalkyl" refers to a cyclic, bicyclic, tricyclic, or polycyclic non-aromatic hydrocarbon group having 3 to 12 carbon atoms. Examples of cycloalkyl moieties include, but are not limited to, cyclopropyl, cyclopentyl, and cyclohexyl. "Heterocyclyl" refers to a monovalent radical of a heterocyclic ring system.Representative heterocyclyls include, but are not limited to, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, pyrrolidonyl, piperidinyl, pyrrolinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, and morpholinyl. "Heteroaryl" refers to a monovalent radical of a heteroaromatic ring system. Examples of heteroaryl moieties include imidazolyl, oxazolyl, thiazolyl, triazolyl, pyrrolyl, furanyl, indolyl, thiophenylpyrazolyl, pyridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, indolizinyl, purinyl, naphthyridinyl, quinolyl, and pteridinyl.

[0101] In some embodiments, the phosphate group of a chemically modified nucleotide can be modified by replacing one or more oxygen atoms with different substituents. In some embodiments, the chemically modified nucleotide can include replacing the unmodified phosphate moiety with a modified phosphate as described herein. In some embodiments, the modification of the phosphate backbone can include a change that results in either an uncharged linker or a charged linker with an asymmetric charge distribution. Examples of modified phosphate groups can include phosphorothioate, phosphonothioacetate, phosphoroselenate, boranophosphate, boranophosphate ester, hydrogen phosphonate, phosphoramidate, alkyl or aryl phosphonate, and phosphotriester. In some embodiments, one of the non-bridging phosphate oxygen atoms in the phosphate backbone moiety can be replaced with any of the following groups: sulfur (S), selenium (Se), BR (R can be, for example, hydrogen, alkyl, or aryl), C (e.g., an alkyl group, an aryl group, etc.), H, NR (R can be, for example, hydrogen, alkyl, or aryl), or (R can be, for example, alkyl or aryl). The phosphorus atom in an unmodified phosphate group can be achiral. However, replacing one of the non-bridging oxygens with one of the above atoms or groups of atoms can make the phosphorus atom chiral. The phosphorus atom in such modified phosphate groups is a stereocenter. The asymmetric phosphorus atom can have either the "R" configuration (referred to herein as Rp) or the "S" configuration (referred to herein as Sp). In some cases, the engineered polynucleotides include stereomerically pure nucleotides comprising a phosphorothioate S configuration or a phosphorothioate R configuration. In some embodiments, the chiral phosphate product is present in a diastereomeric excess of 50%, 60%, 70%, 80%, 90% or more. In some embodiments, the chiral phosphate product is present in a diastereomeric excess of 95%. In some embodiments, the chiral phosphate product is present in a diastereomeric excess of 96%.In some embodiments, the chiral phosphate product is present in 97% diastereomeric excess. In some embodiments, the chiral phosphate product is present in 98% diastereomeric excess. In some embodiments, the chiral phosphate product is present in 99% diastereomeric excess. In some embodiments, both non-bridging oxygens of the phosphorodithioate can be replaced with sulfur. The phosphorus center in the phosphorodithioate can be achiral, preventing the formation of oligoribonucleotide diastereomers. In some embodiments, modifications to one or both non-bridging oxygens can further include replacing the non-bridging oxygen with a group independently selected from S, Se, B, C, H, N, and OR (R can be, for example, alkyl or aryl). In some embodiments, the phosphate linker can be further modified by replacing the bridging oxygen (i.e., the oxygen connecting the phosphate to the nucleoside) with nitrogen (bridging phosphoramidate), sulfur (bridging phosphorothioate), and carbon (bridging methylene phosphonate). Substitutions can occur at either or both of the linking oxygens.

[0102] In certain embodiments, nucleic acids include linked nucleic acids. Nucleic acids can be linked to each other using any internucleic acid linkage. Two major classes of internucleic acid linkage groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleic acid linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates (P=S). Representative non-phosphorus-containing internucleic acid linkages include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiesters (-OC(O)-S-), thionocarbamate (-OC(O)(NH)-S-), siloxane (-O-Si(H)2-O-), and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)). In certain embodiments, nucleic acid linkages having chiral atoms can be prepared as racemic mixtures or as separate enantiomers, such as alkylphosphonates and phosphorothioates.Non-natural nucleic acids can contain a single modification.Non-natural nucleic acids can contain multiple modifications in one of the multiple moieties or between different moieties.

[0103] Backbone phosphate modifications to nucleic acids include, but are not limited to, methylphosphonates, phosphorothioates, phosphoramidates (bridged or non-bridged), phosphotriesters, phosphorodithioates, phosphodithioates, and boranophosphates, which may be used in any combination. Other non-phosphate linkages may also be used.

[0104] In some embodiments, backbone modifications (e.g., methylphosphonate, phosphorothioate, phosphoramidate, and phosphorodithioate internucleotide linkages) can confer immunomodulatory activity to the modified nucleic acids and / or enhance their stability in vivo.

[0105] In some examples, the phosphorus derivative (or modified phosphate group) is attached to a sugar or sugar analog moiety and can be a monophosphate, diphosphate, triphosphate, alkylphosphonate, phosphorothioate, phosphorodithioate, phosphoramidate, etc.

[0106] In some cases, backbone modifications include replacing phosphodiester linkages with alternative moieties such as anionic, neutral, or cationic groups. Examples of such modifications include cationic internucleotide linkages, N3' to P5' phosphoramidate modifications, boranophosphate DNA, prooligonucleotides, neutral internucleotide linkages such as methylphosphonates, amide-linked DNA, methylene (methylimino) linkages, formacetal and thioformacetal linkages, backbones containing sulfonyl groups, morpholino oligos, peptide nucleic acids (PNAs), and positively charged deoxyribonucleic acid guanidine (DNG) oligos. Modified nucleic acids can include chimeric or mixed backbones containing one or more modifications, for example, a combination of phosphate linkages, such as a combination of phosphodiester and phosphorothioate linkages.

[0107] Phosphate substitutions include, for example, short-chain alkyl or cycloalkyl internucleotide linkages, mixed heteroatom and alkyl or cycloalkyl internucleotide linkages, or one or more short-chain heteroatom or heterocyclic internucleotide linkages. These include those with morpholino linkages (partially formed from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and other backbones with mixed N, O, S, and CH2 moieties. It is also understood that in nucleotide substitutions, both the sugar portion and the phosphate portion of the nucleotide can be replaced by, for example, amide-type linkages (aminoethylglycine) (PNA). For example, other types of molecules (conjugates) can be linked to nucleotides or nucleotide analogs to enhance cellular uptake. Conjugate can be chemically linked to nucleotide or nucleotide analogue.Such conjugate includes but is not limited to lipid moiety such as cholesterol moiety, thioether such as hexyl-S-tritylthiol, thiocholesterol, aliphatic chain such as dodecanediol or undecyl residue, phospholipid such as di-hexadecyl-rac-glycerol or triethylammonium l-di-O-hexadecyl-rac-glycero-SH-phosphonate, polyamine or polyethylene glycol chain, or adamantane acetic acid, palmityl moiety, or octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.

[0108] In some embodiments, the chemical modifications described herein include modifications of the phosphate backbone. In some embodiments, the engineered polynucleotides described herein include at least one chemically modified phosphate backbone. Examples of chemical modifications of the phosphate group or backbone include replacing one or more oxygens with different substituents. Furthermore, modified nucleotides present in the engineered polynucleotide can include replacing unmodified phosphate moieties with modified phosphates described herein. In some embodiments, the phosphate backbone modification can include changes that result in either an uncharged linker or a charged linker with an asymmetric charge distribution. Examples of modified phosphate groups include phosphorothioates, phosphonothioacetates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, alkyl or aryl phosphonates, and phosphotriesters. In some embodiments, one of the non-bridging phosphate oxygen atoms in the phosphate backbone moiety can be replaced with any of the following groups: sulfur (S), selenium (Se), BR (R can be, for example, hydrogen, alkyl, or aryl), C (e.g., an alkyl group, an aryl group, etc.), H, NR (R can be, for example, hydrogen, alkyl, or aryl), or (R can be, for example, alkyl or aryl). The phosphorus atom in an unmodified phosphate group is achiral. However, replacing one of the non-bridging oxygens with one of the above atoms or groups of atoms can make the phosphorus atom chiral, i.e., the phosphorus atom in the phosphate group modified in this manner is a stereogenic center. The asymmetric phosphorus atom can have either the "R" configuration (herein Rp) or the "S" configuration (herein Sp). In such cases, the chemically modified engineered polynucleotide can be stereochemically pure (e.g., S or R configuration). Optionally, the chemically modified engineered polynucleotide comprises stereogenic phosphate modifications.For example, chemically modified engineered polynucleotides include phosphorothioate S configurations or phosphorothioate R configurations.

[0109] In phosphorodithioates, both non-bridging oxygens are replaced by sulfur. The phosphorus center in phosphorodithioates is achiral, preventing the formation of oligoribonucleotide diastereomers. In some embodiments, modifications to one or both non-bridging oxygens can further include replacing the non-bridging oxygens with a group independently selected from S, Se, B, C, H, N, and OR (R can be, for example, alkyl or aryl).

[0110] The phosphate linker can be further modified by substituting the bridging oxygen (i.e., the oxygen that connects the phosphate to the nucleoside) with nitrogen (bridging phosphoramidates), sulfur (bridging phosphorothioates), and carbon (bridging methylene phosphonates). Substitutions can occur at either or both of the linking oxygens.

[0111] Phosphate moiety replacement

[0112] In some embodiments, at least one phosphate group of the engineered polynucleotide can be chemically modified. In some embodiments, the phosphate group can be replaced with a non-phosphorus-containing connector. In some embodiments, the phosphate moiety can be replaced with a dephosphorylated linker. In some embodiments, the charged phosphate group can be replaced with a neutral group. Optionally, the phosphate group can be replaced with methylphosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo, and methyleneoxymethylimino. In some embodiments, the nucleotide analogs described herein can also be modified at the phosphate group. Modified phosphate groups can include phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl, and other alkylphosphonates, including 3'-alkylenephosphonates and chiral phosphonates, phosphinate, phosphoramidate (for example, 3'-aminophosphoramidate and aminoalkylphosphoramidate), thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, and boranophosphate, which modify the linkage between two nucleotides.The phosphate or modified phosphate linkage between two nucleotides can be via a 3'-5' linkage or a 2'-5' linkage, and the linkage can include reverse polarity, such as 3'-5' to 5'-3' or 2'-5' to 5'-2'.

[0113] Phosphate group substitution

[0114] In some embodiments, the chemical modifications described herein include modifications by substitution of a phosphate group. In some embodiments, the engineered polynucleotides described herein include at least one chemical modification comprising a phosphate group substitution or replacement. Exemplary phosphate group substitutions can include non-phosphorus-containing connectors. In some embodiments, the phosphate group substitution or replacement can include replacing a charged phosphate group with a neutral moiety. Examples of moieties that can replace a phosphate group include methylphosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo, and methyleneoxymethylimino.

[0115] Modification of the ribophosphate backbone

[0116] In some embodiments, the chemical modification described herein comprises modifying the ribophosphate backbone of the engineered polynucleotide. In some embodiments, the engineered polynucleotide described herein comprises at least one chemically modified ribophosphate backbone. Examples of chemically modified ribophosphate backbones include scaffolds that mimic nucleic acids and can be constructed so that the phosphate linker and ribose sugar are replaced by nuclease-resistant nucleoside or nucleotide surrogates. In some embodiments, the nucleobases can be tethered by the surrogate backbone. Examples include morpholino, cyclobutyl, pyrrolidine, and peptide nucleic acid (PNA) nucleoside surrogates.

[0117] Sugar modifications

[0118] In some embodiments, the chemical modifications described herein include sugar modifications. In some embodiments, the engineered polynucleotides described herein comprise at least one chemically modified sugar. Examples of chemically modified sugars include 2' hydroxyl groups (OH) modified or substituted with a number of different "oxy" or "deoxy" substituents. In some embodiments, modifications to the 2' hydroxyl group can enhance nucleic acid stability because the hydroxyl cannot further deprotonate to form a 2'-alkoxide ion. 2'-alkoxides can catalyze decomposition by intramolecular nucleophilic attack on the linker atom. Examples of "oxy"-2' hydroxyl group modifications include alkoxy or aryloxy (OR, where "R" can be, for example, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or a sugar), polyethylene glycol (PEG), O(CHCHO), and hydroxyl groups. n Examples include CH2CH2OR (wherein R can be, for example, H or an optionally substituted alkyl, and n can be an integer from 0 to 20 (e.g., 0 to 4, 0 to 8, 0 to 10, 0 to 16, 1 to 4, 1 to 8, 1 to 10, 1 to 16, 1 to 20, 2 to 4, 2 to 8, 2 to 10, 2 to 16, 2 to 20, 4 to 8, 4 to 10, 4 to 16, and 4 to 20)). In some embodiments, the "oxy"-2' hydroxyl group modification is a hydroxyl group selected from the group consisting of LNA (where the 2' hydroxyl can be connected to the 4' carbon of the same ribose sugar, e.g., by a Ci-6 alkylene or Cj-6 heteroalkylene bridge, e.g., methylene, propylene, ether, or amino bridge, O-amino (amino can be, e.g., NH, alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino), and aminoalkoxy, O(CH) n-amino (amino can be, for example, NH2, alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino). In some embodiments, the "oxy"-2' hydroxyl group modification can include a methoxyethyl group (MOE), (OCH2CHOCH3, e.g., PEG derivatives). Optionally, the deoxy modification can include hydrogen (i.e., deoxyribose sugars, e.g., in partial overhanging portions of dsRNAs), halo (e.g., bromo, chloro, fluoro, or iodo), amino (amino can be, for example, NH2, alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid), NH(CH2CH2NH) nThe sugar group can include CH2CH2-amino (amino can be, for example, as described herein), NHC(O)R (R can be, for example, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar), cyano, mercapto, alkyl-thio-alkyl, thioalkoxy, and alkyl, cycloalkyl, aryl, alkenyl, and alkynyl, which can be optionally substituted with, for example, amino, as described herein. In some examples, the sugar group can also contain one or more carbons having the opposite stereochemical configuration to that of the corresponding carbon in ribose. Thus, modified nucleic acids can include nucleotides containing, for example, arabinose as the sugar. A nucleotide "monomer" can have an alpha linkage at the Γ position of the sugar, e.g., an alpha-nucleoside. Modified nucleic acids can further include "abasic" sugars lacking a nucleobase at C-. Abasic sugars can also be further modified at one or more of the constituent sugar atoms. Modified nucleic acids can also include one or more sugars that are in the L-form, e.g., an L-nucleoside. In some embodiments, the engineered polynucleotides described herein contain a sugar group, ribose, which is a five-membered ring containing oxygen. Exemplary modified nucleosides and nucleotides can include substitution of oxygen in ribose (e.g., with sulfur (S), selenium (Se), or an alkylene, such as methylene or ethylene), addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl), ring contraction of ribose (e.g., to form a four-membered cyclobutane or oxetane ring), or ring expansion of ribose (e.g., to form a six- or seven-membered ring with additional carbon or heteroatoms, such as anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino, which also have a phosphoramidate backbone). In some embodiments, modified nucleotides can include polycyclic forms (e.g., tricyclic), and "unlocked" forms, such as glycol nucleic acids (GNAs) (e.g., R-GNAs or S-GNAs in which the ribose is replaced with a glycol unit linked to a phosphodiester bond), threose nucleic acids.In some embodiments, modifications to the sugar of the engineered polynucleotide comprise modifying the engineered polynucleotide to comprise a locked nucleic acid (LNA), an unlocked nucleic acid (UNA), or a bridged nucleic acid (BNA).

[0119] Modification of the ribose sugar components

[0120] In some embodiments, the engineered polynucleotides described herein comprise at least one chemical modification of the ribose sugar moiety. In some embodiments, the chemical modification of the ribose sugar moiety can include 2'-O-methyl, 2'-O-methoxy-ethyl (2'-MOE), 2'-fluoro, 2'-aminoethyl, 2'-deoxy-2'-fuloarabinou-cleic acid, 2'-deoxy, 2'-O-methyl, 3'-phosphorothioate, 3'-phosphonoacetate (PACE), or 3'-phosphonothioacetate (thioPACE). In some embodiments, the chemical modification of the ribose sugar moiety comprises a non-natural nucleic acid. In some examples, the non-natural nucleic acid comprises modifications at the 5' and 2' positions of the sugar ring, such as a 5'-CH2-substituted 2'-O-protected nucleoside. In some cases, non-natural nucleic acids containing amide-linked nucleoside dimers have been prepared for incorporation into oligonucleotides, with the 3'-linked nucleosides (5' to 3') in the dimers containing 2'-OCH3 and 5'-(S)-CH3. The non-natural nucleic acids can contain 2'-substituted 5'-CH2 (or O) modified nucleosides. The non-natural nucleic acids can contain 5'-methylene phosphonate DNA and RNA monomers and dimers. The non-natural nucleic acids can contain 5'-phosphonate monomers with 2'-substitutions and other modified 5'-phosphonate monomers. The non-natural nucleic acids can contain 5'-modified methylene phosphonate monomers. The non-natural nucleic acids can contain 5'- or 6'-phosphonate ribonucleoside analogs containing hydroxyl groups at the 5' and / or 6' positions. Non-natural nucleic acids can include 5'-phosphonate deoxyribonucleoside monomers and dimers having a 5'-phosphate group. Non-natural nucleic acids can include nucleosides having a 6'-phosphonate group, where the 5' and / or 6' positions are unsubstituted or substituted with a thio-tert-butyl group (SC(CH)) (and its analogs), a methyleneamino group (CHNH) (and its analogs), or a cyano group (CN) (and its analogs).

[0121] In some embodiments, the non-natural nucleic acid further comprises a modification of the sugar moiety. Optionally, the nucleic acid contains one or more nucleosides in which the sugar group has been modified. Such sugar-modified nucleosides may confer enhanced nuclease stability, increased binding affinity, or some other beneficial biological property. In certain embodiments, the nucleic acid comprises a chemically modified ribofuranose ring moiety. Examples of chemically modified ribofuranose rings include, but are not limited to, the addition of substituents (including 5' and / or 2' substituents), bridging of two ring atoms to form a bicyclic nucleic acid, S, N(R), or C(R1)(R2) (R = H, C1-C 12 and substitution of the ribosyl ring oxygen atom with an alkyl or protecting group, as well as combinations thereof.

[0122] In some examples, the engineered polynucleotides described herein contain modified sugars or sugar analogs. Thus, in addition to ribose and deoxyribose, the sugar moiety can be a pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose, lyxose, or a sugar "analog" cyclopentyl group. The sugar can be in pyranosyl or furanosyl form. The sugar moiety can be a furanoside of ribose, deoxyribose, arabinose, or 2'-O-alkylribose, and the sugar can be attached to the respective heterocyclic base in either the [α] or [β] anomeric configuration. Sugar modifications include, but are not limited to, 2'-alkoxy-RNA analogs, 2'-amino-RNA analogs, 2'-fluoro-DNA, and 2'-alkoxy- or amino-RNA / DNA chimeras. For example, sugar modifications can include 2'-O-methyl-uridine or 2'-O-methyl-cytidine. Sugar modifications include 2'-O-alkyl-substituted deoxyribonucleosides and 2'-O-ethylene glycol-like ribonucleosides.

[0123] Modifications to the sugar moiety include natural modifications of the ribose and deoxyribose, as well as non-natural modifications. Sugar modifications include, but are not limited to, the following modifications at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl (alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C6). 10 Alkyl or C2-C 10 2' sugar modifications include, but are not limited to, -O[(CH2) n O] m CH3, -O(CH2) n OCH3, -O(CH2) n NH2, -O(CH2) n CH3, -O(CH2) n ONH2, and -O(CH2) n O-N[(CH2)nCH3)]2, where n and m are from 1 to about 10. Other chemical modifications at the 2'-position include, but are not limited to, C1-C 10Examples of suitable sugars include lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, intercalators, groups for improving the pharmacokinetic or pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. Similar modifications can also be made at other positions on the sugar, specifically the 3' position of the sugar on 3'-terminal nucleotides, or the 5' position of 2'-5' linked oligonucleotides and 5'-terminal nucleotides. Chemically modified sugars further include those containing modifications to the bridging ring oxygen, such as CH2 and S. Nucleotide sugar analogs can also have sugar mimetics, such as a cyclobutyl moiety, in place of the pentofuranosyl sugar. Examples of nucleic acids with modified sugar moieties include, but are not limited to, nucleic acids containing 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH, and 2'-O(CH)OCH substituents. Substituents at the 2' position include allyl, amino, azido, thio, O-allyl, O-(C-C 10 alkyl), OCF3, O(CH2)2SCH3, O(CH2)2-ON(R m )(R n ), and O-CH2-C(=O)-N(R m )(R n ) and R m and R n are each independently H or substituted or unsubstituted C-C 10 It is alkyl.

[0124] In certain embodiments, the nucleic acids described herein comprise one or more bicyclic nucleic acids. In certain such embodiments, the bicyclic nucleic acid comprises a bridge between the 4' ribosyl ring atom and the 2' ribosyl ring atom. In certain embodiments, the nucleic acids provided herein comprise one or more bicyclic nucleic acids, wherein the bridge comprises a 4'-2' bicyclic nucleic acid. Examples of such 4'-2' bicyclic nucleic acids include, but are not limited to, one of the formulas: 4'-(CH2)-O-2' (LNA), 4'-(CH2)-S-2', 4'-(CH2)2-O-2' (ENA), 4'-CH(CH3)-O-2', and 4'-CH(CHOCH3)-O-2' and analogs thereof, 4'-C(CH3)(CH3)-O-2' and analogs thereof.

[0125] Modifications to the base of a nucleotide

[0126] In some embodiments, the chemical modifications described herein include modifications of the bases (e.g., nucleobases) of nucleotides. Examples of nucleobases include adenine (A), thymine (T), guanine (G), cytosine (C), and uracil (U). These nucleobases can be modified or substituted in the engineered polynucleotides described herein. The nucleobases of the nucleotides can be independently selected from purines, pyrimidines, and purine or pyrimidine analogs. In some embodiments, the nucleobases can be naturally occurring or synthetic derivatives of the bases.

[0127] In some embodiments, the chemical modifications described herein comprise uracil modifications. In some embodiments, the engineered polynucleotides described herein comprise at least one chemically modified uracil. Examples of chemically modified uracils include pseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine, 4-thio-uridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine, 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine, 5-methoxy-uridine, uridine 5-oxyacetic acid, uridine 5-oxyacetic acid methyl ester, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine, 5-carboxyhydroxymethyl-uridine methyl ester, 5-methoxycarbonylmethyl-uridine, 5-methoxycarbonylmethyl-2-thio-uridine, 5-aminomethyl-2-thio-uridine, 5-methylaminomethyl- Uridine, 5-methylaminomethyl-2-thio-uridine, 5-methylaminomethyl-2-seleno-uridine, 5-carbamoylmethyl-uridine, 5-carboxymethylaminomethyl-uridine, 5-carboxymethylaminomethyl-2-thio-uridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurino Methyl-4-thio-pseudouridine, 5-methyl-uridine, 1-methyl-pseudouridine, 5-methyl-2-thio-uridine, 1-methyl-4-thio-pseudouridine, 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydroundine, dihydropseudouridine, 5,6-Dihydrouridine, 5-methyl-dihydrouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine, 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine pseudouridine), 5-(isopentenylaminomethyl)uridine, 5-(isopentenylaminomethy)-2-thio-uridine, α-thio-uridine, 2'-O-methyl-uridine, 5,2'-O-dimethyl-uridine, 2'-O-methyl-pseudouridine, 2-thio-2'-O-methyl-uridine, 5-methoxycarbonylmethyl-2'-O-methyl-uridine, 5-carbamoylmethyl-2'-O-methyl-uridine, 5-carboxymethylaminomethy Examples of suitable uridines include 2'-O-methyl-uridine, 3,2'-O-dimethyl-uridine, 5-(isopentenylaminomethyl)-2'-O-methyl-uridine, 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, 5-[3-(1-E-propenylamino)uridine], pyrazolo[3,4-d]pyrimidine, xanthine, and hypoxanthine.

[0128] In some embodiments, the chemical modifications described herein comprise modifications of cytosines. In some embodiments, the engineered polynucleotides described herein comprise at least one chemically modified cytosine. Examples of chemically modified cytosines include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetyl-cytidine, 5-formyl-cytidine, N4-methyl-cytidine, 5-methyl-cytidine, 5-halo-cytidine, 5-hydroxymethyl-cytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zeb Examples include lysidine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine, a-thio-cytidine, 2'-O-methyl-cytidine, 5,2'-O-dimethyl-cytidine, N4-acetyl-2'-O-methyl-cytidine, N4,2'-O-dimethyl-cytidine, 5-formyl-2'-O-methyl-cytidine, N4,N4,2'-O-trimethyl-cytidine, 1-thio-cytidine, 2'-F-ara-cytidine, 2'-F-cytidine, and 2'-OH-ara-cytidine.

[0129] In some embodiments, the chemical modifications described herein comprise modifications of adenine. In some embodiments, the engineered polynucleotides described herein comprise at least one chemically modified adenine. Examples of chemically modified adenines include 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro(chloi)-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diamino ... Aza-8-aza-2,6-diaminopurine, 1-methyl-adenosine, 2-methyl-adenine, N6-methyl-adenosine, 2-methylthio-N6-methyl-adenosine, N6-isopentenyl-adenosine, 2-methylthio-N6-isopentenyl-adenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyl-adenosine, N6-threonylcarbamoyl-adenosine, N 6-Methyl-N6-threonylcarbamoyl-adenosine, 2-methylthio-N6-threonylcarbamoyl-adenosine, N6,N6-dimethyl-adenosine, N6-hydroxynorvalylcarbamoyl-adenosine, 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine, N6-acetyl-adenosine, 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, α-thio-adenosine, 2'-O-methyl-adenosine, N6,2'-O-dimethyl- Examples include adenosine, N6-methyl-2'-deoxyadenosine, N6,N6,2'-O-trimethyl-adenosine, 1,2'-O-dimethyl-adenosine, 2'-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2'-F-ara-adenosine, 2'-F-adenosine, 2'-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

[0130] In some embodiments, the chemical modifications described herein comprise modifications of guanine. In some embodiments, the engineered polynucleotides described herein comprise at least one chemically modified guanine. Examples of chemically modified guanosine include inosine, 1-methyl-inosine, wyosine, methylwyosine, 4-demethyl-wyosine, isowyosine, wybutosine, peroxywybutosine, hydroxywybutosine, unmodified hydroxywybutosine, 7-deaza-guanosine, queuosine, epoxyqueuosine, galactosyl-queuosine, mannosyl-queuosine, 7-cyano-7-deaza-guanosine, and 7-cyano-7-deaza-guanosine. Aza-guanosine, 7-aminomethyl-7-deaza-guanosine, archeosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine, N2-methyl-guanosine, N2,N2-dimethyl-guanosine, N2,7-dimethyl-guanosine, N2,N2 7-dimethyl-guanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-meththio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, α-thio-guanosine, 2'-O-methyl-guanosine, N2-methyl-2'-O-methyl-guanosine, N2,N2-dimethyl-2'-O-methyl-guanosine, 1-methyl-2'-O-methyl-guanosine, N2,7-dimethyl-2'-O-methyl-guanosine, 2'-O-methyl-inosine, 1,2'-O-dimethyl-inosine, 6-O-phenyl-2'-deoxyinosine, 2'-O-ribosylguanosine, 1-thio-guanosine, 6-O-methyguanosine, O 6 Examples include 2'-methyl-2'-deoxyguanosine, 2'-F-ara-guanosine, and 2'-F-guanosine.

[0131] In some cases, chemical modification of an engineered polynucleotide can include introducing or substituting a nucleic acid analog or a non-natural nucleic acid into the engineered polynucleotide. In some embodiments, the nucleic acid analog can be any one of the chemically modified nucleic acids described herein. Examples of nucleic acid analogs can be found in International Patent Applications PCT / US2015 / 025175, PCT / US2014 / 050423, PCT / US2016 / 067353, PCT / US2018 / 041503, PCT / US18 / 041509, PCT / US2004 / 011786, or PCT / US2004 / 011833, all of which are expressly incorporated by reference in their entirety. Chemically modified nucleotides described herein can include variants of guanosine, uridine, adenosine, thymidine, and cytosine, including any naturally occurring or non-naturally occurring guanosine, uridine, adenosine, thymidine, or cytidine that has been chemically altered by, for example, acetylation, methylation, or hydroxylation. Examples of chemically modified nucleotides include 1-methyl-adenosine, 1-methyl-guanosine, 1-methyl-inosine, 2,2-dimethyl-guanosine, 2,6-diaminopurine, 2'-amino-2'-deoxyadenosine, 2'-amino-2'-deoxycytidine, 2'-amino-2'-deoxyguanosine, 2'-amino-2'-deoxyuridine, 2-amino-6-chloropurine riboside, 2-aminopurine riboside, 2'-araadenosine, 2'-aracytidine, 2'-arauridine, 2'-azido-2'-deoxyadenosine, 2'-azido-2'-deoxycytidine, thymidine, 2'-azido-2'-deoxyguanosine, 2'-azido-2'-deoxyuridine, 2-chloroadenosine, 2'-fluoro-2'-deoxyadenosine, 2'-fluoro-2'-deoxycytidine, 2'-fluoro-2'-deoxyguanosine, 2'-fluoro-2'-deoxyuridine, 2'-fluorothymidine, 2-methyl-adenosine, 2-methyl-guanosine, 2-methyl-thio-N6-isopentenyl-adenosine, 2'-O-methyl-2-aminoadenosine, 2'-O-methyl-2'-deoxyadenosine, 2'-O-methyl-2'-deoxycytidine,2'-O-methyl-2'-deoxyguanosine, 2,-O-methyl-2'-deoxyuridine, 2'-O-methyl-5-methyluridine, 2'-O-methylinosine, 2'-O-methylpseudouridine, 2-thiocytidine, 2-thio-cytidine, 3-methyl-cytidine, 4-acetyl-cytidine, 4-thiouridine, 5-(carboxyhydroxymethyl)-uridine, 5,6-dihydrouridine, 5-aminoallylcytidine, 5-aminoallyl-deoxyuridine, 5-bromouridine, 5-carboxymethylaminomethyl-2-thio-uracil, 5-carboxymethylaminomethyl-uracil, 5-chloro-ara-cytosine, 5-fluoro-uridine, 5-iodouridine, 5-Methoxycarbonylmethyl-uridine, 5-methoxy-uridine, 5-methyl-2-thio-uridine, 6-azacytidine, 6-azauridine, 6-chloro-7-deaza-guanosine, 6-chloropurine riboside, 6-mercapto-guanosine, 6-methyl-mercaptopurine-riboside, 7-deaza-2'-deoxy-guanosine, 7-deazaadenosine, 7-methyl-guanosine, 8-azaadenosine, 8-bromo-adenosine, 8-bromo-guanosine, 8-mercapto-guanosine, 8-oxoguanosine, benzimidazole-riboside, β-D-mannosyl-queuosine, dihydro-uridine, inosine, N1-methyladenosine, N6-([6-aminohexyl] In some embodiments, the chemically modified nucleic acids described herein include 2-amino-6-chloropurine riboside-5'-triphosphate, 2-aminopurine-riboside-5'-triphosphate, 2-aminoadenosine-5'-triphosphate, 2'-amino-2'-deoxycytidine-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate,2'-Fluorothymidine-5'-triphosphate, 2'-O-methyl-inosine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5-aminoallyluridine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, 5-bromouridine-5'-triphosphate, 5-bromo-2'-deoxycytidine-5'-triphosphate, 5-bromo-2' -deoxyuridine-5'-triphosphate, 5-iodocytidine-5'-triphosphate, 5-iodo-2'-deoxycytidine-5'-triphosphate, 5-iodouridine-5'-triphosphate, 5-iodo-2'-deoxyuridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5-methyluridine-5'-triphosphate, 5-propynyl-2'-deoxycytidine-5'-triphosphate, 5- Propynyl-2'-deoxyuridine-5'-triphosphate, 6-azacytidine-5'-triphosphate, 6-azauridine-5'-triphosphate, 6-chloropurine riboside-5'-triphosphate, 7-deazaadenosine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 8-azaadenosine-5'-triphosphate, 8-azidoadenosine-5'-triphosphate, benzimidazole-riboside- In some embodiments, the chemically modified nucleic acid described herein comprises at least one chemically modified nucleotide selected from pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine,The nucleotides include at least one chemically modified nucleotide selected from 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thiopseudouridine. In some embodiments, the artificial nucleic acids described herein are selected from the group consisting of 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, The compound comprises at least one chemically modified nucleotide selected from 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine. In some embodiments, the chemically modified nucleic acids described herein are selected from the group consisting of 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyl adenosine,The nucleotides include at least one chemically modified nucleotide selected from N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine. In other embodiments, the chemically modified nucleic acids described herein comprise at least one chemically modified nucleotide selected from inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine. In certain embodiments, the chemically modified nucleic acids described herein are selected from the group consisting of 6-aza-cytidine, 2-thio-cytidine, α-thio-cytidine, pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine, α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine, pyrrolo-cytidine, inosine, and the like. , α-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytidine, 8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2-amino-purine, pseudo-iso-cytidine, 6-chloro-purine, N6-methyl-adenosine, α-thio-adenosine, 8-azido-adenosine, 7-deaza-adenosine.

[0132] Modified bases of non-natural nucleic acids include, but are not limited to, uracil-5-yl, hypoxanthine-9-yl (I), 2-aminoadenine-9-yl, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine. uracil, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and adenine, 8-azaguanine and adenine, 7-deazaguanine and adenine, and 3-deazaguanine and adenine. Certain unnatural nucleic acids, such as 5-substituted pyrimidines, 6-azapyrimidines and N-2 substituted purines, N-6 substituted purines, O-6 substituted purines, 2-aminopropyladenine, 5-propynyluracil, 5-propynylcytosine, 5-methylcytosine, those that increase the stability of duplex formation, universal nucleic acids, hydrophobic nucleic acids, promiscuous nucleic acids, size-expanded nucleic acids, fluorinated nucleic acids, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6, and O-6 substituted purines, 2-aminopropyladenine, 5-propynyluracil, and 5 those containing -propynylcytosine, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, other alkyl derivatives of adenine and guanine, 2-propyl, and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyl (-C≡C-CH3)uracil, 5-propynylcytosine, other alkynyl derivatives of pyrimidine nucleic acids, 6-azouracil, 6-azocytosine,6-azothymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl, other 5-substituted uracils and cytosines, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-Deazaadenine, tricyclic pyrimidine, phenoxazine cytidine ([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamp, phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyrimidine Indole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one), those in which the purine or pyrimidine base is replaced by other heterocycles, 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, 2-pyridone, azacytosine, 5-bromocytosine, bromouracil, 5-chlorocytosine, chlorinated cytosine, cyclocytosine, cytosine arabinoside, 5-fluorocytosine, fluoropyrimidine, fluorouracil, 5,6-dihydrocytosine, 5-iodocytosine, hydroxyurea, iodouracil, 5-nitrocytosine, 5-bromouracil, 5-chlorouracil, 5-fluorouracil, and 5-iodouracil, 2-amino-adenine, 6-thio-guanine, 2-thio-thymine, 4-thio-thymine, 5-propynyl-uracil, 4-thio-uracil, N4-ethylcytosine, 7-deazaguanine, 7-deaza-8-azaguanine, 5-hydroxycytosine, 2'-deoxyuridine, or 2-amino-2'-deoxyadenosine.

[0133] Optionally, the at least one chemical modification may include chemically modifying the 5' or 3' end of the engineered polynucleotide, such as the 5' cap or 3' tail. In some embodiments, the engineered polynucleotide includes a chemical modification comprising a 3' nucleotide, which may be stabilized against degradation, for example, by incorporating one or more of the modified nucleotides described herein. In this embodiment, uridine may be substituted with modified uridine, e.g., 5-(2-amino)propyluridine and 5-bromouridine, or any of the modified uridines described herein, and adenosine and guanosine may be substituted with modified adenosine and guanosine, e.g., with a modification at the 8th position, e.g., 8-bromoguanosine, or any of the modified adenosines or guanosines described herein. In some embodiments, deazanucleotides, e.g., 7-deaza-adenosine, may be incorporated into the gRNA. In some embodiments, O- and N-alkylated nucleotides, e.g., N6-methyladenosine, may be incorporated into the gRNA. In some embodiments, sugar-modified ribonucleotides can be incorporated, e.g., where the 2'OH group is replaced with a group selected from H, -OR, -R (where R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or a sugar), halo, -SH, -SR (where R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or a sugar), amino (wherein amino can be, e.g., NH, alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or an amino acid), or cyano (-CN). In some embodiments, the phosphate backbone can be modified, e.g., with a phosphothioate group, as described herein.In some embodiments, the nucleotides in the overhang region of the gRNA can each independently be modified or unmodified nucleotides, including, but not limited to, 2'-sugar modifications, such as 2-F2'-O-methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine (Teo), 2'-O-methoxyethyladenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo), or any combination thereof.

[0134] In some embodiments, all nucleotides of the targeting moiety have a 2'O-methyl modification. This is believed to increase the affinity of the engineered polynucleotide for its pre-mRNA target and / or prevent the engineered polynucleotide from being degraded by nucleases. In some embodiments, all nucleotides of the targeting moiety have a phosphorothioate modification.

[0135] Adjustment part In some embodiments of the engineered polynucleotides described herein, the post-transcriptional regulatory portion (or regulatory portion) (e.g., spliceosome portion) is selected from a spliceosomal ribonucleoprotein complex, a spliceosomal small nuclear ribonucleic acid (snRNA), a spliceosomal protein, a functional variant thereof, or a functional fragment thereof. In some embodiments, the spliceosomal portion comprises a U1 snRNA and a spliceosomal protein. In some embodiments, the spliceosomal portion comprises a U2 snRNA and a spliceosomal protein. In some embodiments, the spliceosomal snRNA is selected from U1, U2, U4, U5, U6, U11, U12, U14atac, U6atac, and combinations thereof. In some embodiments, the spliceosomal snRNA is U1 or U2. In some embodiments, the spliceosomal protein is selected from Sm, U1-70k, U1A, U1C, and combinations thereof. Non-limiting examples of spliceosome portions include SmD1, SmD2, SmD3, SmE, SmF, SmG, U1, U2, U4, U5, U6, U11, U12, U14, or U16.

[0136] In some embodiments described herein, upon association with an engineered polynucleotide and an RNA (e.g., an mRNA, such as a pre-mRNA), the spliceosome portion cleaves or splices the RNA (e.g., an mRNA, such as a pre-mRNA) in the target sequence. In some embodiments, the spliceosome portion further facilitates modification of the cleaved RNA (e.g., a cleaved mRNA, such as a cleaved pre-mRNA).

[0137] In some embodiments, the engineered polynucleotide binds to the target sequence via base pairing, such as Watson-Crick base pairing. The binding of the engineered polynucleotide to the recruitment moiety provided herein can be used to regulate the expression or activity of a target gene. In some embodiments, the binding of the engineered polynucleotide to the recruitment moiety allows the recruitment moiety to splice the pre-mRNA encoding the target gene with increased specificity, thereby regulating the target gene. In some embodiments, the binding of the engineered polynucleotide to the recruitment moiety allows the recruitment moiety to splice the pre-mRNA encoding the target gene with increased efficiency, thereby regulating the target gene. Regulation can refer to increasing or decreasing the expression or activity of the target gene. A non-limiting example of a target gene can be microtubule-associated protein tau (MAPT). In some embodiments, the expression or activity of the target gene increases by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more when the engineered polynucleotide is bound to the recruitment moiety, compared to the expression or activity of the target gene when the engineered polynucleotide is not bound to the recruitment moiety. In some embodiments, the expression or activity of the target gene decreases by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more when the engineered polynucleotide is bound to the recruitment moiety, compared to the expression or activity of the target gene when the engineered polynucleotide is not bound to the recruitment moiety.

[0138] In some embodiments, modulating target expression or activity comprises correcting aberrant expression of the target gene due to a splice variant, in some embodiments, the expression or activity of the misfolded target gene or protein due to the aberrant splice variant is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more when the engineered polynucleotide is bound to the recruitment moiety, compared to the expression or activity of the misfolded target gene or protein due to the aberrant splice variant when the engineered polynucleotide is not bound to the recruitment moiety. In some embodiments, the amount of misfolded protein aggregates due to aberrant splice variants is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more when the engineered polynucleotide is bound to the recruitment moiety, compared to the amount of misfolded protein aggregates due to aberrant splice variants when the engineered polynucleotide is not bound to the recruitment moiety. In some embodiments, the amount of plaques containing misfolded protein due to aberrant splice variants is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more when the engineered polynucleotide is bound to the recruitment moiety, compared to the amount of plaques containing misfolded protein due to aberrant splice variants when the engineered polynucleotide is not bound to the recruitment moiety.

[0139] molecular interactions In various aspects of the present disclosure, the engineered polynucleotide may be capable of participating in molecular interactions with polypeptides (e.g., U1-C) or other polynucleotides (e.g., pre-mRNA, U1 snRNA). The engineered polynucleotide may be configured to interact with, or otherwise capable of interacting with, other polypeptides or polynucleotides via one or more targeting or recruitment moieties. In some embodiments described herein, the targeting moiety comprises free 5' and 3' ends that interact with a conserved site in the constitutive splice donor. This interaction can silence the U1 snRNA RNA-binding domain. In some embodiments, the targeting moiety comprises 2'-modified nucleotides that increase affinity for the pre-mRNA target.

[0140] In some embodiments, the binding of the targeting moiety to the pre-mRNA is stabilized by hydrogen bonds and electrostatic interactions between U1-C and the pre-mRNA backbone around the splice junction region of the pre-mRNA. In such embodiments, U1-C may not make specific base contact with the pre-mRNA. 2' nucleotide modifications may favor hydrogen bonding between the targeting moiety and U1-C. Thus, the binding of the targeting moiety (free 5' and 3' ends) to the pre-mRNA duplex allows U1-C to recognize and stabilize the targeting interaction.

[0141] U1-C can stabilize the central core of the spliceosome. U1-C enhances the affinity of incompatible 5'-splices and stabilizes the central core of the spliceosome machinery through the interaction bridge between U1-70KD and the Sm ring.

[0142] In some embodiments, the targeting moiety can interact with the zinc finger of U1-C. Phosphorothioate internucleotide linkages in the targeting moiety can facilitate the interaction between the targeting moiety and the zinc finger. The engineered polynucleotide can contain phosphorothioate internucleotide linkages at specific or particular positions that interact with the zinc finger.

[0143] In some embodiments, the recruitment moiety forms a hydrogen bond with stem-loop II of U1-A, and such an interaction can regulate polyadenylation and acetylation signaling by U1-A, since stem-loop II of U1-A cannot be silenced by the recruitment moiety.

[0144] In some embodiments described herein, the engineered polynucleotide does not contain any intramolecular disulfide bonds.

[0145] In some embodiments of the engineered polynucleotides described herein, upon association with the engineered polynucleotide and spliceosome moiety, the RNA (e.g., mRNA, such as pre-mRNA) exhibits substantially no base pairing with the RNA binding domain (RBD) of U1 snRNA.

[0146] In some embodiments of the engineered polynucleotides described herein, when associated with the engineered polynucleotide and the spliceosome moiety, the RNA (e.g., mRNA, such as pre-mRNA) does not exhibit substantially base-specific interactions with the U1-C protein.

[0147] In some embodiments of the engineered polynucleotides described herein, the engineered polynucleotide is configured to specifically interact with a zinc finger of a U1-C protein, e.g., comprising the amino acid sequence YYCDYCDTYLTHDSPSVRKTHCTGRKHRDNVKF (SEQ ID NO: 5). In some embodiments, the 5'-targeting portion of the engineered polynucleotide is configured to specifically interact with a zinc finger of a U1-C protein. In some embodiments, the engineered polynucleotide (e.g., its 5'-targeting portion) is configured to covalently interact with a zinc finger of a U1-C protein (e.g., via a disulfide bond). In some embodiments, the engineered polynucleotide (e.g., its 5'-targeting portion) is configured to non-covalently interact with a zinc finger of a U1-C protein (e.g., via a hydrogen bond).

[0148] In some embodiments, an engineered polynucleotide (e.g., ASMO1 described herein) comprises a nucleotide sequence complementary to U1 snRNA. In some embodiments of the engineered polynucleotides described herein, the engineered polynucleotide comprises a nucleotide sequence complementary to a subsequence of stem-loop II (SL2) of U1 snRNA. In some embodiments, the side of the stem-loop structure of the engineered polynucleotide comprises a nucleotide sequence complementary to a subsequence of stem-loop II (SL2) of U1 snRNA. In some embodiments, the subsequence comprises a sequence corresponding to 5'-GGCCU-3' of SL2 of U1 snRNA. In some embodiments, the subsequence does not comprise a sequence corresponding to 5'-CACGUUA-3' of SL2 of U1 snRNA.

[0149] In some embodiments of the engineered polynucleotides described herein, the engineered polynucleotide exhibits substantially no base pairing with the anchor sequence of SL2 of U1 snRNA. In some embodiments, the anchor sequence comprises a sequence corresponding to 5'-CACGUUA-3'. In some embodiments, the internal loop of the engineered polynucleotide exhibits substantially no base pairing with the anchor sequence of SL2 of U1 snRNA. In some embodiments, the lower stem of the engineered polynucleotide exhibits substantially no base pairing with the anchor sequence of SL2 of U1 snRNA.

[0150] In some embodiments of the engineered polynucleotides described herein, the engineered polynucleotide exhibits substantially no base pairing with the H helix of U1 snRNA.

[0151] In some embodiments, the engineered polynucleotide does not contain any intramolecular disulfide bonds. In some embodiments, when the engineered polynucleotide recruits a spliceosome portion described in a target sequence, such as a target pre-mRNA, it does not exhibit base pairing with the RNA binding domain (RBD) of the spliceosome portion, such as the U1 snRNA. Figure 4 illustrates this lack of base pairing between the engineered polynucleotide and the RBD of the spliceosome portion, where the RBD portion of the U1 snRNA has the following sequence: 3'-GUCCAUUCAUA-5', which forms base pairing with the target sequence. In some examples, when the engineered polynucleotide and the spliceosome portion are combined, the engineered polynucleotide exhibits substantially no base-specific interaction with the U1-C spliceosome portion. In some embodiments, the engineered polynucleotide is configured to specifically interact with the zinc finger of the U1-C protein or the U1-1 spliceosome portion. FIG. 10C shows a representative sequence of the U1-C snRNP, which contains 145 amino acids, with the highlighted 36 amino acids (YYCDYCDTYLTHDSPSVRKTHCTGRKHRDNVKF (SEQ ID NO: 5)) comprising the zinc finger domain.

[0152] In some embodiments, the engineered polynucleotide is configured to covalently interact with a zinc finger of the U1-C protein or U1-1 spliceosome portion (e.g., via a disulfide bond). In some embodiments, the engineered polynucleotide is configured to non-covalently interact with a zinc finger of the U1-C protein or U1-1 spliceosome portion (e.g., via a hydrogen bond). In some embodiments, the engineered polynucleotide (e.g., ASMO1 described herein) comprises a nucleotide sequence complementary to U1 snRNA. In some embodiments, the engineered polynucleotide comprises a nucleotide sequence complementary to a subsequence of stem-loop II (SL2) of U1 snRNA. In some embodiments, the flank of the stem-loop secondary structure of the engineered polynucleotide comprises a nucleotide sequence complementary to a subsequence of stem-loop II (SL2) of U1 snRNA. In some embodiments, the subsequence comprises a sequence corresponding to 5'-GGCCU-3' of SL2 of U1 snRNA. In some embodiments, the subsequence does not include a sequence corresponding to 5'-CACGUUA-3' of SL2 of U1 snRNA. In some embodiments, the engineered polynucleotide exhibits substantially no base pairing with the anchor sequence of SL2 of U1 snRNA. Optionally, the internal loop of the engineered polynucleotide exhibits substantially no base pairing with the anchor sequence of SL2 of U1 snRNA. In some aspects, the lower stem of the engineered polynucleotide exhibits substantially no base pairing with the anchor sequence of SL2 of U1 snRNA. In some aspects, the anchor sequence includes a sequence corresponding to 5'-CACGUUA-3'. Optionally, the engineered polynucleotide exhibits substantially no base pairing with the H helix of U1 snRNA, and the engineered polynucleotide does not contain any intramolecular disulfide bonds. For example, Figure 2A shows the absence of intramolecular disulfide bonds due to the presence of chemical modifications having phosphorothioate internucleotide linkages.Stabilization of the U1 snRNP complex can be observed through the strong ionic attraction of the zinc fingers of U1-C, induced by disulfide bridges with thiols of the engineered polynucleotide (ASMO) targeting moiety at the 5' and / or 3' ends. The pre-mRNA / engineered polynucleotide (ASMO) duplex can be stabilized by hydrogen bonds and electrostatic interactions between U1-C and the backbone of the pre-mRNA around the seam joint, but U1-C does not make base-specific contacts with the pre-mRNA. This structure demonstrates that nucleotide selection for 5'-splice by U1 snRNP is achieved primarily through interactions between the 5' / 3' stem and the pre-mRNA. Meanwhile, U1-C adjusts the relative affinity of the 5'-splice mismatch site and stabilizes the central core of the spliceosome machinery through bridging interactions between U1-70KDa and the Sm ring (see Figures 7-9). Among the U1 snRNP-specific proteins, U1-70k and U1-C play an important role in assisting in the recognition of pre-mRNA transcripts. U1-70k has a highly conserved but predicted unstructured N-terminus (residues 2–60), an RNA-binding domain (i.e., RBD) (residues 92–202) that mediates interaction with the stem-loop of U1 snRNA, and a C-terminus rich in arginine and serine residues (the RS "domain") as well as repeats of R-(D / E) residues. Although this C-terminal domain is not conserved, the RS "domain" is important for interaction with non-snRNP splicing factors such as ASF / SF2. The serines in this region are subject to post-translational modification (phosphorylation) and are therefore important for splicing activity. U1-C consists of an N-terminal zinc finger domain and a C-terminal region rich in repeats of RG residues. The arginines in this U1-C region are subject to post-translational modification (methylation). In contrast to U1-70k, U1-C does not bind to free U1 snRNA but requires the prior binding of Sm proteins and U1-70k. Mutations in the zinc finger region of U1-C have a significant effect on 5' splice site recognition by U1-snRNP, indicating that this protein has a direct role in this activity.Knowledge of the assembly and function of the U1 snRNP has been significantly enhanced, first by cryo-electron microscopy studies and more recently by the elucidation of its three-dimensional structure by X-ray crystallography. Previously, crystal structures of four of the seven Sm proteins were used to model the remaining three (Sm-F, Sm-E, and Sm-G) and propose that they interact to form a seven-membered ring. The crystal structure of the complete recombinant human U1 snRNP reveals that the Sm proteins form a heptameric ring composed of a single copy of each Sm protein, with the Sm region of U1 snRNA passing through its center. In the crystal structure, U1-C is positioned to recognize the duplex formed when the 5' end of U1 snRNA base-pairs with the 5' splice site. Therefore, the observation that the N terminus of U1-70k extends 180 Å from the RBD, traversing Sm-D2 and Sm-D3 / B and wrapping around one face of the Sm ring may ensure the correct structure and positioning of U1-C in its interaction with the U1 snRNA:5′ splice site duplex (Fig. 9 ).

[0153] In some embodiments, the engineered polynucleotide, when associated with the spliceosome components described herein, causes the pre-mRNA to exhibit substantially no base-pairing with the RNA-binding domain (RBD) of U1 snRNA. Optionally, when associated with the spliceosome components, the pre-RNA exhibits substantially no base-specific interaction with the U1-C protein. Figure 4 shows that in the absence of the engineered polynucleotide, the RBD of U1 snRNA binds at a conserved region of the constitutive donor. In contrast, in the presence of the engineered polynucleotide, the 5' / 3' stem prevented RBD interaction between U1 snRNA and pre-RNA (Figures 3 and 6).

[0154] In some embodiments, the engineered polynucleotide is configured to specifically interact with the zinc finger of the U1-C protein. In some embodiments, the 5' targeting portion of the engineered polynucleotide is configured to specifically interact with the zinc finger of the U1-C protein. In some embodiments, the engineered polynucleotide is configured to covalently interact with the zinc finger of the U1-C protein (e.g., via a disulfide bond). In some embodiments, the engineered polynucleotide is configured to non-covalently interact with the zinc finger of the U1-C protein (e.g., via a hydrogen bond). Formation of a pre-RNA / engineered polynucleotide (ASMO) duplex that interacts with amino acid residues from the U1-C zinc finger stabilizes the 5' region (Figure 9). Molecular dynamics can then be observed that favor formation by the disulfide bond formed by atoms in the main chain and side chain of the U1-C zinc finger and the 5' stem of the engineered polynucleotide. Because ASMO exhibits interactions with all cysteines present in the U1-C zinc finger, it can also form strong ionic bonds (Figures 9 and 10). Additional exemplary interactions between U1-C and pre-mRNA in the presence or absence of the engineered polynucleotides described herein are shown in Table 5.

[0155] [Table 5-1]

[0156] [Table 5-2]

[0157] In some embodiments, an engineered polynucleotide (e.g., ASMO1 as described herein) comprises a nucleotide sequence complementary to U1 snRNA. In some aspects, the engineered polynucleotide comprises a nucleotide sequence complementary to a subsequence of stem-loop II (SL2) of U1 snRNA. Other aspects described herein describe flanking portions of the stem-loop structure of an engineered polynucleotide, the flanking portions comprising a nucleotide sequence complementary to a subsequence of stem-loop II (SL2) of U1 snRNA. In some instances, the subsequence comprises a sequence corresponding to 5'-GGCCU-3' of SL2 of U1 snRNA, where the subsequence does not comprise a sequence corresponding to 5'-CACGUUA-3' of SL2 of U1 snRNA, and the engineered polynucleotide does not substantially base pair with the anchor sequence of SL2 of U1 snRNA. In some aspects, the engineered polynucleotide comprises an internal loop of the engineered polynucleotide that does not substantially base pair with the anchor sequence of SL2 of U1 snRNA. In some embodiments, the engineered polynucleotide comprises a lower stem of the engineered polynucleotide that exhibits substantially no base pairing with an anchor sequence of SL2 of U1 snRNA. In some embodiments, the anchor sequence comprises a sequence corresponding to 5'-CACGUUA-3', in which case the engineered polynucleotide exhibits substantially no base pairing with the H helix of U1 snRNA.

[0158] Engineered polynucleotide sets It is described herein that in some embodiments, each set of engineered polynucleotides independently comprises the polynucleotides described herein.For example, the polynucleotides of the set independently comprise: (i) one or more targeting moieties (such as those described herein) that are configured to bind to ribonucleic acid (RNA) (such as those described herein) (e.g., messenger ribonucleic acid (mRNA) such as pre-messenger ribonucleic acid (pre-RNA)) at a target sequence (such as those described herein), and (ii) a recruitment moiety (such as those described herein) that is configured to recruit a post-transcriptional regulatory moiety (e.g., spliceosome moiety) (such as those described herein), and the set of engineered polynucleotides is configured to specifically bind to RNA (e.g., mRNA such as pre-RNA) at a plurality of target sequences, including the target sequence (such as those described herein).

[0159] vector In some embodiments described herein, the invention includes vectors or plasmids that contain nucleic acid sequences that encode the engineered polynucleotides described herein.

[0160] In some embodiments described herein, multiple vectors or multiple plasmids are described, each containing multiple nucleic acid sequences encoding an engineered polynucleotide described herein. In some embodiments, the multiple vectors or multiple plasmids contain multiple nucleic acid sequences encoding more than one engineered polynucleotide described herein. In some embodiments, the multiple vectors or multiple plasmids contain multiple nucleic acid sequences encoding multiple engineered polynucleotides (each independently described herein).

[0161] Pharmaceutical Composition In some embodiments, the present specification describes pharmaceutical compositions comprising an engineered polynucleotide described herein, or a plasmid, vector, or isolated DNA encoding the sequence. As used herein, a pharmaceutical composition refers to a mixture of at least one engineered polynucleotide or a vector encoding at least one engineered polynucleotide with other chemical components (i.e., pharmaceutically acceptable inactive ingredients), such as carriers, excipients, binders, fillers, suspending agents, flavoring agents, sweeteners, disintegrants, dispersing agents, surfactants, lubricants, colorants, diluents, solubilizers, humectants, plasticizers, stabilizers, penetration enhancers, wetting agents, antifoaming agents, antioxidants, preservatives, or one or more combinations thereof. Optionally, the composition comprises two or more pharmaceutical compositions discussed herein. In practicing the methods of treatment or use provided herein, a therapeutically effective amount of a pharmaceutical composition described herein is administered in a pharmaceutical composition to a mammal suffering from the disease, disorder, or condition being treated. In some embodiments, the mammal is a human. The therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the pharmaceutical composition used, and other factors. The pharmaceutical composition can be used alone or in combination with one or more pharmaceutical compositions as a component of a mixture. The pharmaceutical compositions described herein include engineered polynucleotides, compositions, cells contacted with engineered polynucleotides or with compositions containing engineered polynucleotides, or combinations thereof.

[0162] The pharmaceutical formulations described herein may be administered to a subject by any suitable route of administration, including, but not limited to, intravenous, intrathecal, intraventricular, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intranasal, intravitreal, intraosseous, transmucosal, inhalation, or intraperitoneal routes of administration. The pharmaceutical formulations described herein may include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast-dissolve formulations, tablets, capsules, pills, delayed release formulations, sustained release formulations, pulsatile release formulations, multiparticulate formulations, and combination immediate-controlled release formulations.

[0163] Pharmaceutical compositions, including drug compositions, are manufactured conventionally, such as by way of example only, conventional mixing, dissolving, granulating, dragee-making, pulverizing, emulsifying, encapsulating, entrapping, or compressing processes.

[0164] kit In some embodiments, the present specification describes kits for using the engineered polynucleotides, compositions, or pharmaceutical compositions described herein. In some embodiments, the kits disclosed herein may be used to treat a disease or disorder in a subject. In some embodiments, the kits include a collection of materials or components other than the engineered polynucleotides, compositions, or pharmaceutical compositions. In some embodiments, the kits include components for assaying and selecting appropriate oligonucleotides for treating a disease or disorder. In some embodiments, the kits include components for performing assays such as enzyme-linked immunosorbent assay (ELISA), single molecule array (Simoa), PCR, or qPCR. The exact nature of the components configured in the kit depends on its intended purpose. For example, some embodiments are configured for treating a disease or disorder disclosed herein in a subject. In some embodiments, the kits are configured specifically for treating a mammalian subject. In some embodiments, the kits are configured specifically for treating a human subject.

[0165] Instructions for use may be included in the kit. In some embodiments, the kit includes instructions for administering the composition to a subject in need thereof. In some embodiments, the kit includes instructions for further manipulating the engineered polynucleotide. In some embodiments, the kit includes instructions for thawing or otherwise restoring biological activity of an engineered polynucleotide that may be frozen or lyophilized during storage or transport. In some embodiments, the kit includes instructions for measuring efficacy for its intended purpose (e.g., therapeutic efficacy when used to treat a subject).

[0166] Optionally, the kit also contains other useful components, such as diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, bandages, or other useful equipment. The materials or components assembled into the kit can be stored and provided to medical professionals in any convenient and appropriate form that preserves their operability and usefulness. For example, the engineered polynucleotide, composition, or pharmaceutical composition may be in dissolved, dehydrated, or lyophilized form. The components are typically contained in suitable packaging materials.

[0167] method Described herein are methods for utilizing engineered polynucleotides (such as those described herein), such as methods for altering ribonucleic acid (RNA) (e.g., messenger ribonucleic acid (mRNA) such as pre-messenger ribonucleic acid (pre-mRNA)) in a cell. The method can include contacting a cell with an engineered polynucleotide (such as those described herein) comprising one or more targeting moieties and a recruitment moiety. The one or more targeting moieties can bind to an RNA (e.g., an mRNA such as a pre-mRNA) (e.g., such as those described herein) at an internal target sequence (e.g., such as those described herein), and the recruitment moiety recruits a post-transcriptional regulatory moiety (e.g., a spliceosome moiety) (e.g., such as those described herein) near the target sequence of the RNA (e.g., an mRNA such as a pre-mRNA) to alter the RNA (e.g., an mRNA such as a pre-mRNA) in the cell, thereby resulting in one or more altered RNAs (e.g., one or more altered mRNAs, such as one or more altered pre-mRNAs). In some embodiments, the method alters the expression or activity of a target gene. In some embodiments, prior to contact, the cells exhibit abnormal messenger ribonucleic acid (mRNA) or protein corresponding to the target gene. In some embodiments, one targeting moiety of the one or more targeting moieties is sufficiently identical to or complementary to a consensus sequence in the target sequence of the target gene (e.g., microtubule-associated protein tau (MAPT)).

[0168] Described herein are methods for altering ribonucleic acid (RNA) (e.g., messenger ribonucleic acid (mRNA) such as pre-messenger ribonucleic acid (pre-mRNA)) at multiple locations in a cell. The methods can include contacting a cell with a set of engineered polynucleotides (e.g., each independently, such as those described herein). The engineered polynucleotides can include one or more targeting moieties and recruitment moieties. The one or more targeting moieties can bind to an RNA (e.g., an mRNA, such as a pre-mRNA) (e.g., as described herein) at multiple target sequences (e.g., as described herein) therein. Each recruitment moiety can recruit a post-transcriptional regulatory moiety (e.g., a spliceosome moiety) (e.g., as described herein) near a target sequence of the RNA (e.g., an mRNA, such as a pre-mRNA) to alter the RNA (e.g., an mRNA, such as a pre-mRNA) in the cell, thereby resulting in one or more altered RNAs (e.g., one or more altered mRNAs, such as one or more altered pre-mRNAs). In some embodiments, the method alters the expression or activity of a target gene by altering (e.g., cleaving and / or chemically modifying) RNA (e.g., mRNA, such as pre-mRNA) at multiple locations. In some embodiments, prior to contacting, the cell exhibits aberrant messenger ribonucleic acid (mRNA) or protein corresponding to the target gene. In some embodiments, one or each of the one or more targeting moieties is sufficiently identical to or complementary to a consensus sequence in the target sequence of the target gene (e.g., microtubule-associated protein tau (MAPT)).

[0169] In some embodiments, the method comprises delivering an engineered polynucleotide to a cell. In some embodiments, the method comprises delivering a polynucleotide encoding the engineered polynucleotide into a cell, and then expressing the engineered polynucleotide to regulate expression or activity of a gene encoded by a target sequence described herein. In some embodiments, the method comprises using the engineered polynucleotide to treat a disease or condition in a subject in need thereof. The disease or condition may be associated with aberrant expression or activity of a target gene encoded by an RNA (e.g., an mRNA, such as a pre-mRNA). In some embodiments, the RNA (e.g., an mRNA, such as a pre-mRNA) corresponds to a target gene (e.g., microtubule-associated protein tau (MAPT)).

[0170] Figure 1 shows a schematic diagram for identifying splice donors and acceptors for designing the nucleotide sequence of an engineered polynucleotide, and the engineered polynucleotides or methods described herein offer improvements over currently available approaches for modulating gene expression or activity to treat a disease or condition. In some embodiments, the methods described herein modulate gene expression or activity with an engineered polynucleotide that targets a transcript of the disease or condition-causing gene. In some embodiments, the methods described herein comprise administering an engineered polynucleotide described herein to a subject in need thereof. Optionally, the methods described herein comprise utilizing an engineered polynucleotide to recruit a regulatory moiety to modulate the expression or activity of the disease or condition-causing gene, thereby treating the disease or condition. In some aspects, the methods described herein comprise utilizing an engineered polynucleotide to stabilize the assembly of a regulatory moiety to modulate the expression or activity of the disease or condition-causing gene, thereby treating the disease or condition.

[0171] Described herein, in some embodiments, are methods for delivering an engineered polynucleotide described herein to a cell. In some embodiments, the method comprises directly or indirectly delivering the engineered polynucleotide to a cell. In some embodiments, the method comprises contacting the cell with a composition comprising the engineered polynucleotide described herein. In some embodiments, the method comprises expressing the engineered polynucleotide described herein in the cell. In some embodiments, the engineered polynucleotide, or a vector encoding the engineered polynucleotide, can be delivered to a cell by any of the transfection methods described herein. In some embodiments, the engineered polynucleotide can be delivered to a cell by the use of an expression vector. In the context of an expression vector, the vector can be readily introduced into a cell described herein by any method in the art. For example, the expression vector can be transferred to a cell by physical, chemical, or biological means.

[0172] Physical methods for introducing engineered polynucleotides or vectors encoding engineered polynucleotides into cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, gene guns, electroporation, etc. Methods for generating cells containing vectors and / or exogenous nucleic acids are suitable for the methods herein. One method for introducing engineered polynucleotides or vectors encoding engineered polynucleotides into host cells is calcium phosphate transfection.

[0173] Chemical means for introducing engineered polynucleotides or vectors encoding engineered polynucleotides into cells include colloidal dispersion systems such as macromolecule complexes, nanocapsules, microspheres, and beads, as well as lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, spherical nucleic acids (SNAs), liposomes, or lipid nanoparticles. An example of a colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., artificial membrane vesicle). Other cutting-edge methods for targeted delivery of nucleic acids are available, such as delivering engineered polynucleotides or vectors encoding engineered polynucleotides with targeted nanoparticles.

[0174] When a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for introducing engineered polynucleotides or vectors encoding engineered polynucleotides into cells (in vitro, ex vivo, or in vivo). In another embodiment, the engineered polynucleotides or vectors encoding engineered polynucleotides can be associated with lipids. The lipid-associated engineered polynucleotides or vectors encoding engineered polynucleotides can be encapsulated in the aqueous interior of the liposome, interspersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule associated with both the liposome and the engineered polynucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained as a suspension in lipids, contained with or complexed to micelles, or otherwise associated with lipids. The lipid, lipid / DNA, or lipid / expression vector association compositions are not limited to any particular structure in solution. For example, in some embodiments, they exist in bilayer structures, as micelles, or in "folded" structures. Alternatively, they are simply dispersed in solution, sometimes forming aggregates that are not uniform in size or shape. In some embodiments, lipids are fatty substances that are naturally occurring or synthetic lipids. For example, lipids include the lipid droplets that naturally occur in the cytoplasm, as well as a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

[0175] Lipids suitable for use are obtained from commercial sources. Stock solutions of lipids in chloroform or chloroform / methanol are often stored at approximately -20°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol. "Liposome" is a generic term that encompasses a variety of unilamellar and multilamellar lipid vesicles formed by the formation of enclosed lipid bilayers or aggregates. Liposomes are often characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before forming a closed structure, trapping water and dissolved solutes between the lipid bilayers. However, compositions with structures in solution that differ from the typical vesicular structure are also encompassed. For example, in some embodiments, lipids are present in micellar structures or simply as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0176] In some cases, non-viral delivery methods include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, exosomes, polycation or lipid:cargo conjugates (or aggregates), naked polypeptides (e.g., recombinant polypeptides), naked DNA, artificial virions, and drug-enhanced polypeptide or DNA uptake. In some embodiments, the delivery method includes conjugating or encapsulating the compositions or engineered polynucleotides described herein with at least one polymer, such as a natural polymer or a synthetic material. The polymer may be biocompatible or biodegradable. Non-limiting examples of suitable biocompatible, biodegradable synthetic polymers include aliphatic polyesters, poly(amino acids), copoly(ether-esters), polyalkylene oxalates, polyamides, poly(iminocarbonates), polyorthoesters, polyoxaesters, polyamide esters, polyoxaesters containing amine groups, and poly(anhydrides). Such synthetic polymers can be homopolymers or copolymers (e.g., random, block, segmented, graft) of two or more of several different monomers, such as lactic acid, lactide, glycolic acid, glycolide, ε-caprolactone, trimethylene carbonate, p-dioxanone, etc. In one example, the scaffold can be composed of a polymer containing glycolic acid and lactic acid, such as a 90 / 10 or 5 / 95 ratio of glycolic acid to lactic acid. Non-limiting examples of naturally occurring biocompatible, biodegradable polymers can include glycoproteins, proteoglycans, polysaccharides, glycosaminoglycans (GAGs) and fragments derived from these components, elastin, laminin, dechlorins, fibrinogen / fibrin, fibronectin, osteopontin, tenascin, hyaluronic acid, collagen, chondroitin sulfate, heparin, heparan sulfate, ORC, carboxymethylcellulose, and chitin.

[0177] In some cases, the engineered polynucleotides described herein or vectors encoding the engineered polynucleotides can be packaged and delivered to cells via extracellular vesicles. Extracellular vesicles can be any membrane-bound particle. In some embodiments, extracellular vesicles can be any membrane-bound particle secreted by at least one cell. In some cases, extracellular vesicles can be any membrane-bound particle synthesized in vitro. In some cases, extracellular vesicles can be any membrane-bound particle synthesized without cells. In some cases, extracellular vesicles can be exosomes, microvesicles, retrovirus-like particles, apoptotic bodies, apoptosomes, oncosomes, exophers, enveloped viruses, exomers, or other very large extracellular vesicles.

[0178] In some embodiments, the present specification describes methods for modulating or altering the expression or activity of a gene encoded by a target sequence in a cell. In some embodiments, the target sequence is a pre-messenger ribonucleic acid (pre-mRNA) in the cell. In some embodiments, the method includes contacting the cell with an engineered polynucleotide comprising one or more targeting moieties and a recruitment moiety. In some embodiments, the one or more targeting moieties bind to the pre-mRNA at an internal target sequence. In some embodiments, the recruitment moiety recruits a post-transcriptional regulatory moiety (e.g., a spliceosome moiety) near the target sequence of the pre-mRNA to alter the pre-mRNA in the cell, thereby resulting in one or more altered pre-mRNAs. In some embodiments, the pre-mRNA corresponds to a target gene, such as microtubule-associated protein tau (MAPT). In some embodiments, the method increases the expression or activity of the target gene when the engineered polynucleotide binds to and recruits the spliceosome moiety to the target sequence. In some embodiments, the method decreases the expression or activity of the target gene when the engineered polynucleotide binds to and recruits the spliceosome moiety to the target sequence. In some embodiments, the method corrects aberrant messenger ribonucleic acid (mRNA) or protein corresponding to the target gene when the engineered polynucleotide binds to and recruits a spliceosome moiety to the target sequence.

[0179] In some embodiments, the method includes contacting or delivering two or more engineered polynucleotides to a single cell, each engineered polynucleotide comprising one or more targeting moieties configured to bind to two or more target sequences. The two or more target sequences may be located on the same strand of a pre-mRNA encoding a target gene. The two or more target sequences may be located on different strands of a pre-mRNA encoding the same target gene. The two or more target sequences may be located on different strands of a pre-mRNA, each strand of the pre-mRNA encoding a different target gene. In some embodiments, the method includes two or more engineered polynucleotides configured to specifically bind to pre-mRNAs at multiple target sequences, including the target sequence.

[0180] Disclosed herein, in some embodiments, are methods for treating a disease or condition by modulating the expression or activity of a target gene in a cell, thereby treating the disease or condition. In some embodiments, the method includes treating the disease or condition by correcting aberrant messenger ribonucleic acid (mRNA) or protein corresponding to the target gene. In some embodiments, the disease or condition is associated with increased expression or activity of any one of the target genes described herein. In some embodiments, the disease or condition is associated with decreased expression or activity of any one of the target genes described herein. In some embodiments, the disease or condition is associated with aberrant splicing of messenger ribonucleic acid (mRNA) or protein corresponding to any one of the target genes described herein.

[0181] In some embodiments, methods for treating a disease or condition can include modulating the expression or activity of a gene in a cell. The gene can include a gene that is not a target gene and / or does not directly interact with the engineered polynucleotide. Administration of an engineered polynucleotide or a pharmaceutical composition containing the engineered polynucleotide can alter gene expression in a cell or subject, generating a different gene expression profile. For example, a gene in a cell or subject can be downregulated or upregulated as a result of administration of the engineered polynucleotide. The alteration of the gene in a cell can provide a benefit that alleviates the symptoms or cause of the disease or condition.

[0182] In some embodiments, the engineered polynucleotide or pharmaceutical composition comprising the engineered polynucleotide can be administered alone to a subject (e.g., stand-alone treatment). In some embodiments, the engineered polynucleotide or pharmaceutical composition comprising the engineered polynucleotide is administered in combination with an additional agent. Optionally, the additional agent used herein is administered alone. The engineered polynucleotide or pharmaceutical composition comprising the engineered polynucleotide and an additional agent can be administered together or sequentially. The combination therapy can be administered within the same day, or can be administered one day or several days, weeks, months, or years apart.

[0183] In some embodiments, the engineered polynucleotide, or pharmaceutical composition comprising the engineered polynucleotide, is administered at a concentration of at least 5 nM. In some embodiments, the engineered polynucleotide, or pharmaceutical composition comprising the engineered polynucleotide, is administered at a concentration of at least 10 nM. In some embodiments, the engineered polynucleotide, or pharmaceutical composition comprising the engineered polynucleotide, is administered at a concentration of at least 15 nM. In some embodiments, the engineered polynucleotide, or pharmaceutical composition comprising the engineered polynucleotide, is administered at a concentration of at least 20 nM. In some embodiments, the engineered polynucleotide, or pharmaceutical composition comprising the engineered polynucleotide, is administered at a concentration of at least 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 11 nM, 12 nM, 13 nM, 14 nM, 15 nM, 16 nM, 17 nM, 18 nM, 19 nM, 20 nM, 21 nM, 22 nM, 23 nM, 24 nM, 25 nM, 26 nM, 27 nM, 28 nM, 29 nM, 30 nM, or more. In some embodiments, the engineered polynucleotide, or pharmaceutical composition comprising the engineered polynucleotide, is administered at a concentration of 1 nM or less, 2 nM or less, 3 nM or less, 4 nM or less, 5 nM or less, 6 nM or less, 7 nM or less, 8 nM or less, 9 nM or less, 10 nM or less, 11 nM or less, 12 nM or less, 13 nM or less, 14 nM or less, 15 nM or less, 16 nM or less, 17 nM or less, 18 nM or less, 19 nM or less, 20 nM or less, 21 nM or less, 22 nM or less, 23 nM or less, 24 nM or less, 25 nM or less, 26 nM or less, 27 nM or less, 28 nM or less, 29 nM or less, 30 nM or less, or less. In some embodiments, the engineered polynucleotide, or pharmaceutical composition comprising the engineered polynucleotide, is administered at a concentration of about 5 nM to 20 nM.

[0184] In some embodiments, the engineered polynucleotide, or pharmaceutical composition comprising the engineered polynucleotide, is a first-line treatment for a disease or disorder. In some embodiments, the engineered polynucleotide, or pharmaceutical composition comprising the engineered polynucleotide, is a second-line, third-line, or fourth-line treatment. In some embodiments, the engineered polynucleotide, or pharmaceutical composition comprising the engineered polynucleotide, comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or more oligonucleotides. Generally, the methods disclosed herein involve administering the engineered polynucleotide, or pharmaceutical composition comprising the engineered polynucleotide, by intrathecal or intravenous ("iv") administration. However, in some examples, the methods involve administering the engineered polynucleotide, or pharmaceutical composition comprising the engineered polynucleotide, by intraperitoneal injection. In some examples, the methods involve administering the engineered polynucleotide, or pharmaceutical composition comprising the engineered polynucleotide, by oral or intranasal administration. It is contemplated that the engineered polynucleotides disclosed herein, or pharmaceutical compositions comprising engineered polynucleotides, can also be administered by other routes, such as subcutaneous injection, intramuscular injection, intracerebroventricular injection, intravitreal injection, intradermal injection, transdermal administration, intranasal administration, intralymphatic injection, rectal administration, intragastric administration, or any other suitable parenteral administration. In some embodiments, routes for local delivery closer to the site of injury or inflammation are preferred over systemic routes. The route, dosage, time point, and duration of administration of the therapeutic agent can be adjusted. In some embodiments, administration of the therapeutic agent occurs before or after the onset of either or both acute and chronic symptoms of the disease or disorder.

[0185] Suitable doses and dosages to be administered to a subject will be determined by factors including, but not limited to, the particular engineered polynucleotide, composition, or pharmaceutical composition, the disease state and its severity, the identity of the subject requiring treatment (e.g., weight, sex, age), and can be determined according to the particular circumstances surrounding the case, including, for example, the particular agent being administered, the route of administration, the disease being treated, and the subject being treated.

[0186] This paper describes a method for treating neurodegenerative diseases using engineered polynucleotides or vectors encoding engineered polynucleotides.In some embodiments, the neurodegenerative disease is Alzheimer's disease.The engineered polynucleotide can be the engineered polynucleotide described in PCT / US2022 / 037391, which is incorporated by reference in its entirety.

[0187] Described herein are methods for treating a neurodegenerative disease in a subject in need thereof, the methods comprising administering to the subject a pharmaceutical composition comprising an engineered polynucleotide or a vector encoding the engineered polynucleotide. In some embodiments, the engineered polynucleotide comprises one or more targeting moieties configured to specifically bind to a pre-messenger ribonucleic acid (pre-mRNA) at an internal target sequence, and (ii) a recruitment moiety configured to recruit a spliceosome moiety, wherein upon association with the pre-mRNA and the engineered polynucleotide, the spliceosome moiety alters the pre-mRNA at or near the target sequence.

[0188] In some embodiments, the method comprises contacting a cell of a subject with an engineered polynucleotide or a vector encoding the engineered polynucleotide. In some embodiments, the cell is a microglia. In some embodiments, the cell is an astrocyte. In some embodiments, the cell is a neuron. In some embodiments, the cell is an excitatory neuron. In some embodiments, the cell is from a patient diagnosed with Alzheimer's disease. In some embodiments, the cell is a neuron from a healthy patient. In some embodiments, the cell is a neuron from a patient diagnosed with Alzheimer's disease. In some embodiments, the cell is an excitatory neuron from a patient diagnosed with Alzheimer's disease. In some embodiments, the cell is from the hippocampus or cortical region.

[0189] In some embodiments, the neurodegenerative disease is a neurodegenerative disease associated with the presence of tau in the subject's brain. For example, the neurodegenerative disease can be a tauopathy or other disease in which the presence of tau indicates a pathological condition. As described elsewhere in this disclosure, when administered to a subject or cell, the engineered polynucleotide can reduce the amount of tau, alter tau expression, or reduce tau expression. When the presence of tau is altered, administration of the engineered polypeptide can alleviate or ameliorate the symptoms of the neurodegenerative disease or otherwise treat the neurodegenerative disease. In some embodiments, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Pick's disease, chronic traumatic encephalopathy, progressive supranuclear palsy, argyrophilic grain disease, amyotrophic lateral sclerosis, and frontotemporal dementia.

[0190] In some embodiments, contacting cells with an engineered polynucleotide or a vector encoding an engineered polynucleotide results in an increase in fiber width, number of branches, branch junctions, neurite straightness, and total axonal material in the cells. In some embodiments, contacting cells with an engineered polynucleotide or a vector encoding an engineered polynucleotide results in improved axonal function related to synapses and cognitive function. In some embodiments, contacting cells with an engineered polynucleotide or a vector encoding an engineered polynucleotide results in a decrease in the level of tau protein in the cells. In some embodiments, contacting cells with an engineered polynucleotide or a vector encoding an engineered polynucleotide results in differential expression of genes associated with regulating neuronal generation, neurogenesis, neuronal differentiation, and synapse assembly. In some embodiments, contacting cells with an engineered polynucleotide or a vector encoding an engineered polynucleotide results in enrichment of genes associated with the steroid biosynthesis pathway, MAPK signaling, cell cycle, PI3K-Akt signaling, cellular senescence pathway, fatty acid metabolism and biosynthesis, cholesterol metabolism, AMPK signaling pathway, and Ras signaling pathway. In some embodiments, contacting a cell with an engineered polynucleotide or a vector encoding an engineered polynucleotide results in the enrichment of genes associated with increased mRNA transport. In some embodiments, contacting a cell with an engineered polynucleotide or a vector encoding an engineered polynucleotide results in the prevention of Alzheimer's disease-related phenotypes in the cell. In some embodiments, contacting a cell with an engineered polynucleotide or a vector encoding an engineered polynucleotide results in increased synapse formation. In some embodiments, contacting a cell with an engineered polynucleotide or a vector encoding an engineered polynucleotide results in improved synaptic integrity and synaptic plasticity.In some embodiments, contacting a cell with an engineered polynucleotide reduces premature polyadenylation of one or more transcripts in the cell. In some embodiments, contacting a cell with an engineered polynucleotide or a vector encoding the engineered polynucleotide results in reduced excitotoxicity. In some embodiments, contacting a cell with an engineered polynucleotide or a vector encoding the engineered polynucleotide results in improved synapse-related neuronal pathways and morphology in the cell. In some embodiments, contacting a cell with an engineered polynucleotide or a vector encoding the engineered polynucleotide results in increased neuronal activity. In some embodiments, contacting a cell with an engineered polynucleotide or a vector encoding the engineered polynucleotide results in reduced U1-70K aggregation or mislocalization in the cell. In some embodiments, contacting a cell with an engineered polynucleotide or a vector encoding the engineered polynucleotide results in reduced amyloid-beta expression or aggregation in the cell. In some embodiments, contacting a cell with an engineered polynucleotide or a vector encoding the engineered polynucleotide modulates astrogliosis.

[0191] In some embodiments, the cells are neurons obtained from induced pluripotent stem cells. In some embodiments, the cells are excitatory neurons obtained from induced pluripotent stem cells. In some embodiments, the cells are derived from a healthy patient. In some embodiments, the cells are excitatory neurons derived from a healthy patient. In some embodiments, the cells are derived from the hippocampus or cortical region.

[0192] In some embodiments, the engineered polynucleotide is ASMO1. In some embodiments, ASMO1 interacts directly or indirectly with U1-C and U1-70K. In some embodiments, ASMO1 interacts directly or indirectly with the U1 complex and MAPT pre-mRNA.

[0193] The use of absolute or sequential terms, such as "will," "will not," "shall," "shall not," "must," "must not," "first," "initially," "next," "successfully," "before," "after," "lastly," and "finally," is intended as an example and not as a limitation on the scope of the embodiments of the invention disclosed herein.

[0194] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, the terms "including," "includes," "having," "has," "with," or variations thereof, when used in either the detailed description and / or claims, are intended to be as inclusive as the term "comprising."

[0195] As used herein, the phrases "at least one," "one or more," and "and / or" are open-ended expressions that are both conjunctive and disjunctive in operation. For example, the phrases "at least one of A, B, and C," "at least one of A, B, or C," "one or more of A, B, and C," "one or more of A, B, or C," and "A, B, and / or C" each mean A alone, B alone, C alone, A and B, A and C, B and C, or A, B, and C.

[0196] As used herein, "or" can refer to "and," "or," or "and / or," and can be used both exclusively and inclusively. For example, the term "A or B" can refer to "A or B," "A but not B," "B but not A," and "A and B." In some cases, the context may dictate a specific meaning.

[0197] Any systems, methods, software, and platforms described herein are modular, and therefore terms such as "first" and "second" do not necessarily imply a priority, order of importance, or sequence of actions.

[0198] The term "about" when referring to a number or numerical range means that the referenced number or numerical range is an approximation within experimental variation (or statistical experimental error), and that the number or numerical range may vary, for example, by 1% to 15% of the stated number or numerical range. For example, the term "about" refers to ±10% of the stated number or value.

[0199] The terms "increased," "increasing," or "increase" are used herein collectively to mean an increase by a statistically significant amount. In some embodiments, the term "increased" or "increase" refers to an increase of at least 10% compared to a reference level, e.g., an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to a 100% increase, or any increase between 10 and 100%, compared to a reference level, standard, or control. Other examples of "increase" include an increase of at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold, or more, compared to a reference level.

[0200] The terms "decreased," "decreasing," or "decrease" are generally used herein to mean a statistically significant decrease. In some embodiments, "decreased" or "decreasing" means a decrease of at least 10% compared to a reference level, e.g., a decrease of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to a 100% decrease (e.g., a nonexistent or undetectable level compared to a reference level), or any decrease between 10 and 100%. In the context of a marker or symptom, these terms refer to a statistically significant decrease in such level. A decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40%, or more, preferably to a level that is accepted as being within the normal range for individuals without a given disease.

[0201] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the present invention be limited by the specific examples provided herein. While the present invention has been described with reference to the foregoing specification, the description and illustration of the embodiments herein are not intended to be construed in a limiting sense. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. Furthermore, it is to be understood that all aspects of the present invention are not limited to the specific descriptions, configurations, or relative proportions set forth herein, depending upon a variety of conditions and variables. It is to be understood that various alternatives to the embodiments of the present invention described herein may be employed in practicing the invention. Therefore, it is contemplated that the present invention shall encompass any and all such alternatives, modifications, variations, or equivalents. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0202] Enumeration of Embodiments Embodiment 1. An engineered polynucleotide comprising one or more targeting moieties configured to specifically bind to a pre-messenger ribonucleic acid (pre-mRNA) at an internal target sequence, and a recruitment moiety configured to recruit a post-transcriptional regulatory moiety (e.g., a spliceosome moiety), wherein upon association with said pre-mRNA and said engineered polynucleotide, said post-transcriptional regulatory moiety alters the pre-mRNA at or near said target sequence.

[0203] Embodiment 2. The engineered polynucleotide of embodiment 1, wherein one targeting moiety of said one or more targeting moieties is sufficiently identical to or complementary to a consensus sequence in said target sequence of the target gene.

[0204] Embodiment 3. The engineered polynucleotide of embodiment 1 or 2, wherein the one or more targeting moieties comprise: (1) a first targeting moiety configured to specifically bind to a first targeting sequence in the target sequence of the pre-mRNA; and (2) a second targeting moiety configured to specifically bind to a second targeting sequence in the target sequence of the pre-mRNA.

[0205] Embodiment 4. The engineered polynucleotide of embodiment 3, wherein said first targeting sequence comprises a consensus sequence in said target sequence.

[0206] Embodiment 5. The engineered polynucleotide of embodiment 3 or 4, wherein the second targeting sequence comprises a consensus sequence in the target sequence.

[0207] Embodiment 6. The engineered polynucleotide of any one of embodiments 3 to 5, wherein the first targeting sequence and the second targeting sequence are separated in the target sequence by a spacing sequence of 5 nucleotides or less (e.g., 1 or 2 nucleotides).

[0208] Embodiment 7. The engineered polynucleotide of any one of embodiments 1 to 6, wherein the target sequence comprises an exon-intron boundary in the pre-mRNA.

[0209] Embodiment 8 The engineered polynucleotide of embodiment 7, wherein the first targeting sequence and the second targeting sequence are both 5' or 3' to the exon-intron boundary.

[0210] Embodiment 9. The engineered polynucleotide of embodiment 7, wherein one of the first targeting sequence and the second targeting sequence is 5' to the exon-intron boundary, and the other of the first targeting sequence and the second targeting sequence is 3' to the exon-intron boundary.

[0211] Embodiment 10. The engineered polynucleotide of any one of embodiments 1 to 9, wherein the target sequence comprises a splice site in the pre-mRNA.

[0212] Embodiment 11. The engineered polynucleotide of embodiment 10, wherein the first targeting sequence or the second targeting sequence comprises a splice site (e.g., a 5'ss) in the pre-mRNA.

[0213] Embodiment 12. The engineered polynucleotide of any one of embodiments 1 to 11, wherein one of the first targeting moiety and the second targeting moiety is 5' to the recruitment moiety, and the other of the first targeting moiety and the second targeting moiety is 3' to the recruitment moiety.

[0214] Embodiment 13. The engineered polynucleotide of embodiment 12, wherein the first targeting moiety or the second targeting moiety comprises a sequence identical to or complementary to a sequence set forth in Table 1.

[0215] Embodiment 14. The engineered polynucleotide of embodiment 12, wherein said first targeting moiety comprises a sequence identical to or complementary to a sequence selected from the exon sequence column of Table 1, and said second targeting moiety comprises a sequence identical to or complementary to a sequence set forth in the intron sequence column of Table 1.

[0216] Embodiment 15. The engineered polynucleotide of embodiment 12, wherein said first targeting portion comprises a sequence identical to or complementary to a sequence set forth in the intron sequence column of Table 1, and said second targeting portion comprises a sequence identical to or complementary to a sequence set forth in the exon sequence column of Table 1.

[0217] Embodiment 16. The engineered polynucleotide of embodiment 12, wherein the first targeting moiety or the second targeting moiety comprises a sequence identical to or complementary to a consensus sequence of an intron donor site (e.g., selected from GU, GT, GC, and CA).

[0218] Embodiment 17. The engineered polynucleotide of embodiment 12, wherein the first targeting moiety or the second targeting moiety comprises a sequence identical to or complementary to a consensus sequence (e.g., G) of an exon donor site.

[0219] Embodiment 18. The engineered polynucleotide of embodiment 12, wherein the first targeting moiety or the second targeting moiety comprises a sequence identical to or complementary to a consensus sequence selected from GU, GC, G, and CA.

[0220] Embodiment 19. The engineered polynucleotide of any one of embodiments 1 to 18, wherein the spliceosome portion is selected from a spliceosomal ribonucleoprotein complex, a spliceosomal small nuclear ribonucleic acid (snRNA), a spliceosomal protein, a functional variant thereof, or a functional fragment thereof.

[0221] Embodiment 20 The engineered polynucleotide of embodiment 19, wherein said spliceosome portion comprises U1 snRNA and spliceosome proteins.

[0222] Embodiment 21. The engineered polynucleotide of any one of embodiments 19-20, wherein the spliceosomal snRNA is selected from U1, U2, U4, U5, U6, U11, U12, U14atac, U6atac, and combinations thereof.

[0223] Embodiment 22. The engineered polynucleotide of any one of embodiments 19 to 21, wherein the spliceosome protein is selected from Sm, U1-70k, U1A, U1C, and combinations thereof.

[0224] Embodiment 23. The engineered polynucleotide of any one of embodiments 1 to 22, wherein the recruitment portion comprises a nucleotide sequence that is at least 70%, 80%, 85%, or 90% identical to or complementary to any one of SEQ ID NOs: 1 to 2.

[0225] Embodiment 24. The engineered polynucleotide of embodiment 23, wherein the recruitment portion comprises a nucleotide sequence identical to or complementary to any one of SEQ ID NOs: 1-2.

[0226] Embodiment 25. The engineered polynucleotide of any one of embodiments 1 to 24, wherein the engineered polynucleotide comprises a (e.g., secondary) structural feature.

[0227] Embodiment 26 The engineered polynucleotide of embodiment 25, wherein said engineered polynucleotide comprises an apical loop, an upper stem, an internal loop, a lower stem, or a combination thereof.

[0228] Embodiment 27. The engineered polynucleotide of embodiment 25, wherein the engineered polynucleotide comprises a loop (e.g., an internal loop) adjacent to a stem (e.g., a lower stem or an upper stem) comprising two complementary stem sequences.

[0229] Embodiment 28. The engineered polynucleotide of embodiment 27, wherein the stem sequence of said stem (e.g., lower stem or upper stem) comprises about 5 or less, 4 or less, or 3 or less nucleotides.

[0230] Embodiment 29. The engineered polynucleotide of embodiment 27 or 28, wherein the loop is an internal loop flanked by a stem (e.g., a lower stem) and a further stem (e.g., an upper stem) comprising two complementary stem sequences.

[0231] Embodiment 30 The engineered polynucleotide of embodiment 29, wherein said internal loop comprises a nucleic acid sequence of 10 or fewer, 9 or fewer, or 8 or fewer nucleotides.

[0232] Embodiment 31 The engineered polynucleotide of embodiment 29 or 30, wherein the stem sequence of said additional stem (e.g., upper stem) comprises no more than about 5, no more than 4, or no more than 3 nucleotides.

[0233] Embodiment 32 The engineered polynucleotide of any one of embodiments 29 to 31, wherein the engineered polynucleotide further comprises an apical loop.

[0234] Embodiment 33 The engineered polynucleotide of embodiment 32, wherein the apical loop comprises a nucleic acid sequence of 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, or 5 or less nucleotides.

[0235] Embodiment 34. The engineered polynucleotide of any one of embodiments 1 to 33, wherein the engineered polynucleotide does not contain any intramolecular disulfide bonds.

[0236] Embodiment 35. The engineered polynucleotide of any one of embodiments 1 to 34, wherein, upon association with said engineered polynucleotide and said spliceosome portion, said pre-mRNA exhibits substantially no base pairing with the RNA binding domain (RBD) of U1 snRNA.

[0237] Embodiment 36. The engineered polynucleotide of any one of embodiments 1 to 35, wherein, upon association with the engineered polynucleotide and the spliceosome portion, the pre-mRNA exhibits substantially no base-specific interaction with U1-C protein.

[0238] Embodiment 37. The engineered polynucleotide of any one of embodiments 1 to 36, wherein the engineered polynucleotide is configured to specifically interact with a zinc finger of a U1-C protein.

[0239] Embodiment 38 The engineered polynucleotide of embodiment 37, wherein the 5' targeting portion of said engineered polynucleotide is configured to specifically interact with a zinc finger of a U1-C protein.

[0240] Embodiment 39. The engineered polynucleotide of embodiment 37 or 38, wherein the engineered polynucleotide (e.g., the 5' targeting portion thereof) is configured to covalently interact with a zinc finger of a U1-C protein (e.g., via a disulfide bond).

[0241] Embodiment 40. The engineered polynucleotide of any one of embodiments 37 to 39, wherein the engineered polynucleotide (e.g., the 5' targeting portion thereof) is configured to non-covalently interact (e.g., via hydrogen bonding) with a zinc finger of a U1-C protein.

[0242] Embodiment 41. The engineered polynucleotide of any one of embodiments 1 to 40, wherein the engineered polynucleotide comprises a nucleotide sequence complementary to a subsequence of stem loop II (SL2) of U1 snRNA.

[0243] Embodiment 42. The engineered polynucleotide of embodiment 41, wherein the sides of the stem-loop structure of the engineered polynucleotide comprise a nucleotide sequence complementary to a partial sequence of stem-loop II (SL2) of U1 snRNA.

[0244] Embodiment 43. The engineered polynucleotide of embodiment 41 or 42, wherein the subsequence comprises a sequence corresponding to 5'-GGCCU-3' of SL2 of U1 snRNA.

[0245] Embodiment 44. The engineered polynucleotide of any one of embodiments 41 to 43, wherein the partial sequence does not include a sequence corresponding to 5'-CACGUUA-3' of SL2 of U1 snRNA.

[0246] Embodiment 45. The engineered polynucleotide of any one of embodiments 1 to 44, wherein said engineered polynucleotide exhibits substantially no base pairing with the anchor sequence of SL2 of U1 snRNA.

[0247] Embodiment 46 The engineered polynucleotide of embodiment 45, wherein the internal loop of said engineered polynucleotide exhibits substantially no base pairing with said anchor sequence of said SL2 of U1 snRNA.

[0248] Embodiment 47. The engineered polynucleotide of embodiment 45 or 46, wherein the lower stem of said engineered polynucleotide exhibits substantially no base pairing with said anchor sequence of said SL2 of U1 snRNA.

[0249] Embodiment 48. The engineered polynucleotide of any one of embodiments 45 to 47, wherein the anchor sequence comprises a sequence corresponding to 5'-CACGUUA-3'.

[0250] Embodiment 49. The engineered polynucleotide of any one of embodiments 1 to 48, wherein said engineered polynucleotide exhibits substantially no base pairing with the H helix of U1 snRNA.

[0251] Embodiment 50. The engineered polynucleotide of any one of embodiments 1 to 49, wherein said engineered polynucleotide comprises at least one chemical modification.

[0252] Embodiment 51. The engineered polynucleotide of embodiment 50, wherein said engineered polynucleotide comprises at least one 2'-modified (e.g., 2'-methoxy, 2'-methoxymethyl, or 2'-methoxyethyl) nucleotide.

[0253] Embodiment 52 The engineered polynucleotide of embodiment 50 or 51, wherein at least about 50%, 60%, 70%, 80%, or 90% of the nucleotides in said engineered polynucleotide are chemically modified nucleotides.

[0254] Embodiment 53. The engineered polynucleotide of embodiment 50 or 51, wherein at least about 50%, 60%, 70%, 80%, or 90% of the nucleotides of said engineered polynucleotide are 2'-modified (e.g., 2'-methoxy, 2'-methoxymethyl, or 2'-methoxyethyl) nucleotides.

[0255] Embodiment 54. The engineered polynucleotide of any one of embodiments 50 to 53, wherein the engineered polynucleotide comprises at least one phosphorothioate internucleotide linkage.

[0256] Embodiment 55. The engineered polynucleotide of any one of embodiments 50 to 54, wherein at least about 50%, 60%, 70%, 80%, or 90% of the internucleotide linkages of said engineered polynucleotide are chemically modified.

[0257] Embodiment 56. The engineered polynucleotide of any one of embodiments 50 to 54, wherein at least about 50%, 60%, 70%, 80%, or 90% of the internucleotide linkages of said engineered polynucleotide are phosphorathioate.

[0258] Embodiment 57. The engineered polynucleotide of any one of embodiments 1 to 56, wherein the engineered polynucleotide comprises from about 10 to about 40 nucleotides, from about 10 to about 35 nucleotides, from about 10 to about 30 nucleotides, or from about 10 to about 25 nucleotides.

[0259] Embodiment 58. The engineered polynucleotide of any one of embodiments 1 to 57, wherein the recruitment portion comprises from about 10 to about 30 nucleotides, or from about 10 to about 20 nucleotides.

[0260] Embodiment 59. The engineered polynucleotide of any one of embodiments 1 to 58, wherein the one or more targeting moieties each independently comprise from about 2 to about 15 nucleotides, from about 2 nucleotides to about 10 nucleotides, or from about 2 nucleotides to about 8 nucleotides.

[0261] Embodiment 60. The engineered polynucleotide of any one of embodiments 1 to 59, wherein one of the first targeting portion and the second targeting portion comprises about 2 nucleotides, and the other of the first targeting portion and the second targeting portion comprises about 5 or 6 nucleotides.

[0262] Embodiment 61. The engineered polynucleotide of any one of embodiments 1 to 60, wherein, upon association with the engineered polynucleotide and the pre-RNA, the spliceosome portion cleaves or splices the pre-mRNA in the target sequence.

[0263] Embodiment 62 The engineered polynucleotide of any one of embodiments 1 to 61, wherein the spliceosome portion further promotes modification of the cleaved pre-mRNA.

[0264] Embodiment 63. An engineered polynucleotide comprising a nucleotide sequence at least 70%, 80%, 85%, or 90% identical to or complementary to any one of SEQ ID NOs: 1-4, wherein the engineered polynucleotide is characterized by (e.g., secondary) structural features.

[0265] Embodiment 64. The engineered polynucleotide of embodiment 63, wherein the nucleotide sequence is identical to or complementary to any one of SEQ ID NOs: 1 to 4.

[0266] Embodiment 65. The engineered polynucleotide of embodiment 63 or 64, wherein said structural features comprise one or more stem-loop structures.

[0267] Embodiment 66 The engineered polynucleotide of embodiment 63 or 64, wherein said structural feature comprises an apical loop, an upper stem, an internal loop, a lower stem, or a combination thereof.

[0268] Embodiment 67. The engineered polynucleotide of any one of embodiments 63 to 66, wherein the engineered polynucleotide comprises a loop (e.g., an internal loop) adjacent to a stem (e.g., a lower stem or an upper stem) comprising two complementary stem sequences.

[0269] Embodiment 68. The engineered polynucleotide of embodiment 67, wherein the stem sequence of said stem (e.g., lower stem or upper stem) comprises about 5 or less, 4 or less, or 3 or less nucleotides.

[0270] Embodiment 69. The engineered polynucleotide of any one of embodiments 63 to 68, wherein the loop is an internal loop adjacent to the stem (e.g., the lower stem) and a further stem (e.g., an upper stem) comprising two complementary stem sequences.

[0271] Embodiment 70. The engineered polynucleotide of embodiment 69, wherein said internal loop comprises a nucleic acid sequence of 10 or fewer, 9 or fewer, or 8 or fewer nucleotides.

[0272] Embodiment 71. The engineered polynucleotide of embodiment 69 or 70, wherein the stem sequence of the additional stem (e.g., the upper stem) comprises no more than about 5, no more than 4, or no more than 3 nucleotides.

[0273] Embodiment 72 The engineered polynucleotide of any one of embodiments 69 to 71, wherein the engineered polynucleotide further comprises an apical loop.

[0274] Embodiment 73 The engineered polynucleotide of embodiment 72, wherein the apical loop comprises a nucleic acid sequence of 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, or 5 or less nucleotides.

[0275] Embodiment 74. The engineered polynucleotide of any one of embodiments 63 to 73, wherein the engineered polynucleotide further comprises one or more targeting moieties that are sufficiently identical to or complementary to a target sequence of a target gene.

[0276] Embodiment 75. The engineered polynucleotide of embodiment 74, wherein one targeting moiety of the one or more targeting moieties is sufficiently identical to or complementary to a consensus sequence in the target sequence of the target gene.

[0277] Embodiment 76 The engineered polynucleotide of embodiment 74, wherein said target gene is microtubule-associated protein tau (MAPT).

[0278] Embodiment 77. The engineered polynucleotide of any one of embodiments 63 to 75, wherein the engineered polynucleotide comprises at least one chemical modification.

[0279] Embodiment 78. The engineered polynucleotide of embodiment 77, wherein said engineered polynucleotide comprises at least one phosphorothioate internucleotide linkage.

[0280] Embodiment 79. The engineered polynucleotide of embodiment 77, wherein at least about 50%, 60%, 70%, 80%, or 90% of the internucleotide linkages of said engineered polynucleotide are chemically modified.

[0281] Embodiment 80. The engineered polynucleotide of embodiment 77, wherein at least about 50%, 60%, 70%, 80%, or 90% of the internucleotide linkages are phosphorathioate.

[0282] Embodiment 81. The engineered polynucleotide of any one of embodiments 77 to 80, wherein the engineered polynucleotide comprises at least one 2'-modified (e.g., 2'-methoxy, 2'-methoxymethyl, or 2'-methoxyethyl) nucleotide.

[0283] Embodiment 82. The engineered polynucleotide of any one of embodiments 77 to 80, wherein at least about 50%, 60%, 70%, 80%, or 90% of the nucleotides in said engineered polynucleotide are chemically modified nucleotides.

[0284] Embodiment 83. The engineered polynucleotide of any one of embodiments 77 to 80, wherein at least about 50%, 60%, 70%, 80%, or 90% of the nucleotides in said engineered polynucleotide are 2'-modified (e.g., 2'-methoxy, 2'-methoxymethyl, or 2'-methoxyethyl) nucleotides.

[0285] Embodiment 84. The engineered polynucleotide of any one of embodiments 64 to 84, wherein the engineered polynucleotide comprises from about 10 to about 40 nucleotides, from about 10 to about 35 nucleotides, from about 10 to about 30 nucleotides, or from about 10 to about 25 nucleotides.

[0286] Embodiment 85. A method for altering a pre-messenger ribonucleic acid (pre-mRNA) in a cell, the method comprising contacting a cell with an engineered polynucleotide comprising one or more targeting moieties and a recruitment moiety, wherein the one or more targeting moieties bind to the pre-mRNA at an internal target sequence, and the recruitment moiety alters the pre-mRNA in the cell by recruiting a post-transcriptional regulatory moiety (e.g., a spliceosome moiety) near the target sequence of the pre-mRNA, thereby resulting in one or more altered pre-mRNAs.

[0287] Embodiment 86 The method of embodiment 85, wherein one targeting moiety of the one or more targeting moieties is sufficiently identical to or complementary to a consensus sequence in the target sequence of the target gene.

[0288] Embodiment 87 The method of embodiment 85 or 86, wherein the pre-mRNA corresponds to a target gene.

[0289] Embodiment 88 The method of embodiment 87, wherein the target gene is microtubule-associated protein tau (MAPT).

[0290] Embodiment 89. The method of any one of embodiments 85 to 88, wherein the method alters the expression or activity of the target gene.

[0291] Embodiment 90. The method of any one of embodiments 85 to 89, wherein prior to said contacting step, said cells exhibit aberrant messenger ribonucleic acid (mRNA) or protein corresponding to said target gene.

[0292] Embodiment 91. A set of engineered polynucleotides, each independently comprising: (i) one or more targeting moieties configured to specifically bind to pre-messenger ribonucleic acid (pre-mRNA) at a target sequence; and (ii) a recruitment moiety configured to recruit a post-transcriptional regulatory moiety (e.g., a spliceosome moiety), wherein the set of engineered polynucleotides is configured to specifically bind to the pre-mRNA at a plurality of target sequences, including the target sequence. [Example]

[0293] The following illustrative examples represent embodiments of the stimuli, systems, and methods described herein and are not intended to be limiting in any way.

[0294] Example 1. Modulation of target gene expression by engineered polynucleotides Cells obtained from a cell line (e.g., HEK293 cell line) are cultured and maintained in cell culture medium. The cells can then be contacted with an engineered polynucleotide or a vector encoding the engineered polynucleotide for delivery to the cells by any one of the delivery methods described herein. After the engineered polynucleotide is delivered to the cells, the cells can be cultured for a period of time to allow the engineered polynucleotide to regulate the expression or activity of the target gene. The cells can then be harvested and lysed to measure the expression or activity of the target gene. For example, the cells can be harvested and lysed to examine the abundance of pre-mRNA, mRNA, or protein of the target gene regulated by the engineered polynucleotide. In other cases, the cells can be fixed and prepared for microscopic examination. For example, the cells can be examined under a microscope for the presence or changes in abundance of inclusions or amyloid plaques associated with any one of the target genes described herein (e.g., tau neurofibrillary tangles encoded by the MAPT target gene or amyloid plaques encoded by the APP target gene).

[0295] Example 2. Treating neurological disorders by editing RNA A subject is diagnosed with Alzheimer's disease caused by aberrant splicing or polyadenylation activity of pre-mRNA in the subject's brain. The subject is prescribed an administration regimen of a pharmaceutical composition comprising a chemically modified engineered polynucleotide disclosed herein to recruit and stabilize at least one regulatory moiety to reduce the aberrant splicing or polyadenylation activity of multiple different pre-mRNAs, thereby reducing the symptoms of Alzheimer's disease. Some of the pre-mRNAs (among other pre-mRNAs) may include MAPT pre-mRNA. Upon binding to MAPT pre-mRNA, the chemically modified engineered polynucleotide recruits and stabilizes at least one regulatory moiety for accurate splicing of MAPT pre-mRNA. In some embodiments, the chemically modified engineered polynucleotide increases the specificity or efficiency of recruiting and stabilizing at least one regulatory moiety RNA editing entity. Modulation of MAPT pre-mRNA by the engineered polynucleotide reduces the amount of tau plaques in the subject, thereby treating or reducing the symptoms of Alzheimer's disease in the subject.

[0296] Example 3. Assessment of mitochondrial activity in induced pluripotent stem cell (IPSC)-derived neurons after incubation with ASMO1 Mitochondria are central to various cellular processes, including ATP production, intracellular calcium signaling, and reactive oxygen species generation. Neurons critically depend on mitochondrial function to establish membrane excitability and execute the complex processes of neurotransmitter and plasticity. In addition to assessing cellular metabolism, feasibility studies are used to determine whether a particular molecule is toxic to a test system, which is fundamentally important for determining working concentrations and safety profiles. Cell viability, defined as the number of healthy cells in a sample, can be used as a marker of death following exposure to a toxicant.

[0297] The CyQUANT MTT Cell Viability Assay utilizes the MTT reagent to determine mammalian cell viability. Measuring changes in cell viability is a method for assaying cell health and determining genotoxicity. The redox potential in viable mammalian cells converts the water-soluble MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to an insoluble formazan product. After solubilization of the formazan with SDS (sodium dodecyl sulfate) reagent, the concentration of the colorimetric probe is determined by optical density measurement at 540-570 nm (CyQUANT™ MTT Cell Viability Assay).

[0298] Healthy patient-derived cells (HDCs) and cells diagnosed with Alzheimer's disease (ADCs) were cultured for 6 days, after which ASMO1 (6.17, 18.52, 55.56, 166.67, 500 nM) or medium alone was added for 7 days (day 13). The culture medium was replaced with fresh medium containing MTT solution. The cells were maintained in a CO2 incubator (37°C, 5% CO2) for 4 hours, after which SDS-HCl was added. The contents of different wells were homogenized, and absorbance readings were taken at 540 nm on a microplate reader. The median value was obtained for each biological replicate. Three independent experiments were performed with three technical replicates. Values ​​are expressed as the mean ± standard error of the mean (SEM). To assess statistical differences between groups, a one-way ANOVA test was performed, followed by Tukey's post-hoc test. A P value of less than 0.05 was considered significant. Statistical analysis was performed using GraphPad Prism 9 software (GraphPad Software Inc., San Diego, CA, USA).

[0299] As shown in Figure 11A-B, (A) HDC-derived or (B) ADC-derived excitatory neurons were cultured for 6 days and then treated and incubated with APT20TTMG. After 7 days of treatment, neurons were assessed for mitochondrial activity using a commercially available kit.

[0300] No concentration of ASMO1 alters mitochondrial activity, suggesting that it does not alter the viability of neurons derived from iPSCs derived from healthy patients and patients diagnosed with Alzheimer's disease.

[0301] Example 4. Assessment of mitochondrial activity after incubation with ASMO1 in central nervous system cells Mitochondria are increasingly recognized as a key hub in the immune response mediated by astrocytes and microglia. A pathological hallmark of AD is impaired mitophagy, the cellular process by which damaged mitochondria are removed from cells by autophagy. Disruption of cellular energy is a key factor underlying the pathogenesis of AD, supporting the idea that altered mitochondrial function may be a cause or consequence of the pathological features of the disease.

[0302] Mammalian cell viability was determined using the CyQUANT MTT cell viability assay, as in Example 3. Neurons and microglia from healthy patients (HDC) and microglia from patients diagnosed with Alzheimer's disease (ADC) were plated and cultured for 6 days, after which ASMO1 (18.52 and 500 nM) or medium alone was added for 7 days (day 13). Astrocytes were plated and cultured for 1 day, after which ASMO1 (18.52 and 500 nM) or medium alone was added for 24 hours. The culture medium was then replaced with fresh medium containing MTT solution. Cells were maintained in a CO2 incubator (37°C, 5% CO2) for 4 hours, after which SDS-HCl was added. The contents of different wells were homogenized, and absorbance readings were taken at 540 nm in a microplate reader. Two to three independent experiments with three technical replicates were performed, and statistical analysis was performed as described in Example 3.

[0303] As shown in Figure 12, HDC-derived excitatory neurons were cultured for 6 days and then treated and incubated with APT20TTMG. After 7 days of treatment, neurons were evaluated for mitochondrial activity using a commercially available kit. Analysis of variance (ANOVA) followed by Tukey's post-hoc test was performed on data from three independent experiments with three technical replicates. A P value of <0.05 was considered statistically significant compared to the control group (no treated cells).

[0304] As shown in Figure 13A-B, (A) HDC-derived or (B) ADC-derived microglia were cultured for 6 days and then treated and incubated with APT20TTMG. After 7 days of treatment, microglia were assessed using a commercially available kit to measure mitochondrial activity. Analysis of variance (ANOVA) followed by Tukey's post-hoc test was performed. Data from two independent experiments with three technical replicates were analyzed. A p value of <0.05 was considered statistically significant compared to the control group (no treated cells).

[0305] As shown in Figure 14, astrocytes were cultured for 1 day and then treated and incubated with APT20TTMG. After 24 hours of treatment, neurons were evaluated for mitochondrial activity using a commercially available kit. Analysis of variance (ANOVA) followed by Tukey's post-hoc test was performed on data from three independent experiments with three technical replicates. A P value of <0.05 was considered statistically significant compared to the control group (no treated cells).

[0306] After incubation with ASMO1, no changes in mitochondrial activity were observed in neurons, microglia, and astrocytes. However, in neurons and microglia from healthy patients, this parameter was slightly reduced at the highest concentration (500 nM), and in astrocytes at both concentrations evaluated, which may be related to a decreased reactivity in astrocytes and, consequently, a decrease in cellular stress.

[0307] Example 5. Assessment of glutamate levels in induced pluripotent stem cell (iPSC)-derived neurons after incubation with ASMO1 Glutamate is the most abundant excitatory neurotransmitter in the mammalian central nervous system (CNS). Although it is widely distributed in the CNS, it is located almost exclusively intracellularly. The amount of available extracellular glutamate is tightly regulated to allow appropriate levels of signal transduction. Most excitatory neurotransmission in the mammalian CNS is mediated by glutamate and its receptors, primarily ligand-gated ionotropic glutamate receptors (iGluRs). These receptors also play fundamental roles in the molecular mechanisms underlying synaptic plasticity, learning, and memory. One subgroup of iGluRs is selectively gated by the specific agonist N-methyl-d-aspartate (NMDA) and is therefore named NMDA receptors (NMDARs), which are essential for their critical role in synaptic function and plasticity. Insufficient synaptic NMDAR signaling impairs neuronal survival. However, excessive stimulation of glutamatergic signaling leads to excitotoxicity, which damages or kills neurons. In addition to acute effects, numerous studies indicate a role for glutamate excitotoxicity in delayed, slowly evolving neurodegeneration.

[0308] The Glutamate-Glo™ Assay is a bioluminescent assay for detecting glutamate in biological samples. This assay is based on the conversion of glutamine to glutamate by the enzyme glutaminase. Glutamate oxidation and NADH production are then coupled with a bioluminescent NADH detection system. Glutamate dehydrogenase uses glutamate and NAD+ to generate α-ketoglutarate and NADH. In the presence of NADH, the proluciferin reductase substrate is converted to luciferin by the reductase, which is then used by the Ultra-Gio™ recombinant luciferase to generate light.

[0309] Healthy patient-derived cells (HDCs) and cells from patients diagnosed with Alzheimer's disease (ADCs) were cultured for 6 days, after which ASMO1 (6.17, 18.52, 55.56, 166.67 nM, and 500 nM) or medium alone was added for 7 days (day 13), and the extracellular medium was collected for further analysis. Samples were prepared for relative quantification of glutamate using the Glutamate-Glo™ assay, and glutamate levels were reported in relative light units (RLU). Three independent experiments with six technical replicates were performed, and statistical analysis was performed as described in Example 3.

[0310] As shown in Figure 15A-B, (A) HDC-derived or (B) ADC-derived excitatory neurons were cultured for 6 days and then treated and incubated with APT20TTMG. After 7 days of treatment, neurons were assessed for glutamate release using a commercially available kit.

[0311] No significant changes in extracellular glutamate levels were observed after incubation with ASMO1 in neurons derived from iPSCs from healthy patients and patients diagnosed with Alzheimer's disease, confirming previous data in Examples 3-4 showing that ASMO1 appears safe at these concentrations and conditions.

[0312] Example 6. Evaluation of morphological changes in induced pluripotent stem cell (iPSC)-derived neurons after incubation with ASMO1 Neuronal populations affected by Alzheimer's disease (AD) are characterized by abnormal synaptic morphology and a marked reduction in the total number of synapses, which are thought to underlie the memory and cognitive impairments characteristic of this disease. The proper function of axons and axon terminals depends on the transport of proteins, organelles, vesicles, and other elements from their synthesis sites in the cell body. Thus, axonal transport is a critical cellular process underlying axonal and synaptic function. Several studies have demonstrated that dystrophic axons are observed before the detectable deposition of classical tau and amyloid pathology. Understanding the time course of axonal pathology is germane to understanding disease progression over time and defining possible therapeutic targets and time windows during which treatments may be effective.

[0313] Healthy patient-derived cells (HDCs) and cells from patients diagnosed with Alzheimer's disease (ADCs) were cultured in NeuroHTS™ technology plates for 6 days, after which ASMO1 (6.17, 18.52, 55.56, 166.67 nM, and 500 nM) or medium alone was added for 7 days (day 13). All neurons were then evaluated in a 7-factor neuronal morphological profile via the MetaXpress software. As shown in Figure 16, neurons were evaluated in three separate regions and parameters. In the upper compartment of the well, cell number and nuclear aggregates were assessed in the soma and dendrites. In the central compartment of the channel (axon), the thickness of axonal fibers was verified. In the lower compartment, where the axons and dendrites are located, four parameters were evaluated: number of branches, number of branch junctions, neurite straightness, and axonal material. Three independent experiments with up to three technical replicates were performed.

[0314] Values ​​were expressed as the mean ± standard error of the mean. To assess statistical differences between groups, one-way ANOVA tests were performed, followed by Tukey's (parametric) test for cell aggregates, fiber width, and neurite linearity, or by Kruskal-Wallis (nonparametric) post-test tests for cell number, number of branches, number of branches per cell, branch junctions, branch junctions per cell, total axonal material, and axonal material per cell. A P value of less than 0.05 was considered significant. Statistical analysis was performed using GraphPad Prism 9 software (GraphPad Software Inc., San Diego, CA, USA).

[0315] As shown in Figure 17A-B, excitatory neurons were cultured for 6 days and then incubated with ASMO1. After 7 days of treatment, the number of neurons was assessed using the software MetaXpress. (A) Cell counts from healthy patient cells (HDCs) and (B) cell counts from AD patient cells (ADCs). Cell counts are an indirect indicator of cell death. This parameter is crucial for understanding the safety profile of ASMO1 in these cells and helps select the concentrations used in subsequent assays. In addition, to avoid masking the results, some analyzed parameters need to be normalized with respect to the total number of cells at the end of the experiment. Although the concentrations did not significantly reduce cell counts in HDCs and ADCs, there appears to be a significant reduction at the two highest concentrations (166.67 and 500 nM).

[0316] As shown in Figure 18A-B, excitatory neurons were cultured for 6 days and then incubated with ASMO1. After 7 days of treatment, cell aggregates were evaluated using the software MetaXpress. (A) Cell aggregates derived from healthy patient cells (HDCs), (B) cell aggregates derived from AD patient cells (ADCs).

[0317] This parameter may be related to the aggregation of neurons in the culture, i.e., due to the presence of cellular debris and even free DNA. These two factors may appear as indicators of toxicity after the addition of the molecule. Incubation with different concentrations of ASMO1 did not alter cell aggregation, a new safety indicator in the presence of ASMO1.

[0318] As shown in Figure 19A-B, excitatory neurons were cultured for 6 days and then incubated with ASMO1. After 7 days of treatment, fiber width was assessed using the software MetaXpress. (A) Fiber width from healthy patient cells (HDCs) and (B) fiber width from AD patient cells (ADCs).

[0319] Fiber width / thickness is an indicator of axonal bundles, which are composed of intertwined groups of axons. Any impairment of fiber thickness can lead to alterations in axonal transport, which plays an important role in the pathogenesis of AD. In both HDC and ADC, fiber thickness tended to increase after treatment with all concentrations of ASMO1. Furthermore, ADC neurons treated with ASMO1 at all evaluated concentrations showed a profile similar to that of negative HDC controls. Even in HDC, incubation with ASMO1 appeared to increase fiber thickness. Although the results did not show statistical differences, this parameter was taken into consideration when selecting concentrations in subsequent studies. These combined results support the hypothesis that ASMO1 can improve axonal transport.

[0320] As shown in Figure 20A-D, the number of branches is an indicator of neuronal growth, regeneration, and / or proliferation rate. Neurons output information through a wide variety of axon morphologies. One process that generates this diversity is axon branching. From reaching multiple targets to defining innervation territories within specific targets, axon branching allows neurons to establish unique patterns of connectivity. Changes in axon branching patterns are achieved through the regulation of various morphological processes, including branch initiation, branch elongation, the development of branching complexity, branch retraction / pruning, and terminal branching. One class of proteins involved in the regulation of axon branching is cytoskeleton-associated proteins. Axon degeneration, i.e., a decrease in the level of axon branching, is directly related to neurodegeneration. Molecules that can prevent degeneration or even regenerate neurons susceptible to degeneration, are closely related to AD. Indirectly, this parameter may be related to synapse gain at later stages. For two analyses (number of branches and branches per cell), HDC neurons at a concentration of 18.52 nM showed an interesting profile, with an approximately 80% increase in branch number and a two-fold increase in branch number per cell. In ADC neurons, this same concentration increased both the number of branches and the number of branches per cell by approximately 50% and 60%, respectively, suggesting protection from neuronal degeneration or increased axonal growth. (A) Number of branches from healthy patient cells (HDC), (B) Number of branches from AD patient cells (ADC), (C) Number of branches per cell from healthy patient cells (HDC), and (D) Number of branches per cell from AD patient cells (ADC). As shown in Figure 21A-D, branch junctions are an indicator of the complexity of branching structures and the development of potential synapses in neurons. It is a complementary parameter to branch number and therefore indicates the likelihood that the axon of one neuron will communicate with the dendrites of another neuron. Therefore, the greater the number of branch junctions, the greater the potential for communication and synapse formation. The most interesting profiles were for the 6.17 and 18.52 nM treatments for both test systems (HDC and ADC): for total branch junctions, HDC treatment at a concentration of 6.17 nM doubled this parameter.For ADCs, it increased by approximately 3.5-fold. (A) Branch junctions from healthy patient cells (HDCs); (B) Branch junctions from AD patient cells (ADCs), (C) Branch junctions per cell from healthy patient cells (HDCs), (D) Branch junctions per cell from AD patient cells (ADCs).

[0321] Neurites are any projections from the cell body of a neuron, a process of differentiation into neuronal dendrites and axons, which may be important for the exchange of information between different neurons. Therefore, the straightness of neurites is a parameter for assessing the quality of neurons with regard to the transport of molecules and action potentials within the neuron. The straighter this area is, the greater the possibility that information will pass along the neuron. Figure 22A-B shows the straightness of neurites for different cells. When compared to the negative control (no treatment) in the same system, a statistically significant increase ( ** p=0.0028). (A) Linearity of neurites derived from healthy patient cells (HDCs), (B) Linearity of neurites derived from AD patient cells (ADCs).

[0322] As shown in Figure 23A-D, at concentrations of 6.17 and 18.52 nM, both total axonal material and axonal material per cell indicate increased yields of overall neuronal content. In the case of axonal material per cell (normalized by total cell number), ADCs treated with the lowest ASMO1 concentration increased this parameter by approximately 160%, and at 18.52 nM, it increased by 130%. In HDC cells, treatment with the same concentrations increased the axonal material content per cell by approximately 80% and 3.5-fold, respectively. It is worth noting that in addition to several concentrations that have the potential to increase axonal material, parameters tended to improve for ADCs treated with the first three concentrations. (A) Total axonal material from healthy patient cells (HDCs), (B) Total axonal material from AD patient cells (ADCs), (C) Axonal material per cell from healthy patient cells (HDCs), and (D) Axonal material per cell from AD patient cells (ADCs).

[0323] As shown in Figures 24-25, excitatory neurons were cultured for 6 days and then incubated with ASMO1. After 7 days of treatment, all morphological changes were evaluated. Parameters evaluated included soma and dendrites (cell number and nuclear aggregates), axons (fiber width), and axons and dendrites (number of branches, number of branch junctions, neurite straightness, and axonal material).

[0324] Incubation with ASMO1 did not cause toxicity in HDC and ADC neurons, as demonstrated by cell count and cell aggregation parameters. Several analyzed parameters showed a tendency to increase after incubation with ASMO1, which appears favorable for preventing neurodegeneration and improving axonal function, directly related to synaptic and cognitive function, such as fiber width, branch number, branch junctions, neurite straightness, and total axonal material. Therefore, it is possible to confirm that ASMO1 exposure has the potential to be beneficial in both healthy neurons and neurons with AD-related phenotypes, helping to prevent the degeneration of vulnerable neurons in AD.

[0325] Example 7. Assessment of complex U1 protein binding by immunoprecipitation in neuroblastoma cell lines after incubation with ASMO-1 For protein immunoprecipitation assays, cell lysates (300 μg / IP) from SK-H-SH were added to 0.5 mg streptavidin beads pre-conjugated with either 90 pmol biotinylated APT20TTMG or beads alone (negative control) and incubated overnight at 4°C. The flow-through was collected, and the beads were washed 10 times with ice-cold IP lysis buffer. Proteins were eluted from the beads in 30 μl of 4× Laemmli Sample Buffer / 2-mercaptoethanol at 90°C for 5 min. Samples (5 μg input, 2.5% flow-through, and 50% eluate fractions) were separated on NuPAGE and electrophoresed. The separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes, blocked, and then incubated overnight at 4°C with the following primary antibodies: U1-70K (Abecam, catalog no. ab83306), U1-A (Abecam, catalog no. ab166890), U1-C (Abecam, catalog no. ab192028), and GAPDH (Abecam, catalog no. ab8245). Then, they were incubated with HRP-conjugated secondary antibodies. Proteins were visualized with enhanced chemiluminescence (ECL) reagents (Thermo Fisher Scientific, USA), and images were captured using the Jess Simple Western Imaging suite. Evaluation was performed using three biological replicates (N = 3).

[0326] For RNA immunoprecipitation using biotinylated APT20TTMG, cells were rinsed once with ice-cold PBS, collected, and pelleted by centrifugation at 500 g for 5 minutes. Each sample was resuspended and lysed in 150 μl of ice-cold RSB-100 buffer (10 mM Tris-HCl pH 7.4, 100 mM NaCl, 2.5 mM MgCl2, 0.5% Triton X-100) containing Complete Ultra EDTA-free protease inhibitor cocktail mini-tablets (Roche) and 100 U / ml Super-ASE RNase inhibitor (Thermo Fisher Scientific) for 20 minutes at 4°C. To increase protein yield, each sample was homogenized using a 29G × 13 mm needle and syringe. Cell lysates were clarified by centrifugation at 17,000 g for 20 minutes at 4°C. The supernatant was collected, and the total protein concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Each sample was adjusted to 1.00 mg / ml using RSB-100 lysis buffer, and a 100 μl aliquot was taken from each sample (input) and stored at -20°C until needed. For RNA manipulation, 0.5 mg of Dynabeads M-270 streptavidin magnetic beads (Thermo Fisher Scientific) were washed and prepared according to the manufacturer's instructions. The beads were conjugated with 90 pmol of biotinylated APT20TTDC or APT20TTMG (beads without oligo used as a negative control) for 30 minutes at room temperature. After washing, 100 μl of SK-N-SH cell lysate (100 μg) was added to the beads immobilized with biotinylated oligos (and controls) and incubated overnight at 4°C with gentle rotation. RNA was isolated from input, flow-through, and eluate samples using Direct-zol RNA Microprep (Cambridge Bioscience) according to the manufacturer's instructions. RNA (50% of input and flow-through and 100% of eluate fractions) was converted to cDNA using the following:

[0327] The SuperScript IV First-Strand synthesis system (Thermo Fisher) was used according to the manufacturer's instructions. After RNA removal, 1 μl of cDNA was amplified by qPCR using the following Taqman probes (Thermo Fisher): U1 (RNVU1-18, Hs04940459_gH), total tau (MAPT, Assay ID: Hs00902193_m1), and GAPDH (GAPDH, Assay ID: Hs99999905_m1). Gene expression was analyzed using an Applied Biosystems QuantStudio™ 12K Flex Real-Time PCR System.

[0328] Figure 26A-D shows various Western blots for detecting the presence of specific proteins in samples. Cell lysate (100 μg / IP) from SK-H-SH was added to 0.5 mg of streptavidin beads pre-conjugated with either 90 pmol of biotinylated APT20TTMG or beads alone (negative control) and incubated overnight at 4°C. Samples (5 μg input, 2.5% flow-through, and 50% eluate fractions) were evaluated and electrophoresed. Protein immunoprecipitation was assessed by Western blotting using specific antibodies. GAPDH was used as a negative control (n = 3 technical replicates).

[0329] Figure 27A-C shows the relative expression of specific proteins in samples. Human neuroblastoma SK-N-SH cells were lysed and prepared for qRT-PCR immunoprecipitation assays, which were performed using RNA isolated from input (50%), flow-through-FT (50%), and eluate (100%) samples. Relative expression of (A) U1 snRNA, (B) tau pre-mRNA, and (C) GAPDH. Data are presented as mean ± standard error of the mean. Graphs show data from three independent experiments analyzed using ANOVA followed by Tukey's post hoc test or t-test. ** p<0.01, **** p<0.0001.

[0330] Immunoprecipitation assays were performed using SK-N-SH neuroblastoma cells to verify the assembly of the APT20TTMG / U1-pre-mRNA complex. These assays confirmed that APT20TTMG binds to key proteins of the U1-complex, such as U1-70K and U1-C proteins, but not to the U1-A snRNP. The lack of binding to the GAPDH protein was expected, as this protein was used as a negative control in the experiment. qRT-PCR confirmed the binding of APT20TTMG to U1 snRNA as well as to tau and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) pre-mRNA. This binding pattern indicates interaction with a functional U1 snRNP complex, which also contains a set of proteins called the Sm core, which were not evaluated in this study. Furthermore, because U1-70K is involved in crosslinking pre-U1 (U1 snRNA precursor) and U1 snRNA to the SMN complex, which mediates Sm core assembly, the binding of APT20TTMG to U1-70K also provides evidence of putative Sm core assembly within the immunoprecipitated pool. The assembled Sm core plays a critical role in the stable binding of U1-70K to U1-snRNA. Consequently, the binding of APT20TTMG to these proteins, as well as to pre-mRNA and U1 snRNA, strongly suggests the correct assembly of the initial splicing machinery and subsequent spliceosome recruitment. This is noteworthy because the U1 complex must be properly assembled for correct splicing, and this process can be impaired by the typical U1 snRNP splicing dysfunction present in AD.

[0331] Evidence for direct or indirect interaction of ASMO1 with U1-C and U1-70K indicates that the predicted targets of ASMO are correct, i.e., ASMO generally interacts with the U1 complex and pre-mRNAs represented in these assays by MAPT and GAPDH pre-mRNAs, enabling correct U1-pre-mRNA complex assembly.

[0332] Example 8. Evaluation of tau protein regulation after incubation with ASMO1 in induced pluripotent stem cell (iPSC)-derived neurons Along with extracellular deposition of insoluble amyloid-β, which begins before early-onset Alzheimer's disease (AD), early AD is characterized by the intracellular accumulation of neurofibrillary tangles (NFTs) composed of aggregated, hyperphosphorylated tau. Despite this pathological role of tau in AD, under physiological conditions, this protein is a phosphoprotein that promotes microtubule assembly and stabilization in neurons. Tau also plays a role in chromatin structure, signal transduction, synaptic plasticity, and nucleic acid protection. In AD, abnormal tau hyperphosphorylation interferes with its ability to bind to microtubules, leading to abnormal tau self-assembly, accumulation, aggregation, and NFT formation in neurons. Pathogenic forms of tau have also been implicated in disrupting axonal maintenance processes, inducing loss of neuronal connectivity, inhibiting axonal transport, and accumulating in neuropil threads in AD.

[0333] Healthy patient-derived cells (HDCs) and cells from patients diagnosed with Alzheimer's disease (ADCs) were cultured for 6 days, after which ASMO1 (6.17 nM, 18.52 nM, and 500 nM) or medium alone was added for 7 days (day 13). Cells were then lysed, and samples from each experimental group were evaluated using the commercially available ab273617 Human Tau SimpleStep ELISA® kit. Quantitation curves were generated for each biological replicate. Three independent experiments were performed with three technical replicates. Values ​​were expressed as the mean ± standard error of the mean. To assess statistical differences between groups, a one-way ANOVA test was performed, followed by a Dunnett's multiple comparison test. A P value of less than 0.05 was considered significant. Statistical analysis was performed using GraphPad Prism 9 software (GraphPad Software Inc., San Diego, CA, USA).

[0334] As shown in Figure 28A-B, excitatory neurons from HDC (A) or ADC (B) were cultured for 6 days and then treated with medium (negative control) and incubated. After 7 days of treatment, tau levels were assessed using a commercially available kit.

[0335] In neurons derived from healthy patients (HDC), there was no change in tau protein levels after incubation with three concentrations of APT20TTMG. However, the same profile was not observed in neurons obtained from patients diagnosed with AD (ADC). After incubation with APT20TTMG at concentrations of 18.52 nM and 500 nM, the protein levels increased to 41.4 ± 2.9 ( * p=0.0196) and 60.4±0.8% ** p=0.0023), suggesting a concentration-dependent effect. A concentration of 6.17 nM also showed a reduction (33.7 ± 2.7%), but this result was not statistically different. It is noteworthy that APT20TTMG's proposed mechanism of action, in addition to acting on a pathway preceding the formation of amyloid-β plaques, two of the cardinal hallmarks of AD, acts on a pathway preceding the formation of neurofibrillary tangles, which are composed of tau protein aggregates. Thus, throughout this study, it was very clear that ASMO1 reduces the amount of tau protein at a stage and time preceding its increase in cultured neurons. Because levels are already low in cells mimicking some of the pathophysiological features of the disease, we can conclude that APT20TTMG has the potential to act at an earlier stage of the disease and prevent excessive tau protein formation.

[0336] ASMO1 concentration-dependently reduced tau protein only in neurons from patients diagnosed with AD, but not in neurons from healthy patients, suggesting that it may act at an early stage of the disease and reduce its progression.

[0337] Example 9. RNA-seq differential gene expression analysis of induced pluripotent stem cell (iPSC)-derived neurons after incubation with ASMO1 This study involved RNA sequencing (RNA-seq) differential gene expression analysis of neurons from induced pluripotent stem cells (IPSCs) after incubation with APT20TTMG. Gene expression occurs when DNA is transcribed into RNA, primarily mRNA transcripts. The level of mRNA transcripts is often used to infer protein abundance. This set of genes expressed by the genome is called the transcriptome, and measurement of RNA transcript levels can be accessed by RNA-seq, an efficient method for sequencing the transcriptome using next-generation sequencing (NGS). Once transcript levels are quantified and normalized, investigation of up- or down-regulated genes (thus, differential expression is measured) in the transcriptome in response to treatment with ASMO1 is possible. This differential expression analysis allows insight into the molecule's mechanism of action, theoretical safety assessment, and potential side effects underlying therapeutic intervention.

[0338] In this context, identifying genes directly or indirectly affected by ASMO1 (APT20TTMG) may be essential for understanding its potential effects in target cells such as neurons. To this end, RNA-seq and subsequent differential gene expression analysis of APT20TTMG-treated and untreated neurons derived from iPSCs from healthy patients and patients diagnosed with Alzheimer's disease was the methodology chosen to answer the desired biological question. The goal of this methodology was to identify key differences in gene expression and related pathways between iPSC-derived neurons from healthy patients (HDCs) and iPSC-derived neurons from patients diagnosed with Alzheimer's disease (ADCs) incubated with two concentrations of ASMO1 (APT20TTMG) compared to their untreated counterparts. Neurons derived from IPSCs from healthy patients (HDCs) and Alzheimer's disease patients (ADCs) were cultured for 6 days, after which treatment (APT20TTMG-18.52 nM, APT20TTMG-500 nM, or medium alone) was added for 7 days (day 13). It is important to note that this time point of treatment allows for the study of upstream events that lead to disease onset (ADCs increase in relation to HDCs before APP and MAPT) and was therefore chosen to assess the effect of APT20TTMG on ADC cells before the establishment of the disease phenotype.

[0339] After RNA extraction, libraries were generated from 250 ng of total RNA as follows: mRNA enrichment was performed using the NEBNext Poly(A) Magnetic Isolation Module. cDNA synthesis was achieved using the NEBNext RNA First-Strand Synthesis and NEBNext Ultra Directional RNA Second-Strand Synthesis Modules. The remaining steps of library preparation were performed using Illumina's NEBNext Ultra II DNA Library Prep Kit. Libraries were normalized and pooled, then denatured in 0.02 N NaOH and neutralized using HT1 buffer. The pool was loaded at 175 pM onto a NovaSeq S4 lane using the Xp protocol. All sample libraries were then sequenced using a NovaSeq 6000 (Illumina). 2 × 100 cycles (paired-end mode) were run. A phiX library was used as a control and mixed with the libraries at a 1% level. Base calling was performed using RTA v3. The samples were then demultiplexed and FASTQ reads were generated using the program bcl2fastq2 v2.20. Reads were paired at a read length of 101, aiming to achieve 100 million reads per sample. This experiment was performed with three replicates and one identical repeat.

[0340] The quality of raw reads (FASTQ files) was assessed using FASTQC v0.11.8 and combined with MultiQC. After examining the quality of the raw reads, trimming was deemed unnecessary. Reads were aligned to the human reference genome using STAR v2.7.6a. The GTF annotation file for the human reference genome used was downloaded from Ensembl (release 102). Raw counts were calculated using FeatureCounts v1.6.0. Differential expression analysis to determine differentially expressed genes (DEGs) was performed using the DESeq2 R package. In total, five main comparisons were performed, each with its own unique set of DEGs: (i) untreated ADC (medium only) versus ADC treated with 18.52 nM APT20TTMG; (ii) untreated ADC (medium only) versus ADC treated with 500 nM APT20TTMG; (iii) untreated HDC (medium only) versus HDC treated with 18.52 nM APT20TTMG; (iv) untreate...

Claims

1. 1. A method of treating a neurodegenerative disease in a subject in need thereof, comprising administering to said subject a pharmaceutical composition comprising an engineered polynucleotide, said engineered polynucleotide comprising: (i) one or more targeting moieties configured to specifically bind to pre-messenger ribonucleic acid (pre-mRNA) at an internal target sequence; (ii) a recruitment moiety configured to recruit a spliceosome moiety; Upon association with the pre-mRNA and the engineered polynucleotide, the spliceosome portion alters the pre-mRNA at or near the target sequence.

2. 2. The method of claim 1, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Pick's disease, chronic traumatic encephalopathy, progressive supranuclear palsy, argyrophilic grain disease, amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD).

3. 2. The method of claim 1, wherein the neurodegenerative disease is a neurodegenerative disease associated with the presence of tau aggregation or dysfunction or U1 snRNP aggregation or dysfunction in the subject's brain.

4. The method of any one of claims 1 to 3, wherein the method alters the expression of tau.

5. The method of any one of claims 1 to 4, wherein the method modulates the expression of tau.

6. The method of any one of claims 1 to 5, wherein the method reduces the total amount of tau in the brain of the subject.

7. The method according to any one of claims 1 to 8, wherein the neurodegenerative disease is a neurodegenerative disease associated with the presence of amyloid beta in the brain of the subject.

8. The method according to any one of claims 1 to 8, wherein the method alters the expression of amyloid beta.

9. The method of any one of claims 1 to 8, wherein the method reduces expression of amyloid beta.

10. The method of any one of claims 1 to 9, wherein the method reduces the total amount of amyloid beta in the brain of the subject.

11. The method of any one of claims 1 to 10, wherein the method reduces the total amount of amyloid plaques in the brain of the subject.

12. 1. A method of reducing tau expression and / or aggregation in a neuron, the method comprising administering to the neuron an engineered polynucleotide, the engineered polynucleotide comprising: (i) one or more targeting moieties configured to specifically bind to pre-messenger ribonucleic acid (pre-mRNA) at an internal target sequence, which may be a conserved splice site; (ii) a recruitment moiety configured to recruit a spliceosome moiety; Upon association with the pre-mRNA and the engineered polynucleotide, the spliceosome portion alters the pre-mRNA at or near the target sequence.

13. 1. A method for reducing amyloid beta expression and / or aggregation in a neuron, the method comprising administering to the neuron an engineered polynucleotide, the engineered polynucleotide comprising: (i) one or more targeting moieties configured to specifically bind to pre-messenger ribonucleic acid (pre-mRNA) at an internal target sequence, which may be a conserved splice site; (ii) a recruitment moiety configured to recruit a spliceosome moiety; Upon association with the pre-mRNA and the engineered polynucleotide, the spliceosome portion alters the pre-mRNA at or near the target sequence.

14. 1. A method for reducing aggregation and / or mislocalization of U1-70K in a neuron, the method comprising administering to the neuron an engineered polynucleotide, the engineered polynucleotide comprising: (i) one or more targeting moieties configured to specifically bind to pre-messenger ribonucleic acid (pre-mRNA) at an internal target sequence; (ii) a recruitment moiety configured to recruit a spliceosome moiety; Upon association with the pre-mRNA and the engineered polynucleotide, the spliceosome portion alters the pre-mRNA at or near the target sequence.

15. 1. A method of modulating astrogliosis, the method comprising administering to a neuron an engineered polynucleotide, the engineered polynucleotide comprising: (i) one or more targeting moieties configured to specifically bind to pre-messenger ribonucleic acid (pre-mRNA) at an internal target sequence; (ii) a recruitment moiety configured to recruit a spliceosome moiety; Upon association with the pre-mRNA and the engineered polynucleotide, the spliceosome portion alters the pre-mRNA at or near the target sequence.

16. 1. A method for increasing fiber width in a neuron, the method comprising administering to the neuron an engineered polynucleotide, the engineered polynucleotide comprising: (i) one or more targeting moieties configured to specifically bind to pre-messenger ribonucleic acid (pre-mRNA) at an internal target sequence; (ii) a recruitment moiety configured to recruit a spliceosome moiety; Upon association with the pre-mRNA and the engineered polynucleotide, the spliceosome portion alters the pre-mRNA at or near the target sequence.

17. 1. A method for increasing branch number and branch junctions in a neuron, the method comprising administering to the neuron an engineered polynucleotide, the engineered polynucleotide comprising: (i) one or more targeting moieties configured to specifically bind to pre-messenger ribonucleic acid (pre-mRNA) at an internal target sequence; (ii) a recruitment moiety configured to recruit a spliceosome moiety; Upon association with the pre-mRNA and the engineered polynucleotide, the spliceosome portion alters the pre-mRNA at or near the target sequence.

18. 1. A method for increasing total axonal material in a neuron, the method comprising administering to the neuron an engineered polynucleotide, the engineered polynucleotide comprising: (i) one or more targeting moieties configured to specifically bind to pre-messenger ribonucleic acid (pre-mRNA) at an internal target sequence; (ii) a recruitment moiety configured to recruit a spliceosome moiety; Upon association with the pre-mRNA and the engineered polynucleotide, the spliceosome portion alters the pre-mRNA at or near the target sequence.

19. 1. A method for increasing electrical activity in a neuron, the method comprising administering to the neuron an engineered polynucleotide, the engineered polynucleotide comprising: (i) one or more targeting moieties configured to specifically bind to pre-messenger ribonucleic acid (pre-mRNA) at an internal target sequence; (ii) a recruitment moiety configured to recruit a spliceosome moiety; Upon association with the pre-mRNA and the engineered polynucleotide, the spliceosome portion alters the pre-mRNA at or near the target sequence.

20. The method of any of claims 12 to 19, wherein the neurons are derived from an individual suffering from a neurodegenerative disorder.

21. 21. The method of claim 20, wherein the neurodegenerative disorder is selected from the group consisting of Alzheimer's disease, Pick's disease, chronic traumatic encephalopathy, progressive supranuclear palsy, argyrophilic grain disease, amyotrophic lateral sclerosis (ALS), and frontotemporal dementia.

22. The method according to any one of claims 12 to 21, wherein the neurons are healthy neurons.

23. The method of any one of claims 12 to 22, wherein the neurons are derived from a healthy subject.

24. The method of any one of claims 12 to 23, wherein the method alters and / or modulates the expression of a gene in the neuron.

25. 25. The method of claim 24, wherein the method alters and / or modulates the expression of a gene that is part of the MAPK signaling pathway.

26. 25. The method of claim 24, wherein the method alters and / or modulates the expression of a gene that is part of the cholesterol metabolic pathway.

27. 25. The method of claim 24, wherein the method alters and / or modulates the expression of a gene that is part of the steroid biosynthetic pathway.

28. 25. The method of claim 24, wherein the method alters and / or modulates the expression of a gene associated with mRNA transport.

29. 25. The method of claim 24, wherein the method alters and / or modulates the expression of a gene that is part of a cell cycle regulation or cellular senescence pathway.

30. 25. The method of claim 24, wherein the method alters and / or modulates the expression of a gene that is part of the pyruvate metabolic pathway.

31. 25. The method of claim 24, wherein the method alters and / or modulates the expression of a gene that is part of the Ras signaling pathway.

32. 25. The method of claim 24, wherein the method alters and / or modulates the expression of a gene that is part of the AMPK signaling pathway.

33. 25. The method of claim 24, wherein the method alters and / or modulates the expression of a gene that is part of a fatty acid metabolic pathway.

34. 25. The method of claim 24, wherein the method alters and / or modulates the expression of a gene that is part of a fatty acid synthesis pathway.

35. 25. The method of claim 24, wherein the method alters and / or modulates the expression of a gene that is part of the PPAR signaling pathway.

36. 25. The method of claim 24, wherein the method alters and / or modulates the expression of a gene that is part of the PI3K-Akt signaling pathway.

37. The method of any one of claims 12 to 36, wherein the method increases mRNA transport in the neuron.

38. 38. The method of any one of claims 12 to 37, wherein the method reduces excitotoxicity in the neuron.

39. The method of any one of claims 12 to 38, wherein the method increases synaptic integrity of the neuron.

40. 39. The method of any one of claims 12 to 38, wherein the method generates differentially expressed genes associated with the regulation of neurogenesis, neuronal differentiation, and synapse assembly.

41. 40. The method of any one of claims 12 to 39, wherein the method reduces premature polyadenylation of one or more transcripts in the neuron.

42. 40. The method of any one of claims 12 to 39, wherein the method reduces cryptic splicing of one or more transcripts in the neuron.

43. 43. The method of any one of claims 1 to 42, wherein the engineered polynucleotide is administered at a concentration of at least 5 nM.

44. 43. The method of any one of claims 1 to 42, wherein the engineered polynucleotide is administered at a concentration of at least 10 nM.

45. 43. The method of any one of claims 1 to 42, wherein the engineered polynucleotide is administered at a concentration of about 5 nM to about 20 nM.

46. 46. ​​The method of any one of claims 1 to 45, wherein the engineered polynucleotide is administered intratumorally.

47. 46. ​​The method of any one of claims 1 to 45, wherein the engineered polynucleotide is administered intravenously.

48. 46. ​​The method of any one of claims 1 to 45, wherein the engineered polynucleotide is administered intrathecally.

49. 46. ​​The method of any one of claims 1-45, wherein the engineered polynucleotide is administered via subcutaneous injection, intramuscular injection, intradermal injection, transdermal administration, intranasal administration, intralymphatic injection, intrathecal administration, pulmonary administration, rectal administration, intragastric administration, or any other suitable parenteral administration.

50. 50. The method of any one of claims 1 to 49, wherein one targeting moiety of the one or more targeting moieties is sufficiently identical to or complementary to a consensus sequence in the target sequence of the target gene.

51. 51. The method of any one of claims 1 to 50, wherein the targeting moiety is complementary to and / or hybridizes with the target sequence.

52. 52. The method of any one of claims 1 to 51, wherein the targeting moiety is complementary to a consensus sequence of the target sequence and / or hybridizes with the target sequence.

53. 53. The method of any one of claims 1 to 52, wherein the target sequence comprises a splice site.

54. 54. The method of claim 53, wherein the splice site is a conserved splice site.

55. 54. The method of claim 53, wherein the splice site comprises 5'-GU-3'.

56. 56. The method of any one of claims 49 to 55, wherein the pre-mRNA is encoded by the target gene.

57. 57. The method of claim 56, wherein the method alters the expression or activity of the target gene.

58. 58. The method of any one of claims 1-57, wherein the one or more targeting moieties comprise: (1) a first targeting moiety configured to specifically bind to a first targeting sequence in the target sequence of the pre-mRNA; and (2) a second targeting moiety configured to specifically bind to a second targeting sequence in the target sequence of the pre-mRNA.

59. 54. The method of claim 53, wherein the first targeting sequence comprises a consensus sequence in the target sequence.

60. 60. The method of claim 53 or 59, wherein the second targeting sequence comprises a consensus sequence in the target sequence.

61. 61. The method of any one of claims 53-60, wherein the first targeting sequence and the second targeting sequence are separated in the target sequence by a spacing sequence of 5 nucleotides or less (e.g., 1 or 2 nucleotides).

62. 62. The method of any one of claims 1 to 61, wherein the target sequence comprises an exon-intron boundary in the pre-mRNA.

63. 63. The method of claim 62, wherein the first targeting sequence and the second targeting sequence are both 5' or 3' to an exon-intron boundary.

64. 63. The method of claim 62, wherein one of the first targeting sequence and the second targeting sequence is 5' to the exon-intron boundary and the other of the first targeting sequence and the second targeting sequence is 3' to the exon-intron boundary.

65. 65. The method of any one of claims 1 to 64, wherein the target sequence comprises a splice site in the pre-mRNA.

66. 66. The method of claim 65, wherein the first targeting sequence or the second targeting sequence comprises a splice site (e.g., 5' ss) in the pre-mRNA.

67. 67. The method of any one of claims 1-66, wherein one of the first targeting moiety and the second targeting moiety is 5' to the recruitment moiety and the other of the first targeting moiety and the second targeting moiety is 3' to the recruitment moiety.

68. 68. The method of claim 67, wherein the first targeting moiety or the second targeting moiety comprises a sequence identical to or complementary to a sequence shown in Table 1.

69. 68. The method of Claim 67, wherein said first targeting moiety comprises a sequence identical to or complementary to a sequence selected from the exon sequence column of Table 1, and said second targeting moiety comprises a sequence identical to or complementary to a sequence shown in the intron sequence column of Table 1.

70. 68. The method of Claim 67, wherein said first targeting moiety comprises a sequence identical to or complementary to a sequence set forth in the intron sequence column of Table 1, and said second targeting moiety comprises a sequence identical to or complementary to a sequence set forth in the exon sequence column of Table 1.

71. 68. The method of claim 67, wherein the splice site comprises 5'-GU-3'.

72. 68. The method of Claim 67, wherein the first targeting moiety or the second targeting moiety comprises a sequence identical to or complementary to a consensus sequence of an intron donor site (e.g., selected from GU, GT, GC, and CA).

73. 68. The method of Claim 67, wherein the first targeting moiety or the second targeting moiety comprises a sequence identical to or complementary to a consensus sequence of an exon donor site (e.g., CAG or AGG).

74. 68. The method of claim 67, wherein the first targeting moiety or the second targeting moiety comprises a sequence identical to or complementary to a consensus sequence selected from GU, GT, GC, G, and CA.

75. 75. The method of any one of claims 1 to 74, wherein the first targeting moiety comprises a sequence that is at least 80%, 90%, or identical to a ribosome binding site of a spliceosomal snRNA, such as U1 snRNA.

76. 76. The method of any one of claims 1 to 75, wherein the second targeting moiety comprises a sequence that is at least 80%, 90%, or identical to a ribosome binding site of a spliceosomal snRNA, e.g., U1 snRNA.

77. 77. The method of any one of claims 75 or 76, wherein the sequence that is at least 80%, 90%, or identical to the ribosome binding site can be from about 2 nucleotides to about 10 nucleotides.

78. 75. The method of any one of claims 1 to 74, wherein the spliceosome portion is selected from a spliceosomal ribonucleoprotein complex, a spliceosomal small nuclear ribonucleic acid (snRNA), a spliceosomal protein, a functional variant thereof, or a functional fragment thereof.

79. 79. The method of claim 78, wherein the spliceosome portion comprises U1 snRNA and spliceosome proteins.

80. 80. The method of any one of claims 78 to 79, wherein the spliceosomal snRNA is selected from U1, U2, U4, U5, U6, U11, U12, U14atac, U6atac, and combinations thereof.

81. 81. The method of any one of claims 78 to 80, wherein the spliceosome protein is selected from Sm, U1-70k, U1A, U1C, and combinations thereof.

82. 82. The method of any one of claims 1-81, wherein the recruitment moiety comprises a nucleotide sequence that is at least 70%, 80%, 85%, or 90% identical to or complementary to any one of SEQ ID NOs: 1-2.

83. 83. The method of claim 82, wherein the recruitment moiety comprises a nucleotide sequence identical to or complementary to any one of SEQ ID NOs: 1-2.

84. 84. The method of any one of claims 1 to 83, wherein the engineered polynucleotide comprises (e.g., secondary) structural features.

85. 84. The method of Claim 83, wherein said engineered polynucleotide comprises an apical loop, an upper stem, an internal loop, a lower stem, or a combination thereof.

86. 84. The method of Claim 83, wherein the engineered polynucleotide comprises a loop (e.g., an internal loop) adjacent to a stem (e.g., a lower stem or an upper stem) comprising two complementary stem sequences.

87. 87. The method of claim 86, wherein the stem sequence of the stem (e.g., the lower stem or the upper stem) comprises about 5 or less, 4 or less, or 3 or less nucleotides.

88. 87. The method of claim 85 or 86, wherein the loop is an internal loop flanked by the stem (e.g., the lower stem) and a further stem (e.g., an upper stem) comprising two complementary stem sequences.

89. 89. The method of Claim 88, wherein the internal loop comprises a nucleic acid sequence of 10 or fewer, 9 or fewer, or 8 or fewer nucleotides.

90. 90. The method of claim 88 or 89, wherein the stem sequence of the additional stem (e.g., the upper stem) comprises about 5 or less, 4 or less, or 3 or less nucleotides.

91. 91. The method of any one of claims 88-90, wherein the engineered polynucleotide further comprises an apical loop.

92. 92. The method of Claim 91, wherein the apical loop comprises a nucleic acid sequence of 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, or 5 or less nucleotides.

93. 93. The method of any one of claims 1 to 92, wherein the engineered polynucleotide does not contain any intramolecular disulfide bonds.

94. 94. The method of any one of claims 1-93, wherein the pre-mRNA, upon association with the engineered polynucleotide and the spliceosome moiety, exhibits substantially no base pairing with the RNA binding domain (RBD) of U1 snRNA.

95. 95. The method of any one of claims 1 to 94, wherein the pre-mRNA, when associated with the engineered polynucleotide and the spliceosome moiety, exhibits substantially no base-specific interactions with U1-C protein.

96. 96. The method of any one of claims 1 to 95, wherein the engineered polynucleotide is configured to specifically interact with a zinc finger of a U1-C protein.

97. 97. The method of claim 96, wherein the 5' targeting portion of the engineered polynucleotide is configured to specifically interact with a zinc finger of a U1-C protein.

98. 98. The method of claim 96 or 97, wherein the engineered polynucleotide (e.g., its 5' targeting portion) is configured to covalently interact with a zinc finger of a U1-C protein (e.g., via a disulfide bond).

99. 99. The method of any one of claims 96-98, wherein the engineered polynucleotide (e.g., its 5' targeting portion) is configured to non-covalently interact (e.g., via hydrogen bonding) with a zinc finger of a U1-C protein.

100. 100. The method of any one of claims 96 to 99, wherein the recruitment moiety comprises a nucleotide sequence comprising a phosphorothioate internucleotide linkage that binds to a U1-C zinc finger.

101. 101. The method of any one of claims 1 to 100, wherein the engineered polynucleotide comprises a nucleotide sequence complementary to a subsequence of stem loop II (SL2) of U1 snRNA.

102. 102. The method of Claim 101, wherein the sides of the stem-loop structure of the engineered polynucleotide comprise a nucleotide sequence complementary to a partial sequence of stem-loop II (SL2) of U1 snRNA.

103. The method of claim 101 or 102, wherein the partial sequence comprises a sequence corresponding to 5'-GGCCU-3' of SL2 of U1 snRNA.

104. The method according to any one of claims 101 to 103, wherein the partial sequence does not include a sequence corresponding to 5'-CACGUUA-3' of SL2 of U1 snRNA.

105. 105. The method of any one of claims 1 to 104, wherein the recruitment moiety is complementary to the stem-loop-II region of snRNA, for example U1 snRNA.

106. 106. The method of any one of claims 1 to 105, wherein the recruitment moiety hybridizes to the stem-loop-II region of snRNA, for example U1 snRNA.

107. 107. The method of any one of claims 1 to 106, wherein the recruitment moiety comprises AGGCC.

108. 108. The method of any one of claims 1-107, wherein the recruitment moiety comprises a nucleotide sequence that is at least 80%, 90%, or identical to at least 5 consecutive nucleotides of a sequence shown in Tables 2-3.

109. 109. The method of any one of claims 1-108, wherein the recruitment moiety comprises a nucleotide sequence that is at least 80%, 90%, or identical to about 5 to about 10 contiguous nucleotides of a sequence set forth in Tables 2-3.

110. The method of any one of claims 1 to 104, wherein the recruitment nucleotide sequence comprises (i) a nucleotide sequence complementary to at least 4 nucleotides of stem loop II (SL2) of U1 snRNA.

111. 105. The method of any one of claims 1 to 104, wherein the engineered polynucleotide exhibits substantially no base pairing with the anchor sequence of SL2 of U1 snRNA.

112. 112. The method of claim 111, wherein the internal loop of the engineered polynucleotide exhibits substantially no base pairing with the anchor sequence of the SL2 of the U1 snRNA.

113. 113. The method of claim 111 or 112, wherein the lower stem of the engineered polynucleotide exhibits substantially no base pairing with the anchor sequence of the SL2 of the U1 snRNA.

114. 114. The method of any one of claims 111 to 113, wherein the anchor sequence comprises a sequence corresponding to 5'-CACGUUA-3'.

115. 115. The method of any one of claims 1 to 114, wherein the engineered polynucleotide exhibits substantially no base pairing with the H helix of U1 snRNA.

116. 116. The method of any one of claims 1 to 115, wherein the engineered polynucleotide comprises at least one chemical modification.

117. 117. The method of Claim 116, wherein at least about 50%, 60%, 70%, 80%, or 90% of the nucleotides of said engineered polynucleotide are chemically modified nucleotides.

118. 118. The method of claim 116 or 117, wherein the engineered polynucleotide comprises at least one 2' modified nucleotide.

119. 119. The method of any one of claims 116-118, wherein at least about 50%, 60%, 70%, 80%, or 90% of the nucleotides of said engineered polynucleotide are 2'-modified nucleotides.

120. 120. The method of any one of claims 118 or 119, wherein the 2'-modified nucleotides comprise 2'-methoxy, 2'-methoxymethyl, 2'-methoxyethyl, 2'-fluoro, or 2'-aminoethyl nucleotides.

121. 121. The method of any one of claims 116-120, wherein the engineered polynucleotide comprises nucleotides connected by internucleotide linkages, wherein at least one of the internucleotide linkages does not contain a phosphate.

122. 122. The method of any one of claims 116-121, wherein the engineered polynucleotide comprises nucleotides connected by internucleotide linkages, at least one of the internucleotide linkages comprising sulfur (S), selenium (Se), BR3 (where each R is independently selected from the group consisting of hydrogen, alkyl, and aryl), carbon (C), or NR2 (where each R is independently selected from the group consisting of hydrogen, alkyl, and aryl).

123. 123. The method of any one of claims 116 to 122, wherein the engineered polynucleotide comprises at least one phosphorothioate internucleotide linkage.

124. 124. The method of any one of claims 116-123, wherein at least about 50%, 60%, 70%, 80%, or 90% of the internucleotide linkages of the engineered polynucleotide are chemically modified.

125. 125. The method of any one of claims 116-124, wherein at least about 50%, 60%, 70%, 80%, or 90% of the internucleotide linkages are phosphorathioate.

126. 126. The method of any one of claims 116-125, wherein the internucleotide linkage comprises a methylphosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo, or methyleneoxymethylimino.

127. 127. The method of any one of claims 1-126, wherein the engineered polynucleotide comprises from about 10 to about 40 nucleotides, from about 10 to about 35 nucleotides, from about 10 to about 30 nucleotides, or from about 10 to about 25 nucleotides.

128. 128. The method of any one of claims 1 to 127, wherein the recruitment moiety comprises from about 10 to about 30 nucleotides, or from about 10 to about 20 nucleotides.

129. 129. The method of any one of Claims 1-128, wherein said one or more targeting moieties each independently comprise from about 2 to about 15 nucleotides, from about 2 nucleotides to about 10 nucleotides, or from about 2 nucleotides to about 8 nucleotides.

130. 130. The method of any one of claims 1-129, wherein one of the first targeting moiety and the second targeting moiety comprises about 2 nucleotides, and the other of the first targeting moiety and the second targeting moiety comprises about 5 or 6 nucleotides.

131. 131. The method of any one of claims 1-130, wherein upon association with the engineered polynucleotide and the pre-RNA, the spliceosome portion cleaves or splices the pre-mRNA in the target sequence.

132. 132. The method of any one of claims 1 to 131, wherein the spliceosome portion further promotes modification of the cleaved pre-mRNA.

133. 133. The method of any one of claims 1 to 132, wherein the engineered polynucleotide comprises a nucleotide sequence that is at least 70%, 80%, 85%, or 90% identical to or complementary to any one of SEQ ID NOs: 1-4.

134. 134. The method of any one of claims 1 to 133, wherein the engineered polynucleotide comprises a nucleotide sequence identical to or complementary to any one of SEQ ID NOs: 3 or 4.

135. The engineered polynucleotide comprises: (i) a first targeting moiety configured to specifically bind to a pre-messenger ribonucleic acid (pre-mRNA) at an internal first targeting sequence, the first targeting moiety comprising a sequence identical to or complementary to 5'-GTCCA-3'; (ii) a recruitment moiety comprising a sequence at least 90% similar to or complementary to SEQ ID NO: 1 and configured to recruit spliceosome components including U1 snRNA and U1-C protein, the recruitment moiety comprising an apical loop, an upper stem adjacent to the apical loop, a lower stem, and an internal loop located between the upper stem and the lower stem; (iii) a second targeting moiety configured to specifically bind to the pre-mRNA at an internal second targeting sequence, wherein the second targeting moiety comprises a sequence identical to or complementary to 5'-CG-3'.

136. 1. A method of treating a subject having Alzheimer's disease, the method comprising administering to the subject a pharmaceutical composition comprising an engineered polynucleotide having the nucleotide sequence of SEQ ID NO: 3, thereby regulating pre-mRNA affected by U1 snRNP dysfunction in the brain of the subject.

137. 137. The method of Claim 136, wherein all internucleotide linkages of said engineered polynucleotide comprise phosphorothioate linkages.

138. 138. The method of any one of claims 136-137, wherein the nucleotides at positions 1-5 and 20-24 of the engineered polynucleotide comprise 2'-O-methyl moieties.

139. 139. The method of any one of claims 136 to 138, wherein the pre-mRNA comprises tau, and the method reduces tau protein levels in the brain.

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