Polynucleotide compositions and methods for treating cancer

Abstract

The present disclosure provides 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 cancer.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 63 / 480,466, filed January 18, 2023, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Abnormal splicing is involved in many disease states. Proper splicing of pre-messenger RNA (pre-mRNA) is important for proper translation of proteins. Splicing efficiency must be regulated to ensure proper splicing of pre-mRNA as well as proper suppression of premature polyadenylation to avoid the production of misfolded and potentially 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, for example, at therapeutically effective and safe levels. Summary of the Invention

[0003] In some aspects, described herein are methods of treating cancer in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising an engineered polynucleotide, wherein the engineered polynucleotide comprises (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.

[0004] Some aspects of the present disclosure describe a method for treating cancer in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising (a) a taxane and (b) an engineered polynucleotide, wherein the engineered polynucleotide comprises (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. In some embodiments, the method improves the prognosis of the subject compared to a subject administered the taxane but not the engineered polynucleotide. In some embodiments, the method reduces the total level of tau in the subject, thereby reducing the amount of tau bound to the taxane.

[0005] In some aspects herein, a method for improving the prognosis of a subject suffering from cancer and receiving a taxane is described, comprising administering to the subject a pharmaceutical composition comprising an engineered polynucleotide, wherein the engineered polynucleotide comprises (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. In some embodiments, the method reduces the total level of tau in the subject, thereby reducing the amount of tau bound to the taxane in the subject.

[0006] In some embodiments, the cancer is selected from the group consisting of brain cancer, prostate cancer, breast cancer, renal cancer, kidney cancer, lung cancer, and liver cancer. In some embodiments, the cancer is selected from the group consisting of glioblastoma (GBM), neuroblastoma, hepatocellular carcinoma, breast adenocarcinoma, human prostate cancer, renal cell carcinoma, and renal adenocarcinoma. In some embodiments, the cancer is GBM.

[0007] In some embodiments, the methods disclosed herein reduce premature polyadenylation of one or more transcripts of interest. In some embodiments, the methods disclosed herein reduce cryptic splicing of one or more transcripts of interest. In some embodiments, the methods disclosed herein improve scores associated with histopathological findings. In some embodiments, the histopathological findings include tumor grade, lipid content, necrosis, or nucleus-to-cytoplasm (N:C) ratio.

[0008] In some embodiments, the method reduces tumor volume ratio. In some embodiments, the method reduces tumor progression. In some embodiments, the method alters tau expression. In some embodiments, the method reduces tau expression. In some embodiments, the method reduces the total amount of tau in a subject. In some embodiments, the method reduces Akt expression. In some embodiments, the method reduces glial fibrillary acidic protein (GFAP) expression. 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.

[0009]

[0010] In some aspects described herein, a method of reducing cell viability comprises administering to a cell an engineered polynucleotide, wherein the engineered polynucleotide comprises (i) one or more targeting moieties configured to specifically bind to a pre-messenger ribonucleic acid, 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; wherein the engineered polynucleotide comprises (i) one or more targeting moieties configured to specifically bind to a pre-messenger ribonucleic acid (pre-mRNA) at a target sequence in the pre-mRNA, 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.

[0010]

[0010] In some aspects herein, described are methods of reducing the proliferation rate of a cell, comprising administering to a cell an engineered polynucleotide, wherein the engineered polynucleotide comprises (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; wherein the engineered polynucleotide comprises (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.

[0011] In some embodiments, the method increases cell necrosis or apoptosis. In some embodiments, the method increases the propensity of the cell to be in G2 / M phase. In some embodiments, the cell is a tumor cell. In some embodiments, the tumor cell is a glioma, neuroblastoma, or carcinoma.

[0012]

[0010] In some aspects herein, described are methods of altering cell cycle phase distribution in a plurality of cells, comprising administering to the plurality of cells an engineered polynucleotide, wherein the engineered polynucleotide comprises (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; wherein the engineered polynucleotide comprises (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.

[0013] In some embodiments, the method increases the number of cells in G2 / M phase. In some embodiments, the method increases the number of cells in necrotic or apoptotic phase. In some embodiments, the plurality of cells comprises a tumor. In some embodiments, the tumor cells comprise a glioma, neuroblastoma, or carcinoma.

[0014] In some aspects herein, a method for reducing tau expression by a neuron is described, comprising administering an engineered polynucleotide to the neuron, wherein the engineered polynucleotide comprises (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. In some embodiments, the neuron is derived from an individual suffering from cancer. In some embodiments, the method reduces cryptic splicing of one or more transcripts in the neuron.

[0015]

[0010] In some aspects, described herein are methods of treating a subject having cancer, the method comprising administering to the subject a pharmaceutical composition comprising an engineered polynucleotide having the nucleotide sequence of SEQ ID NO:3, wherein all internucleotide linkages of the engineered polynucleotide comprise phosphorothioate linkages, and wherein the nucleotides at positions 1-5 and 20-24 of the engineered polynucleotide comprise 2'-O-methyl moieties, and wherein the administration modulates U1 snRNP complex formation.

[0016] Some aspects described herein include a method of treating a subject with cancer, the method comprising administering to the subject a pharmaceutical composition comprising an engineered polynucleotide having the nucleotide sequence of SEQ ID NO: 3. In some embodiments, the internucleotide linkages of the engineered polynucleotide comprise phosphorothioate linkages, and in some embodiments, the nucleotides at positions 1-5 and 20-24 of the engineered polynucleotide comprise 2'-O-methyl moieties. In some embodiments, the method further comprises administering a taxane to the subject.

[0017] In some aspects herein, a method is described for treating a subject having cancer, wherein the subject has been administered a taxane, the method comprising the step of: (i) administering to the subject a pharmaceutical composition comprising an engineered polynucleotide having the nucleotide sequence of SEQ ID NO: 3, wherein the internucleotide linkages of the engineered polynucleotide comprise phosphorothioate linkages, and wherein nucleotides at positions 1-5 and 20-24 of the engineered polynucleotide comprise 2'-O-methyl moieties, and wherein administration reduces tau protein levels and tau binding to the taxane in the subject.

[0018] In some embodiments, the methods disclosed herein modulate the formation of the U1 snRNP complex.

[0019] In some aspects herein, engineered polynucleotides are described 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.

[0020] 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 a target sequence therein 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. In some embodiments, the 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 a 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 a target gene.

[0021] 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, such as U1 snRNA. In some embodiments, the first 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 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. The sequence that is at least 80%, 90%, or identical to a 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 U1 snRNA.

[0022] In some embodiments, the first targeting moiety or the second targeting moiety comprises a sequence identical or complementary to a sequence shown in Table 1. In some embodiments, the first targeting moiety comprises a sequence identical 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 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 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 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 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 for 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 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 moiety comprises a U1 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 protein is selected from Sm, U1-70k, U1A, U1C, and combinations thereof. In some embodiments, the recruitment moiety comprises a nucleotide sequence 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 moiety is complementary to the stem-loop II region of a snRNA, e.g., U1 snRNA. In some embodiments, the recruitment moiety hybridizes with the stem-loop II region of a snRNA, e.g., U1 snRNA.In some embodiments, the recruitment moiety comprises AGGCC. In some embodiments, the recruitment moiety 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 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. In some embodiments, the recruitment moiety 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) that comprises 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, 4, or 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., a stem) comprising two complementary stem sequences. In some embodiments, the internal loop comprises a nucleic acid sequence of 10, 9, or 8 nucleotides or less. In some embodiments, the stem sequence of the additional stem (e.g., an upper stem) comprises about 5, 4, or 3 nucleotides or less. In some embodiments, the engineered polynucleotide further comprises an apical loop. In some embodiments, the apical loop comprises a nucleic acid sequence of 10, 9, 8, 7, 6, or 5 nucleotides or less. In some embodiments, the engineered polynucleotide does not comprise 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 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. 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 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, 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, an 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). 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. In some embodiments, 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.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. 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-RNA, 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.

[0023] Some aspects described herein include engineered polynucleotides comprising a nucleotide sequence at least 70%, 80%, 85%, or 90% identical to or complementary to a sequence set forth in Tables 2-3, wherein the engineered polynucleotide is characterized by a (e.g., secondary) structural feature. In some embodiments, the nucleotide sequence is identical to or complementary to a sequence set forth in Tables 2-3. In some embodiments, the structural feature comprises one or more stem-loop structures. In some embodiments, the structural feature 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, 4, or 3 nucleotides or less. In some embodiments, the loop is an internal loop adjacent to a stem (e.g., a lower stem) and an additional stem (e.g., a stem) comprising two complementary stem sequences. In some embodiments, the internal loop comprises a nucleic acid sequence of 10, 9, or 8 nucleotides or less. In some embodiments, the stem sequence of the additional stem (e.g., the upper stem) comprises about 5, 4, or 3 nucleotides or less. In some embodiments, the engineered polynucleotide further comprises an apical loop. In some embodiments, the apical loop comprises a nucleic acid sequence of 10, 9, 8, 7, 6, or 5 nucleotides or less. 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, the targeting moieties of the one or more targeting moieties are 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 an 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, 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, 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.

[0024] Some aspects of the present disclosure describe a method for altering pre-messenger ribonucleic acid (pre-mRNA) in a cell, 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 a target sequence therein, 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, the targeting moiety of the one or more targeting moieties is sufficiently identical to or complementary to a consensus sequence in the target sequence of a gene (e.g., a 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.

[0025] In some embodiments, the engineered polynucleotide comprises: (i) a first targeting moiety configured to specifically bind to pre-messenger ribonucleic acid (pre-mRNA) at a first targeting sequence in the first targeting moiety, the first targeting moiety comprising a sequence identical 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 pre-mRNA at a second targeting sequence in the second targeting moiety, the second targeting moiety comprising a sequence identical or complementary to 5'-CG-3'.

[0026] In some aspects, described herein is a set of engineered polynucleotides, each independently comprising one or more targeting moieties configured to specifically 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.

[0027] Another embodiment described herein is an engineered polynucleotide comprising a first targeting portion configured to specifically bind to pre-messenger ribonucleic acid (pre-mRNA) at a first targeting sequence therein, a recruitment portion configured to recruit a spliceosome portion, and a second targeting portion configured to specifically bind to the pre-mRNA at a second targeting sequence therein, 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 the targeting sequence comprising the first and second targeting sequences. In some embodiments, the first targeting portion is complementary to and / or hybridizes with the first targeting sequence. In some embodiments, the second targeting portion is complementary to and / or hybridizes with the second targeting sequence. In some aspects, the first targeting sequence and the second targeting sequence are separated by a spacing sequence of 5 nucleotides or less in the target sequence. In some aspects, 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 aspects, the first targeting moiety comprises a sequence identical or complementary to a sequence listed in the exon sequence column of Table 1, and the second targeting moiety comprises a sequence identical or complementary to a sequence listed in the intron sequence column of Table 1. In some embodiments, the first 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 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 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 moiety is complementary to the stem-loop II region of a snRNA, such as the U1 snRNA.In some embodiments, the recruitment portion hybridizes with the stem-loop II region of a snRNA, such as 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 five consecutive 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 consecutive 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 aspects, the upper stem or lower stem comprises two complementary sequences, each of which comprises 5 or fewer nucleotides; the internal loop comprises two nucleic acid sequences, each of which comprises 5 or fewer nucleotides; and the apical loop comprises a nucleic acid sequence of 8 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 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 a 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 U1 snRNA. In another embodiment, the sequence of SL2 of 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 joined by internucleotide linkages, wherein at least one of the internucleotide linkages does not contain a phosphate.At least one, or 50%, 60%, 70%, 80%, or 90% of the internucleotide linkages may be phosphorothioate. [Brief explanation of the drawings]

[0028] The patent or patent application file contains at least one drawing which shall be in color. Copies of this patent or patent application including color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0029] [Figure 1]Schematic diagram for identifying splice donor and splice acceptor sites. 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, as "GU" and "AG," respectively, and that the underlined sequence can be any sequence. However, as used 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 branchpoint sequence containing an adenine that ligates to the ribonucleotide at the 5' splice site to form the intron lariat, and a polypyrimidine tract (C or U) between the branchpoint and the splice acceptor sequence. The short GU_AG consensus sequence of an intron is clearly not sufficient to distinguish between many alternative splicing events, but surprisingly, it is not widely known that other sequence information is required to regulate alternative RNA splicing. One or two nucleotides flanking either side 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 intron downstream) and the acceptor site (3' exon and intron downstream). 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] FIG. 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] FIG. 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] FIG. 1 shows the interaction of an exemplary engineered polynucleotide with a target pre-mRNA sequence. [Figure 2D] FIG. 1 shows the interactions of an exemplary engineered polynucleotide with various components of the U1 RNP complex. [Figure 3A] Figure 3A shows anchors generated through engineered polynucleotide "Stem 5' / 3" (also known as 5'-targeting moieties and / or 3'-targeting moieties) designed to interact with conserved moieties present in constitutive donor sites. In Figure 3A, Stem 5' / 3" (GTCCA and CG), such as internucleotide phosphorothioate linkages and 2'O-methyl (2'O-ME) substitutions for the molecular sugar, increase resistance to endonucleases and increase the molecular strength of the interaction between the bases of the Stem 5' / 3' and the conserved region from the constitutive donor. [Figure 3B]Figure 3B shows the anchoring generated 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 a constitutive donor site. Figure 3B illustrates the interaction of the engineered polynucleotides described herein with constitutive donor splicing and the interaction of the silencing RNA-binding portion (RBD) of U1 snRNA with a constitutive donor splicing exon. [Figure 4] This is a schematic diagram of the human U1 snRNP. 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's 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 U1-70 K and Sm proteins. Note the interaction between the U1C ring and the Sm ring. [Figure 5A]This figure shows 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 engineered crystal structures of the U1 substructure are shown, 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. The binding of the RNA duplex is stabilized by hydrogen bonds and electrostatic interactions between U1-C and the RNA backbone surrounding the splice junction; however, U1-C does not make base-specific contacts with the pre-mRNA. This structure, together 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 figure shows 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 engineered crystal structures of the U1 substructure are shown, 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. The binding of the RNA duplex is stabilized by hydrogen bonds and electrostatic interactions between U1-C and the RNA backbone surrounding the splice junction; however, U1-C does not make base-specific contacts with the pre-mRNA. This structure, together with RNA binding assays, demonstrates that 5'-splice site nucleotide selection by U1 snRNP is achieved primarily 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 figure shows 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 engineered crystal structures of the U1 substructure are shown, 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. The binding of the RNA duplex is stabilized by hydrogen bonds and electrostatic interactions between U1-C and the RNA backbone surrounding the splice junction; however, U1-C does not make base-specific contacts with the pre-mRNA. This structure, together with RNA binding assays, demonstrates that 5'-splice site nucleotide selection by U1 snRNP is achieved primarily by base pairing with U1 snRNA, but U1-C fine-tunes the relative affinity of mismatched 5'-splice sites. In Figure 5C, U1-C binds to U1 small nuclear ribonucleoprotein A and 70 kDa. [Figure 6] FIG. 1 shows 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]Schematic diagram of the modulation of the spliceosome machinery by the engineered polynucleotide described herein (ASMO1, also known as APT20TTMG). The 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 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 finger of U1-C, induced by a disulfide bridge with the thiol of 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 surrounding the junction, 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 stem 5' / 3' 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 U1-70kDa 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 change in the molecular dynamics of the U1-snRNA, promoting a conformational change in U1-snRNA and the access of stem-loop II to 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]This figure shows U1-70K complexed with the U1 snRNA stem-loop and U1-A RRM complexed with stem-loop 2, 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 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, inducing regulation of gene expression and acetylation levels. In addition, the presence of a 2'-OME group induces a conformational change in U1-snRNA and a change in the molecular dynamics of the medium, facilitating the access of stem-loop II to the engineered polynucleotide (ASMO1) described herein. [Figure 9A] FIG. 1 shows that U1-C is located on SmD3 and that its binding can be stabilized by the N-terminus of U1-70K. [Figure 9B] Figure 1 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] Schematic diagram of 5'-splice site recognition. 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] FIG. 1 shows the fingerprint Z1 U1-C snRNP represented by 36 amino acid residues in blue. [Figure 10B]FIG. 1 shows the Z1 finger portion of U1-C snRNP, showing the key residues in the 5′ constitutive donor region that interact with the pre-mRNA / ASMO1 duplex. [Figure 10C] A representative sequence of the U1-C snRNP containing 145 aa, with the 36 aa highlighted in green indicating the zinc finger portion. [Figure 11] Figure 11 shows cell viability of the U-87 cell line at four time points after incubation in ASMO1 (also known as "APT20TTMG"). Figure 11A shows results from a 24-hour incubation. Figure 11B shows results from a 48-hour incubation. Figure 11C shows results from a 72-hour incubation. Figure 11D shows results from a 96-hour incubation. [Figure 12] Figure 12 shows cell viability of MCF-7 cell line at four time points after incubation in ASMO1. Figure 12A shows results from 24 hours of incubation. Figure 12B shows results from 48 hours of incubation. Figure 12C shows results from 72 hours of incubation. Figure 12D shows results from 96 hours of incubation. [Figure 13] Figure 13 shows cell viability of the SHSY5Y cell line at four time points after incubation in ASMO1. Figure 13A shows results from 24 hours of incubation. Figure 13B shows results from 48 hours of incubation. Figure 13C shows results from 72 hours of incubation. Figure 13D shows results from 96 hours of incubation. [Figure 14] Figure 14 shows cell viability of PC-3 cell line at three time points after incubation in ASMO1. Figure 14A shows results from 24 hours of incubation, Figure 14B shows results from 48 hours of incubation, and Figure 14C shows results from 72 hours of incubation. [Figure 15]Figure 15 shows cell viability of the 786-O cell line at three time points after incubation in ASMO1. Figure 15A shows results from 24 hours of incubation, Figure 15B shows results from 48 hours of incubation, and Figure 15C shows results from 72 hours of incubation. [Figure 16] FIG. 1 shows the kinetics of internalization of compound ASMO1 by breast cancer cell line MCF-7. [Figure 17] FIG. 1 shows the assessment of mitochondrial activity in MCF-7 cell lines after incubation with ASMO1. [Figure 18] FIG. 1 shows the cell cycle phase distribution of MCF-7 cell line after incubation with ASMO1. [Figure 19] FIG. 1 shows the distribution of MCF-7 stages after incubation with ASMO1. [Figure 20] FIG. 1 shows mitochondrial membrane potential in MCF-7 cell line after incubation with ASMO1. [Figure 21] FIG. 1 shows the kinetics of internalization of compound ASMO1 by the neuroblastoma cell line SH-SY5Y. [Figure 22] Figure 22 shows the evaluation of mitochondrial activity in SH-SY5Y cell line after incubation in ASMO1. MTT assays were performed after 24 hours (Figure 22A) and 48 hours (Figure 22B) of incubation. [Figure 23] FIG. 1 shows the cell cycle phase distribution of the SH-SY5Y cell line after incubation with ASMO1. [Figure 24] FIG. 1 shows the distribution of SH-SY5Y stages after incubation with ASMO1. [Figure 25] FIG. 1 shows mitochondrial membrane potential in SH-SY5Y cell line after incubation with ASMO1. [Figure 26] FIG. 1 shows cell viability results obtained after 24, 48, 96, and 144 hours of incubation with ASMO1 (0.5 μM) in a neuroblastoma cell line (SK-N-SH). [Figure 27] 27A and 27B show MAPT expression and tau quantification obtained after 24, 48, 96, and 144 hours of incubation with ASMO1 (0.5 μM) in neuroblastoma cell lines. Figure 27A shows MAPT expression after ASMO1 (0.5 μM) at various time points. Figure 27B shows tau quantification after ASMO1 (0.5 μM) at various time points. [Figure 28] FIG. 1 shows the kinetics of internalization of compound ASMO1 by the glioblastoma cell line U87-MG. [Figure 29] FIG. 1 shows the cell cycle phase distribution of the U87-MG cell line after incubation with ASMO1. [Figure 30] FIG. 1 shows the distribution of U87-MG phases after incubation with ASMO1. [Figure 31] FIG. 1 shows quantification of mitochondrial membrane potential in U87-MG cell line after incubation with ASMO1. [Figure 32] FIG. 1 shows the evaluation of mitochondrial activity in U87-MG cell line after incubation with APT20TTMG. [Figure 33] FIG. 1 shows the evaluation of proliferation in U87-MG cell line with APT20TTMG. [Figure 34] FIG. 1 shows the effect of intravenous administration of APT20TTMG in a glioblastoma xenograft model in athymic nude mice. [Figure 35] FIG. 1 shows the effect of intravenous administration of APT20TTMG on proliferation markers (Ki-67) in a glioblastoma xenograft model in athymic nude mice. [Figure 36] FIG. 1 shows the effect of intravenous administration of APT20TTMG on histopathological findings and tumor volume total score. [Figure 37] FIG. 1 shows the effect of administration of APT20TTMG on signaling pathway proteins in a glioblastoma xenograft model in athymic nude mice.

[0030] 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

[0031] 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.

[0032] This specification describes a method for treating cancer using the engineered polynucleotides described in this disclosure.This method is applicable to various cancer types, for example, cancers of different organs or cell types, or cancers associated with or caused by different genetic abnormalities.The engineered polynucleotides described herein can be effective in regulating pre-mRNA splicing by recruiting spliceosome components.Without being bound to a specific mechanism, improving the recruitment of spliceosome components to pre-mRNA can be effective in regulating the cellular mechanisms responsible for cancer.The engineered polynucleotides can be administered to a subject suffering from cancer, or can treat cancer, for example, by reducing the number of cancerous cells or preventing the proliferation or metastasis of cancer cells.

[0033] Under physiological conditions, splicing involves the processing of pre-messenger RNA (pre-mRNA), the removal of intronic regions, and the ligation of exon regions to produce mature mRNA. Splicing is carried out by the formation of a series of complexes between snRNPs and small nuclear RNAs (snRNAs). The U1 snRNP complex is one of five complexes (designated U1, U2, U4, U5, and U6) that make up the human spliceosome. The U1 snRNP complex is composed of U1 snRNA, seven Sm proteins that form a heptameric ring, and three other proteins: U1-70K, U1A, and U1C. In the spliceosome, the U1 snRNP complex plays a specific role in the recognition of pre-mRNA splicing sites (splice donor sites) during the initial stage of spliceosome assembly. Other snRNPs, such as U2 snRNP, are then attracted to the splice site, and interactions between their protein components complete spliceosome assembly and splicing. The mature mRNA, formed after chemical capping and polyadenylation, then leaves the cell nucleus and can be translated into protein. In addition to its role in splicing, U1 snRNP actively suppresses the polyadenylation machinery from using earlier (mostly intronic) polyadenylation signals, which would result in aberrantly truncated mRNAs. Accurate splicing is an essential step in gene expression, allowing for the balancing of precision and flexibility in splice site recognition, as well as alternative splicing, which generates multiple isoforms from a single transcript with diverse, sometimes antagonistic, biological functions.

[0034] In addition to its splicing function, the U1 snRNP complex also has a telescripting function, actively inhibiting the utilization of proximal polyadenylation signals (PASs), primarily found within introns, by the polyadenylation machinery, suppressing a process known as premature polyadenylation. It is important to note that polyadenylation is one of the major processes in mRNA maturation and involves the termination of pre-mRNAs, usually by removal of the 3' end portion in the 3' untranslated region (UTR), and the addition of a poly(A) tail. This process, along with splicing and 5' capping, generates mature mRNAs and affects transcript stability, transport, and translation. Suppression of premature polyadenylation by the U1 snRNP complex prevents the production of aberrant and truncated mRNAs.

[0035] Furthermore, U1 snRNP has been described to be associated with stress granules, i.e., cytoplasmic RNA granules containing mRNAs, associated translation initiation factors, and various RNA-binding proteins (e.g., snRNPs) that arise in response to various stresses, in lysosome and autophagosome biogenesis, and in autophagy.

[0036] Cell cycle re-entry induced by U1 dysfunction in cells has been hypothesized as one of the factors that may lead to excessive proliferation and cancer. In some types of cancer, a fundamental role for U1 snRNPs and snRNAs in suppressing premature cleavage and polyadenylation in RNA transcripts, as well as a possible role for these snRNPs in cell migration and proliferation, has been observed.

[0037] Indeed, numerous studies have recognized the importance of alternative polyadenylation (APA) alterations and dysregulation in cancer and its progression. Because pre-mRNAs can have multiple alternative polyadenylation signals (PAS), polyadenylation is a source of transcript diversity in addition to the splicing process. Several studies have confirmed the potential impact of APA on gene expression regulation and function. For example, well-differentiated cells, such as neurons, often use distal PASs located further downstream from the termination codon, resulting in the expression of transcripts with longer 3'UTRs and potentially lower protein expression levels. Conversely, fast-growing cells, primarily cancer cells, tend to use proximal PASs, resulting in shorter 3'UTRs and potentially higher protein expression levels. Increased proximal 3'UTR PAS usage in multiple cancer cell lines and tissues has been associated with enhanced cancer cell proliferation.

[0038] In addition to premature polyadenylation, there is evidence that the spliceosome is also altered in cancers such as glioblastoma (GBM), resulting in the activation of oncogenic splicing events associated with tumor progression and severity. In a well-defined group of adult diffuse gliomas, primarily GBM, dysfunction in the expression of spliceosome components, such as U1 small nuclear RNA (RNU1) and splicing factors that cooperatively recognize target introns, has been observed. Silencing specific splicing factors, such as RBM22, RBM3, PTBP1, and especially SRSF3, has been shown to reduce cell proliferation and migration, tumorsphere formation, and apoptosis induction. These effects are likely mediated by modulation of key signaling pathways, such as PDGFRB and PI3K-AKT / ERK, which are pro-oncogenic pathways in glioma.

[0039] Various studies have recognized the importance of these mechanisms in cancer, including those involved in snRNP assembly, i.e., U1 snRNP biogenesis. For example, a possible relationship between these snRNPs and cell migration and proliferation has been identified in human cervical epithelial carcinoma (HeLa) cell lines. In this study, inhibition of U1 snRNA using antisense morpholino oligonucleotides (U1 AMOs), designed to target U1 snRNA, resulted in extensive premature transcription termination and shortening of the mRNA 3'-UTR by using proximal PASs in introns and the last exon, leading to the production of shorter mRNA isoforms. Interestingly, lower concentrations of U1 AMO, affecting approximately 15–30% of U1 snRNA expression, improved the migration and invasive properties of cancer cells. Furthermore, this phenomenon correlated with the upregulation of oncogenes and downregulation of tumor suppressor genes. The opposite effect was observed when U1 snRNA was overexpressed, resulting in reduced migration and invasion of cancer cells. Furthermore, in a controlled study using HeLa cells, overexpression of U1 snRNA reversed DNA damage induced specifically by UV treatment. This DNA damage was characterized by reduced U1 snRNA levels, primarily intron-selective cleavage in the 3' UTR, paralleled by substantial regulation of polyadenylation, resulting in both shortening and lengthening of gene 3' UTRs and the expression of truncated transcripts. Overall, these studies highlight the important role of polyadenylation in regulating cancer cell fate.

[0040] In another study of hepatocellular carcinoma (HCC), U1A expression (confirmed by mRNA expression and immunohistochemistry) was positively correlated with tumor stage and grade and was an independent poor prognostic factor for HCC. To further address the role of U1A in HCC, knockdown of U1A inhibited migration and cell cycle progression while promoting apoptosis in HCC cell lines. Furthermore, we observed an association between U1A mRNA expression and tumor-infiltrating immune cells. In another study of HCC cell lines and HeLa cells, knockdown of U1A also significantly reduced CCN2 expression and connective tissue growth factor (CTGF) secretion (the CTGF protein is encoded by the CCN2 oncogene), resulting in decreased cell migration and proliferation. Of note, CTGF is involved in cell proliferation, angiogenesis, and migration, which are important events for epithelial-mesenchymal transition and ultimately metastasis. Similarly, U1A is upregulated in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). High U1A expression was associated with poor survival outcomes both at initial and post-progression in LUAD cases. However, 4% of U1A genetic alterations (e.g., missense mutations and deep deletions) were associated with poor overall survival in LUSC cases but not in LUAD cases. In both LUAD and LUSC, U1A expression negatively correlated with the level of infiltrating M2 macrophages but positively correlated with the level of follicular B helper T cells. Furthermore, in the pathogenesis of LUAD, U1A expression correlated with genes involved in cell cycle and ubiquitin machinery-related problems, whereas correlation with RNA splicing-related cellular problems (e.g., spliceosome, RNA splicing, pre-mRNA binding, alternative mRNA splicing, etc.) was primarily observed in LUSC.

[0041] Taken together, these U1 snRNP studies across different cancer types suggest that U1 homeostasis is important for maintaining the normal balance of gene isoform expression and that imbalances in U1 snRNP activity can affect cancer cell proliferation, migration, and invasion. Consequently, modulation of U1 could be a therapeutic target for tumors. Indeed, modulating U1 snRNP activity in cancer has the potential to correct pathological effects resulting from splicing and premature polyadenylation dysfunction, as U1 snRNP participates in both mechanisms under physiological conditions. U1 snRNP is an essential component of the splicing machinery, involved in splice site selection and promoting spliceosome assembly by binding to the 5' splice site, known as the donor splice site, which typically contains a GU sequence. Furthermore, it plays an important role in suppressing premature cleavage and polyadenylation at the 3' end of pre-mRNAs, specifically within GU-rich regions located within introns. This repression mechanism prevents the polyadenylation machinery from recognizing these cryptic regions, which would otherwise result in the generation of truncated and aberrant mRNA transcripts. Furthermore, it is important to emphasize the role of U1 snRNP subunits, such as U1-A, U1-C, and U1-70K proteins, which have been identified as key regulators of both splicing and polyadenylation. Consequently, the precise function of U1 snRNP and its telescripting function, encompassing the repression of premature polyadenylation and cryptic splicing, protects pre-mRNA and contributes to the regulation of alternative polyadenylation, thereby avoiding pro-oncogenic processes.

[0042] In addition to its effect on regulating the U1 snRNP complex, other evidence highlighting ASMO's potential for cancer treatment is its ability to regulate tau expression. Neuroblastoma SK-N-SH cell lines treated with different concentrations of APT20TTMG (ASMO1) showed reduced tau expression at the mRNA and protein levels, with cell viability results indicating a potential cell growth inhibitory effect of APT20TTMG treatment. Furthermore, in vivo studies using a mouse xenograft of human glioblastoma cancer (U-87MG) also demonstrated reduced tumor tau expression compared to the control group, along with a reduction in tumor volume upon APT20TTMG treatment. These results are intriguing because they provide new evidence for the role of tau protein in cancer. Furthermore, as a microtubule-binding protein, tau can interfere with the binding of taxanes (microtubule-stabilizing drugs) to tubulin; therefore, anti-tau molecules could be a strategy to improve the efficacy of taxane-based chemotherapy.

[0043] For example, a relationship between tau expression and response to taxanes has been demonstrated in breast cancer cell lines. Tau knockdown by RNA interference increased sensitivity to taxanes (paclitaxel and docetaxel) in both ZR75-1 and MCF-7 cell lines, suggesting that this is related to the sub-70 kDa tau protein isoform. Furthermore, estrogen receptor (ER) signaling has been demonstrated to affect sensitivity to taxanes by influencing the expression of sub-70 kDa tau protein isoforms. Furthermore, fulvestrant, a selective estrogen receptor degrader used as a drug to treat metastatic breast cancer, reduced ER and tau expression and, when combined with taxanes, increased the sensitivity of tau- and ER-positive breast cancer cells to taxanes. Despite this, studies have shown that among ER-positive patients, patients with tau-positive tumors had better disease-free and overall survival compared with those with tau-negative tumors, but there was no significant interaction between tau expression and paclitaxel benefit. Among the ER-negative patients in this study, tau expression had no prognostic significance. The question raised by the authors is why preclinical findings were not confirmed in large-scale randomized trials, as previously described. The suggested answer is that the role of tau in resistance to paclitaxel (among other causes of resistance) may be clinically relevant only in a minority of patients. However, this discrepancy could also be due to, for example, differences in chemotherapy regimens. In contrast to what has been observed in breast cancer patients, high tau expression has been associated with a poor prognosis in prostate cancer (PC). Furthermore, tau knockdown suppressed androgen receptor expression (AR plays a crucial role in the progression to castration-resistant PC) and increased sensitivity to bicalutamide, an antiandrogen drug that blocks testosterone from reaching cancer cells. Studies with prostate tumor cell lines, ALVA-31 derivatives (ALVA-NEO and ALVA-hCD40), showed that all six alternatively spliced ​​adult brain tau isoforms, in addition to other isoforms, were expressed and highly phosphorylated, and a significant proportion did not bind to microtubules.Furthermore, ALVA-NEO tau interacts with PI3K / AKT and is therefore associated with cell survival and proliferation in many cancers. Consistent with this, downregulation of tau in taxane-resistant prostate cell lines inhibits cell proliferation via the PI3K / Akt / mTOR signaling pathway and sensitizes them to taxane cytotoxicity.

[0044] In GBM, for example, downregulation of tau by a short hairpin interfering RNA (shRNA) approach in the U87-MG cell line significantly reduced 2D cell motility by inducing inefficient cell tail retraction through ROCK relocation. Rho-ROCK signaling is known to affect cell migration and invasive phenotypes by regulating the actin cytoskeleton. In this study, we observed that tau assists in the remodeling of microtubules and the actin cytoskeleton, both of which are essential for migration. This trait may be generated by the restricted activity of the Rho-ROCK pathway, which is involved in regulating polymerization at the rear of cells. All of these effects are initiated upstream by tau-induced microtubule bundling and stabilization. Tau depletion not only disrupts this equilibrium of microtubule and actin assembly at the rear of U87MG cells, but also disrupts rear cell retraction, which is critical for cell migration. Notably, among GBM cell lines (U118, U138, and U251), U87-MG cells expressed the highest amount of tau. Another recent study using shRNA to knockdown tau in a 3D model of U87-MG multicellular spheroids (MCSs) demonstrated inhibition of MCS growth and cell evasion by affecting cell migration and spheroid aggregation. We also observed a decrease in MCS compactness due to mislocalization of N-cadherin. Furthermore, in a glioblastoma xenograft model, mice injected with U87MG sh-tau cells (cells with reduced levels of tau) showed significantly higher median survival than mice injected with U87MG control cells. These results suggest a role for tau in glioblastoma by regulating 3D cell organization and function via the PI3K / AKT signaling axis.

[0045] This MCS study also proposed a model for the role of tau in regulating PI3K / AKT signaling and N-cadherin-β-catenin signaling. N-cadherin junctions are responsible for cell-cell adhesion, which is necessary for spheroid compaction. It has been previously demonstrated that cadherin-catenin complexes at the membrane recruit PI3K and initiate signaling cascades. Tau may strengthen these interactions by stabilizing the microtubule network. Specifically, in PTEN-null mutant GBM cells (PTEN loss is highly prevalent in GBM, which may stimulate invasive behavior), tau is proposed to promote the stabilization and compaction of the N-cadherin / β-catenin complex through microtubule-dependent interactions, strengthening cell-cell adhesion. Tau has also been suggested to be involved in cell contraction by promoting actin assembly. The indirect activation cascade of the PI3K-AKT signaling pathway is also stimulated by tau through MT-independent interactions or by increased N-cadherin-β-catenin signaling, leading to cell survival and proliferation. In tau-depleted U87MG cells, defective recruitment of the N-cadherin-β-catenin complex to the actin cytoskeleton is proposed. In this case, N-cadherin is not stabilized at the membrane, resulting in a loss of MCS compactness. Furthermore, loss of cell-cell cohesion is associated with sequestration of β-catenin by mislocalized N-cadherin and is proposed to result from reduced PI3K-AKT signaling activity. A decrease in phosphorylated Akt kinase is also observed. As a result of this suggested mechanism, impaired cell proliferation and migration are expected in tau-depleted GBM cells. Furthermore, it is interesting to note that tau protein has been described to have an extensive interactome, including cancer-associated kinase proteins that may also affect cellular pathways such as cell signaling, cell motility, and cell metabolism, involving other cascade mechanisms.

[0046] Unlike glioblastoma studies, a study using a renal cell carcinoma cell line found that downregulation of tau improved cell proliferation and invasion in the 786-O cell line, suggesting that tau may play a tumor-suppressive role in this cancer type. Similarly, in the neuroblastoma cell line SH-SY5Y, tau knockout using CRISPR-Cas9 technology and shRNA knockdown resulted in dysregulation and altered activity of the pro-apoptotic tumor suppressor p53, leading to decreased DNA damage-induced apoptosis and increased cellular senescence. In line with this beneficial effect of tau in neuroblastoma, another study found that patients with higher tau mRNA expression (measured by microarray and RNA-seq data) were associated with significantly increased overall survival in pediatric neuroblastoma. The finding that higher tau expression correlated with better outcomes was consistent with higher expression of certain apoptosis effector genes (mainly CASP3 and CASP9) in samples represented by high tau expression, and lower expression of growth-promoting histone genes in samples with high tau expression. Patients with lower tau expression also had reduced overall survival, but also a significantly higher incidence of MYCN amplification (typically associated with malignant outcomes in neuroblastoma). These cases in which tau expression is associated with better outcomes may be related to microtubule stabilization, which leads to a cytostatic effect, a mechanism similar to that of microtubule-stabilizing agents such as paclitaxel, which are widely used in cancer treatments. Thus, the present disclosure provides engineered polynucleotides that function as tumor therapeutics, and corresponding methods using the engineered polynucleotides. The engineered polynucleotides may function through mechanisms of homeostatic regulation of U1 snRNP function, regulation of tau expression, or a combination (e.g., synergistic) of both, causing cytostatic or cytotoxic effects on selected cancer types, enabling broad application for oncology therapy.To enable this U1 regulation, engineered polynucleotides can be designed to have sizes, conformations, and strategic chemical modifications that allow direct attraction and interaction with U1C and indirect attraction and interaction with U1-70K. The engineered polynucleotide (e.g., ASMO1) can have sequences complementary to a highly conserved region that represents the pre-mRNA at the exon-intron junction at the 5' end of the intron, commonly referred to as the donor splice site. U1 regulation is highly relevant to the splicing process because it ensures the correct assembly (with the correct distance, position, and behavior) of the snRNPs that make up the U1 complex. 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. Modulation of tau expression upon treatment with engineered polynucleotides (e.g., ASMO1) may be a response to this splicing regulation in dysregulated cells and may be useful in cancer therapy. Taken together, the important roles of U1 snRNP and tau protein in cancer migration and progression, and our in vitro and in vivo findings, recognize that the engineered polynucleotides contemplated in this disclosure may function as therapeutic agents for the treatment of cancers such as GBM.

[0047] Engineered Polynucleotides The present disclosure provides engineered polynucleotides and methods for using the engineered polynucleotides. An engineered polynucleotide generally refers to a polynucleotide that does 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). 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 its function. 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 "recruiting moiety" can refer to a moiety that can recruit any one of the regulatory moieties described herein. A "spacing sequence" can refer to a moiety that provides spacing between other moieties. In some cases, the recitation of a moiety name does not limit the moiety to a particular function. For example, a "targeting moiety," at least a portion of which can be complementary to a target RNA, can optionally recruit a regulatory moiety. The various moieties described throughout this disclosure can be combined to generate engineered polynucleotides capable of performing a particular function, for example, an engineered polynucleotide can include a targeting moiety as disclosed herein and a recruitment moiety as disclosed herein.

[0048] In some embodiments described herein, an engineered polynucleotide comprises one or more targeting moieties configured to (e.g., specifically) bind to a ribonucleic acid (RNA) (e.g., a messenger ribonucleic acid (mRNA) such as a pre-messenger ribonucleic acid (pre-mRNA)) at a target sequence in (i). The engineered polynucleotide may further comprise (ii) a recruitment moiety configured, upon association with the RNA (e.g., an mRNA such as a pre-mRNA) and the engineered polynucleotide, to recruit a post-transcriptional regulatory moiety (e.g., a spliceosome moiety) such that the post-transcriptional regulatory moiety alters the RNA (e.g., an mRNA such as a pre-mRNA) at or near the target sequence. In some embodiments, "configured to specifically bind to" refers to hybridizing. In some embodiments, a moiety configured to specifically bind to a pre-mRNA refers to a moiety 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., an mRNA such as a pre-mRNA) encodes a target gene. In some embodiments, a pre-mRNA associated with an engineered polynucleotide refers to a pre-mRNA hybridized with at least one nucleotide (and up to about 100%) of the engineered polynucleotide. In some embodiments, "altering 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 nucleotides or less.

[0049] 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., pre-mRNA). The targeting moiety may comprise a sequence that is complementary or identical to a sequence of a ribonucleic acid (e.g., pre-mRNA) so that the engineered polynucleotide and the ribonucleic acid can interact or hybridize. The targeting moiety may be complementary or identical to a target sequence. In some embodiments of the engineered polynucleotides described herein, the targeting moiety of one or more targeting moieties is sufficiently identical 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 enable the engineered polynucleotide to interact with 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 may allow interaction with a conserved site in the constitutive donor and silence the U1 snRNA RNA-binding domain (RBD). The targeting moiety may contain a sequence nearly identical to that of the U1 snRNA RBD. For example, the RBD may contain the sequence 3'-GUCCAUUCAUA-5', and the targeting moiety may contain GTCCA (or GUCCA). Based on the sequence similarity between the U1 snRNA RBD and the targeting moiety, the targeting moiety may effectively displace the U1 snRNA RBD from binding to the pre-mRNA or prevent the U1 snRNA RBD from binding to the pre-mRNA.

[0050] Consensus sequences can be determined based on the identification of variants of unknown significance (VUS). All 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 intron and exon nucleotides adjacent to these invariant nucleotides that are highly conserved and have been found 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 result in 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 or target ribonucleic acid. 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 or target ribonucleic acid.

[0051] 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.

[0052] In some embodiments of the engineered polynucleotides described herein, the one or more targeting moieties include (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 the target sequence of RNA (e.g., mRNA, such as pre-mRNA). In some embodiments, the first targeting sequence includes a consensus sequence in the target sequence. In some embodiments, the consensus sequence of the first targeting sequence includes 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 includes 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 includes 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.

[0053] 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.

[0054] 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.

[0055] 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 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 exon 3' to 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 exon that is not immediately adjacent to an intron.

[0056] In some embodiments, the (e.g., first or second) targeting moiety comprises a nucleic acid sequence that targets a nucleotide in an 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. 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 within 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.

[0057] 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-mRNAs 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 shows the 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.

[0058] 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 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. 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 set forth 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 set forth 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.

[0059] [Table 1-1]

[0060] [Table 1-2]

[0061] 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.

[0062] The targeting moiety (e.g., first or second) can be specific to a gene or to particular exons and introns 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 for 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.

[0063] 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).

[0064] 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.

[0065] In some embodiments, when the spacing sequence in the target sequence is adjacent to the 5' or 3' end of the targeting sequence in the target sequence, the spacing sequence may not be complementary to the targeting portion of the engineered polynucleotide. In some embodiments, when the spacing sequence in the target sequence is adjacent to the 5' or 3' end of the targeting sequence in the target sequence, the spacing sequence may not be complementary to any targeting portion of the engineered polynucleotide.

[0066] In some embodiments, spacing sequence separates the first targeting sequence and the second targeting sequence described herein.In some embodiments, spacing sequence is not complementary to the targeting portion of engineered polynucleotide and does not bind to it.In some embodiments, spacing sequence is not complementary to all the targeting portions of engineered polynucleotide and does not bind to them.

[0067] 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.

[0068] 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).

[0069] 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 target sequences are spaced apart on the single nucleic acid molecule. In some embodiments, the first and second target sequences span an exon-intron boundary of the single nucleic acid molecule. In some embodiments, the first and second target sequences do not span an exon-intron boundary of the single nucleic acid molecule. In some embodiments, the first and second target sequences are adjacent to an exon-intron boundary of the single nucleic acid molecule. In some embodiments, the first and second target sequences both target an intron of the single nucleic acid molecule. In some embodiments, the first and second target sequences span a splice site of the single nucleic acid molecule. In some embodiments, the first and second target nucleic acid sequences do not span a splice site of the single nucleic acid molecule.

[0070] 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.

[0071] 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 target gene. A non-limiting example of a target gene can include the microtubule-associated protein TAU (MAPT).

[0072] 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 is 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.

[0073] 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 sequence specific to a gene. 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.

[0074] In some embodiments, the target sequence is an endogenous nucleic acid molecule. In some embodiments, the binding of the engineered polynucleotide to the target sequence is by base pairing such as Watson-Crick base pairing.

[0075] Mobilization moiety As described herein, an 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 be interposed by an internal loop. The hairpin structure may consist of 13 to 17 nucleotides. The recruitment moiety may interact with stem-loop II of U1 snRNA. The recruitment moiety can comprise a sequence complementary to the sequence or portion of stem-loop II. For example, stem-loop II of U1 snRNA can comprise the sequence 5'-GUAGGCCUCACGUUACCUAU-3', and the recruitment moiety can comprise 5'-CCGGA-3'. Ul-A can also bind to stem-loop II of U1 snRNA. Hydrogen bridges in stem-loop II with the hairpin / intrinsic 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.

[0076] 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.

[0077] [Table 2]

[0078] 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, 4, or 3 nucleotides or less. In some embodiments, the loop is an internal loop adjacent to a stem (e.g., a lower stem) and an additional stem (e.g., a stem) comprising two complementary stem sequences. In some embodiments, the internal loop comprises a nucleic acid sequence of 10, 9, or 8 nucleotides or less. In some embodiments, the stem sequence of the additional stem (e.g., upper stem) comprises no more than about 5, 4, or 3 nucleotides. In some embodiments, the engineered polynucleotide (e.g., recruitment moiety) further comprises an apical loop. In some embodiments, the apical loop comprises no more than 10, 9, 8, 7, 6, or 5 nucleotides of nucleic acid sequence.

[0079] 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.

[0080] 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 the target sequences described herein. For example, the recruitment moiety is not complementary to and does not bind to the pre-mRNA described herein.

[0081] Structural composition In various embodiments, the engineered polynucleotide comprises one or more portions that can perform a function. These one or more portions can be present in a polypeptide engineered with various structural compositions that enable the engineered polynucleotide to perform a given function (e.g., recruitment of regulatory portions or components of the spliceosome, or binding to pre-mRNA). In some embodiments of the engineered polynucleotides described herein, one of the first targeting portion and the second targeting portion is located 5' to the recruitment portion, and the other of the first targeting portion and the second targeting portion is located 3' to the recruitment portion.

[0082] In some embodiments, the engineered polynucleotide has the following structural arrangement from the 5' end to the 3' end: a first targeting portion, a recruitment portion, and a second targeting portion. In some embodiments, the engineered polynucleotide has the following structural arrangement from the 5' end to the 3' end: a second targeting portion, a recruitment portion, and a first targeting portion.

[0083] 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.

[0084] 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.

[0085] [Table 3]

[0086] In some embodiments, an engineered polynucleotide can be produced 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 produce the engineered polynucleotide.

[0087] 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, and the secondary structure may be any one or any combination of an apical loop, a stem, a stem loop, or an internal loop. One or more secondary structures may perform 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.

[0088] In some embodiments, the recruitment portion of the polynucleotide comprises at least 1, 2, 3, 4, or more secondary structures. In some embodiments, the targeting portion has no 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 upper stem proximal to the apical loop and the other lower stem proximal to the targeting portion. In some embodiments, the upper stem comprises at least 2, 4, 6, 8, 10, or more nucleotides, and 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, and the lower stem comprises at least 2, 4, 6, 8, 10, or more nucleotides, and 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, and 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 the regulatory portion, including snRNA.In some embodiments, the snRNA is a U1 snRNA, such as a U-A snRNA. In some embodiments, the snRNA is a U2 snRNA.

[0089] In some embodiments, the nucleic acid sequence of the at least one secondary structure is partially complementary to a regulatory portion comprising a ribonucleoprotein complex. In some embodiments, the nucleic acid sequence of the at least one secondary structure is not complementary to a target 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, the at least one secondary structure improves binding between a recruitment portion and a regulatory portion. In some embodiments, the at least one secondary structure stabilizes assembly of a regulatory portion. In some embodiments, the at least one secondary structure stabilizes assembly of a regulatory portion with other additional portions. In some embodiments, the at least one secondary structure increases the efficiency of regulating expression or activity of a gene encoded by a target sequence. In some embodiments, the at least one secondary structure increases the specificity of regulating expression or activity of a gene encoded by a target sequence. In some embodiments, the at least one secondary structure increases the resistance of the engineered polynucleotide to hydrolytic degradation. In some embodiments, at least one secondary structure increases the resistance of the engineered polynucleotide to degradation by nuclease digestion. In some embodiments, at least one secondary structure increases the half-life of the engineered polynucleotide. In some embodiments, at least one secondary structure reduces the immunogenicity induced by the engineered polynucleotide.

[0090] 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 embodiments, 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.

[0091] In some embodiments, the at least one secondary structure improves binding between the recruitment moiety and the regulatory moiety, ie, the 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 on the regulatory moiety.

[0092] 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 to the regulatory moiety.

[0093] In some embodiments, at least one chemical modification of the engineered polynucleotide stabilizes the assembly of spliceosomes containing the regulatory moiety when the regulatory moiety is associated with a target sequence. In some embodiments, the assembly of spliceosomes containing the regulatory moiety is stabilized by the engineered polynucleotide containing the 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 engineered polynucleotide stabilizes the assembly of spliceosomes comprising regulatory portion and at least one additional portion.For example, at least one chemical modification of engineered polynucleotide stabilizes the assembly of spliceosomes comprising 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, at least one additional portion is U4, U5, U6, U11, U12, U14 or U16 of spliceosomes.

[0094] 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.

[0095] 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.

[0096] In some embodiments, the at least one chemical modification increases the resistance of the engineered polynucleotide to hydrolytic degradation (e.g., endonuclease-mediated degradation). In some embodiments, the 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.

[0097] In some embodiments, the at least one chemical modification increases the resistance of the engineered polynucleotide to degradation by nuclease digestion. In some embodiments, the 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.

[0098] 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.

[0099] 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.

[0100] chemical modification In some embodiments described herein, the engineered polynucleotide comprises at least one chemical modification. As described in this disclosure, the chemical modification may confer a structural or functional advantage to the engineered polynucleotide (e.g., increased half-life, reduced immunogenicity, increased resistance to hydrolysis or enzymatic degradation, improved reactivity or binding to a polypeptide (e.g., a component of the spliceosome) or polynucleotide (e.g., a pre-mRNA, or a component of the spliceosome)).

[0101] In some embodiments, all nucleotides of the targeting moiety are linked by phosphorothioate bonds. In some embodiments, all nucleotides of the targeting moiety include 2'O-methyl modifications. The 2'-modification can prevent nuclease degradation and / or increase the affinity of the targeting moiety for pre-mRNA targets.

[0102] 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 the conformational change of stem-loop II of U1-snRNA and the binding of the recruitment moiety to U1-snRNA.

[0103] 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.

[0104] 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, the at least one chemical modification improves binding between the recruitment moiety and the regulatory moiety. In some embodiments, the at least one chemical modification stabilizes the assembly of the regulatory moiety. In some embodiments, the at least one chemical modification stabilizes the assembly of the regulatory moiety and other additional moieties. In some embodiments, the at least one chemical modification increases the efficiency of modulating the expression or activity of a gene encoded by a target sequence. In some embodiments, the at least one chemical modification increases the specificity of modulating the expression or activity of a gene encoded by a target sequence. In some embodiments, the at least one chemical modification increases the resistance of the engineered polynucleotide to degradation by hydrolysis. In some embodiments, the at least one chemical modification increases the resistance of the engineered polynucleotide to degradation by nuclease digestion. In some embodiments, the at least one chemical modification increases the half-life of the engineered polynucleotide. In some embodiments, the at least one chemical modification reduces immunogenicity induced by the engineered polynucleotide.

[0105] 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 a substitution for one or more 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 shown 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, including a modification to a sugar component, wherein the sugar is a ribose sugar. In some embodiments, the chemical modification of the engineered polynucleotide comprises at least one chemical modification to the ribose sugar component of a nucleotide of the engineered polynucleotide, including 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 than the 2'-O-methyl-RNA and pre-mRNA duplex, as determined by a higher melting temperature (Tm).

[0106] 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, the 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.

[0107] 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, substitution of a phosphate moiety with a "dephospho" linker, modification or substitution 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, substitution 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.

[0108] [Table 4-1]

[0109] [Table 4-2]

[0110] Modification of the phosphate backbone

[0111] In some embodiments, the chemical modification includes 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 straight-chain 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). The alkyl group 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, aryl groups have 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, or 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 more than one hydrogen atom is replaced by an aryl group. Examples of "arylalkyl" or "aralkyl" include benzyl, 2-phenylethyl, 3-phenylpropyl, 9-fluorenyl, benzhydryl, and trityl groups. "Cycloalkyl" refers to a cyclic, bicyclic, tricyclic, or polycyclic non-aromatic hydrocarbon group having 3 to 12 carbons. 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.

[0112] In some embodiments, the phosphate group of a chemically modified nucleotide can be modified by replacing one or more oxygens with different substituents. In some embodiments, a chemically modified nucleotide can include replacing an unmodified phosphate moiety with a modified phosphate as described herein. In some embodiments, the modification of the phosphate backbone can include alterations 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 phosphates, 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), BR3 (R can be, for example, hydrogen, alkyl, or aryl), C (e.g., alkyl group, aryl group, etc.), H, NR2 (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 atoms or groups of atoms described above can make the phosphorus atom chiral. The phosphorus atom in such a modified phosphate group is a stereogenic center. 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 polynucleotide contains a stereogenic nucleotide containing 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 a diastereomeric excess of 97%.In some embodiments, the chiral phosphate product is present in a diastereomeric excess of 98%. In some embodiments, the chiral phosphate product is present in a diastereomeric excess of 99%. In some embodiments, both non-bridging oxygens of the phosphorodithioate can be substituted 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 also include substitution of 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 also be modified by substituting the bridging oxygen (i.e., the oxygen linking the phosphate to the nucleoside) with nitrogen (bridging phosphoramidate), sulfur (bridging phosphorothioate), and carbon (bridging methylene phosphonate). Substitution can occur at either or both of the linking oxygens.

[0113] 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 within one of the moieties or between different moieties.

[0114] Backbone phosphate modifications for nucleic acids include methylphosphonates, phosphorothioates, phosphoramidates (bridged or unbridged), phosphotriesters, phosphorodithioates, phosphodithioates, and boranophosphates, which may be used in any combination. Other non-phosphate linkages may also be used.

[0115] 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.

[0116] 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, or the like.

[0117] In some cases, backbone modifications include replacing phosphodiester linkages with alternative moieties such as anionic, neutral, or cationic groups. Examples of such modifications include anionic 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.

[0118] 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 and phosphate moieties of the nucleotide can be replaced by, for example, amide-type linkages (aminoethylglycine) (PNA). It is also possible to link other types of molecules (conjugates) to nucleotides or nucleotide analogs, for example, to enhance cellular uptake. Conjugate can be chemically linked to nucleotide or nucleotide analogue.This 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 1-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.

[0119] 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 alterations that result in either uncharged linkers or charged linkers with asymmetric charge distribution. Examples of modified phosphate groups include phosphorothioates, phosphonothioacetates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphates, 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 by 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 is, for example, hydrogen, alkyl, or aryl), or 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 (referred to herein as Rp) or the "S" configuration (referred to herein as Sp). In such cases, the chemically modified engineered polynucleotide can be stereochemically pure (e.g., S or R configuration). In some cases, the chemically modified engineered polynucleotide comprises a stereochemically pure phosphate modification. For example, chemically modified engineered polynucleotides include phosphorothioate S configurations or phosphorothioate R configurations.

[0120] In phosphorodithioates, both non-bridging oxygens are replaced by sulfur. The phosphorus center in phosphorodithioates is achiral, which prevents the formation of oligoribonucleotide diastereomers. In some embodiments, modifications to one or both non-bridging oxygens can also 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).

[0121] The phosphate linker can also be modified by replacing 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.

[0122] Phosphate moiety replacement

[0123] 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'-alkylenephosphonate and chiral phosphonate, 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 bond between two nucleotides can be via 3'-5' linkage or 2'-5' linkage, and the linkage has reverse polarity, such as 3'-5' to 5'-3' or 2'-5' to 5'-2'.

[0124] Phosphate group substitution

[0125] 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 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.

[0126] Modification of the ribophosphate backbone

[0127] 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 such that the phosphate linker and ribose sugar are replaced by nuclease-resistant nucleosides 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.

[0128] Sugar modifications

[0129] 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 several 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 2' hydroxyl group, such as an alkoxy group (LNA, where the 2' hydroxyl can be linked 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., a PEG derivative). 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 groups 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 also 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 by 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).

[0130] Modification of the ribose sugar components

[0131] 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'-furoarabinow-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).

[0132] In some embodiments, the non-natural nucleic acid also contains a modified sugar moiety. In some cases, 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 contains a chemically modified ribofuranose ring moiety. Examples of chemically modified ribofuranose rings include 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 These include, but are not limited to, substitution of the ribosyl ring oxygen atom with an alkyl or protecting group, and combinations thereof.

[0133] 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.

[0134] 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, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C 10 Alkyl or C2-C 10 Alkenyl and alkynyl are also included. 2' sugar modifications include -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 Other chemical modifications at the 2' position include, but are not limited to, C1-C[(CH2)nCH3)]2, where n and m are from 1 to about 10. 10Modifications include, but are not limited to, 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 group, reporter group, intercalator, group 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 the 3'-terminal nucleotide, or the 5' position of 2'-5' linked oligonucleotides and 5'-terminal nucleotides. Chemically modified sugars also include those containing modifications on 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 also 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.

[0135] 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' and 2' ribosyl ring atoms. 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 following 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.

[0136] Modifications to the base of a nucleotide

[0137] In some embodiments, the chemical modifications described herein include modifications of the bases (e.g., nucleobases) of nucleotides. Exemplary nucleobases can 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 bases.

[0138] In some embodiments, the chemical modifications described herein include modifications of uracil. In some embodiments, the engineered polynucleotides described herein include 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-taurinomethyl thyl-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-(isopentenylaminomethyl)-2-thiouridine, α-thiouridine, 2'-O-methyluridine, 5,2'-O-dimethyluridine, 2'-O-methyl-pseudouridine, 2-thio-2'-O-methyluridine, 5-methoxycarbonylmethyl-2'-O-methyluridine, 5-carbamoylmethyl-2'-O-methyluridine, 5-carboxymethylaminomethyl 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.

[0139] 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, and 5-aza-zebularine. cytidine, 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.

[0140] 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-diaminopurine, 7-deaza -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, N6 -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.

[0141] 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, and the like. Euosine, 7-cyano-7-deaza-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, N2,7-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 Examples of 2'-O-amino-2'-deoxyguanosine include 2'-O-ribosyl-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, O6-methyl-2'-deoxyguanosine, 2'-F-ara-guanosine, and 2'-F-guanosine.

[0142] In some cases, chemical modification of the engineered polynucleotide may 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 may 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 modified, for example, by 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, 2'-azido-2'-deoxyur ... '-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-bromo- Lysine, 5-carboxymethylaminomethyl-2-thio-uracil, 5-carboxymethylamonomethyl-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]carbamoylmethyl-adenosine, N6-isopentenyl-adenosine, N6-methyl-adenosine, N7-methyl-xanthosine, N-uracil-5-oxyacetic acid methyl ester, puromycin, queusine, uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester, wybutoxosine, xanthosine, and xylo-adenosine. In some embodiments, the chemically modified nucleic acids described herein are selected from the group consisting of 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'-deoxycytidine-5'-triphosphate Xyluridine-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-5'-triphosphate phosphate, N1-methyladenosine-5'-triphosphate, N1-methylguanosine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, 6-methylguanosine-5'-triphosphate, pseudouridine-5'-triphosphate, puromycin-5'-triphosphate, or xanthosine-5'-triphosphate. In some embodiments, the chemically modified nucleic acids described herein are selected from the group consisting of 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, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine,The nucleotides include at least one chemically modified nucleotide selected from 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 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, 4 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-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine,The compound comprises at least one chemically modified nucleotide selected from 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.

[0143] Modified bases of non-natural nucleic acids include 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, and 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, particularly 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine. Certain non-natural nucleic acids include 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, e.g., 2-aminopropyladenine, 5-propynyluracil, and 5-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 cytidine, 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]indole-2 -one), pyridoindole 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.

[0144] Optionally, the at least one chemical modification may include chemically modifying the 5' or 3' end of the engineered polynucleotide, such as a 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., having 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 (where 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 a modified or unmodified nucleotide, and can include, but are 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.

[0145] In some embodiments, all of the nucleotides of the targeting portion have a 2'O-methyl modification. The 2'O-methyl modification is thought to increase the affinity of the engineered polynucleotide for its pre-mRNA target and / or prevent degradation of the engineered polynucleotide by nucleases. In some embodiments, all of the nucleotides of the targeting portion have a phosphorothioate modification.

[0146] Regulatory portion In some embodiments of the engineered polynucleotides described herein, the post-transcriptional regulatory portion (or regulatory portion) (e.g., the spliceosomal 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 includes U1 snRNA and a spliceosomal protein. In some embodiments, the spliceosomal portion includes 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 spliceosomal portions include SmD1, SmD2, SmD3, SmE, SmF, SmG, U1, U2, U4, U5, U6, U11, U12, U14, or U16.

[0147] 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).

[0148] 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, thus 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, thus 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 include the 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.

[0149] In some embodiments, modulation of target expression or activity includes correcting aberrant expression of a target gene resulting from a splice variant. In some embodiments, the expression or activity of a misfolded target gene or protein resulting from an aberrant splice variant is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more reduced when the engineered polynucleotide binds to the mobilizing moiety as compared to the expression or activity of a misfolded target gene or protein resulting from an aberrant splice variant when the engineered polynucleotide does not bind to the mobilizing moiety. In some embodiments, the amount of misfolded protein aggregates resulting from an aberrant splice variant is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more reduced when the engineered polynucleotide binds to the mobilizing moiety as compared to the amount of misfolded protein aggregates resulting from an aberrant splice variant when the engineered polynucleotide does not bind to the mobilizing moiety. In some embodiments, the amount of plaques containing a misfolded protein resulting from an aberrant splice variant is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more reduced when the engineered polynucleotide binds to the mobilizing moiety as compared to the amount of plaques containing a misfolded protein resulting from an aberrant splice variant when the engineered polynucleotide does not bind to the mobilizing moiety.

[0150] Molecular interaction 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.

[0151] In some embodiments, the binding of the targeting moiety to pre-mRNA is stabilized by hydrogen bonds and electrostatic interactions between U1-C and the pre-mRNA backbone around the splice junction region of pre-mRNA.In such embodiments, U1-C may not make specific base contact with pre-mRNA.2' nucleotide modification can favor hydrogen bonding between the targeting moiety and U1-C.Therefore, 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.

[0152] 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.

[0153] In some embodiments, the targeting moiety can interact with the zinc finger of U1-C. The phosphorothioate internucleotide linkage in the targeting moiety can facilitate the interaction between the targeting moiety and the zinc finger. The engineered polynucleotide can include phosphorothioate internucleotide linkages at specific or particular positions that interact with the zinc finger.

[0154] In some embodiments, the recruitment moiety forms hydrogen bonds with stem-loop II of U1-A, and such an interaction can regulate polyadenylation and acetylation signaling by U1-A because stem-loop II of U1-A cannot be silenced by the recruitment moiety.

[0155] In some embodiments described herein, the engineered polynucleotide does not contain any intramolecular disulfide bonds.

[0156] 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) exhibits substantially no base pairing with the RNA binding domain (RBD) of U1 snRNA.

[0157] 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 U1-C protein.

[0158] 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).

[0159] 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.

[0160] 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.

[0161] In some embodiments of the engineered polynucleotides described herein, the engineered polynucleotide exhibits substantially no base pairing with the H helix of U1 snRNA.

[0162] 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 sequence 3'-GUCCAUUCAUA-5' and 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.

[0163] 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 include 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 surrounding the junction, 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 stem 5' / 3' 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 interaction bridges 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 (or 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 profound effect on 5' splice site recognition by U1-snRNP, indicating that this protein has a direct role in this activity.The assembly and function of the U1 snRNP have been greatly enhanced, first by cryo-electron microscopy studies and more recently by the elucidation of its three-dimensional structure by X-ray crystallography. To date, crystal structures of four of the seven Sm proteins have led to the modeling of the remaining three (Sm-F, Sm-E, and Sm-G), which are proposed to 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 portion of the 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 could ensure the correct structure and positioning of U1-C in its interaction with the U1 snRNA:5′ splice site duplex (Fig. 9 ).

[0164] In some embodiments, when the engineered polynucleotide is associated with the spliceosome portion described herein, the pre-mRNA exhibits substantially no base pairing with the RNA binding domain (RBD) of U1 snRNA. In some cases, when the engineered polynucleotide is associated with the spliceosome portion, the pre-mRNA 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 to the 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-mRNA (Figures 3 and 6).

[0165] 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-mRNA / engineered polynucleotide (ASMO) duplex, which interacts with amino acid residues from the U1-C zinc finger, stabilizes the 5' region (Figure 9). Molecular dynamics favoring the formation of a 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 can then be observed. 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.

[0166] [Table 5-1]

[0167] [Table 5-2]

[0168] In some embodiments, an engineered polynucleotide (e.g., ASMO1 as described herein) comprises a nucleotide sequence complementary to U1 snRNA. In some aspects, an 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, whereas in this case, 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, an 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.

[0169] Engineered polynucleotide sets Some embodiments herein describe a set of engineered polynucleotides, each independently as described herein. For example, the polynucleotides of the set independently comprise: (i) one or more targeting moieties (such as those described herein) configured to bind to ribonucleic acid (RNA) (such as those described herein) (e.g., messenger ribonucleic acid (mRNA) such as pre-mRNA) at a target sequence (such as those described herein); and (ii) a recruitment moiety (such as those described herein) configured to recruit a post-transcriptional regulatory moiety (e.g., a spliceosome moiety) (such as those described herein), wherein the set of engineered polynucleotides is configured to specifically bind to RNA (e.g., mRNA such as pre-mRNA) at multiple target sequences, including the target sequence (such as those described herein).

[0170] vector Some embodiments described herein describe vectors or plasmids that include a nucleic acid sequence that encodes an engineered polynucleotide described herein.

[0171] In some embodiments herein, multiple vectors or multiple plasmids are described, each comprising multiple nucleic acid sequences encoding the engineered polynucleotides described herein. In some embodiments, the multiple vectors or multiple plasmids comprise multiple nucleic acid sequences encoding more than one engineered polynucleotide described herein. In some embodiments, the multiple vectors or multiple plasmids comprise multiple nucleic acid sequences encoding multiple engineered polynucleotides (each independently described herein).

[0172] Pharmaceutical Composition Some embodiments described herein include pharmaceutical compositions comprising the engineered polynucleotides described herein, or plasmids, vectors, or isolated DNA encoding the sequences thereof. 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, dispersants, 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 having a disease, disorder, or condition to be 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 contacted with compositions containing engineered polynucleotides, or combinations thereof.

[0173] The pharmaceutical formulations described herein may be administered to a subject by any suitable route of administration, including, but not limited to, intravenous, intrathecal, intraarterial, intratumoral, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, 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- and controlled-release formulations.

[0174] 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.

[0175] kit Some embodiments described herein provide 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 suitable 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.

[0176] 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, which 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).

[0177] 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 provided to the medical professional in any convenient and suitable form that retains 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.

[0178] 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 a target sequence therein (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, before contacting, the cell exhibits abnormal messenger ribonucleic acid (mRNA) or protein corresponding to the target gene. In some embodiments, the targeting moiety of 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)).

[0179] 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 method 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)).

[0180] 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, followed by 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 abnormal 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)).

[0181] 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 a collection of regulatory moieties to modulate the expression or activity of the disease or condition-causing gene, thereby treating the disease or condition.

[0182] In some embodiments, methods of delivering an engineered polynucleotide described herein to a cell are described. 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 the cell by any of the transfection methods described herein. In some embodiments, the engineered polynucleotide can be delivered to the cell by the use of an expression vector. In the context of an expression vector, the vector can be easily introduced into the cell described herein by any method in the art. For example, the expression vector can be transferred to the cell by physical, chemical, or biological means.

[0183] Physical methods for introducing an engineered polynucleotide or a vector encoding the engineered polynucleotide into a cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, gene guns, electroporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are suitable for the methods herein. One method for introducing an engineered polynucleotide or a vector encoding the engineered polynucleotide into a host cell is calcium phosphate transfection.

[0184] 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.

[0185] When a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for introducing the engineered polynucleotide or a vector encoding the engineered polynucleotide into cells (in vitro, ex vivo, or in vivo). In another embodiment, the engineered polynucleotide or a vector encoding the engineered polynucleotide can be associated with a lipid. The lipid-associated engineered polynucleotide or the vector encoding the engineered polynucleotide 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 lipid, mixed with lipid, combined with lipid, contained as a suspension in lipid, contained with or complexed to micelles, or otherwise associated with lipid. The lipid, lipid / DNA, or lipid / expression vector association composition is 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. Lipids are fatty substances that in some embodiments are naturally occurring or synthetic lipids. For example, lipids include the lipid droplets that naturally occur in cytoplasm, as well as a class of compounds that contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

[0186] 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 exist in micellar structures or simply as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0187] 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.

[0188] 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.

[0189] In some aspects herein, a method is described 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 the target sequence therein. 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 the spliceosome moiety and is recruited to the target sequence. In some embodiments, the method reduces the expression or activity of a target gene when the engineered polynucleotide binds to a spliceosome portion and is recruited to the target sequence. In some embodiments, the method corrects an aberrant messenger ribonucleic acid (mRNA) or protein corresponding to the target gene when the engineered polynucleotide binds to a spliceosome portion and is recruited to the target sequence.

[0190] 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 a pre-mRNA encoding a different target gene. In some embodiments, the method includes two or more engineered polynucleotides configured to specifically bind to pre-mRNA at multiple target sequences, including the target sequence.

[0191] In some embodiments, disclosed herein 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 comprises 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.

[0192] 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.

[0193] In some embodiments, the engineered polynucleotide or pharmaceutical composition comprising the engineered polynucleotide is administered at a concentration of at least 0.25 μM. In some embodiments, the engineered polynucleotide or pharmaceutical composition comprising the engineered polynucleotide is administered at a concentration of at least 0.5 μM. In some embodiments, the engineered polynucleotide or pharmaceutical composition comprising the engineered polynucleotide is administered at a concentration of at least 0.75 μM. In some embodiments, the engineered polynucleotide or pharmaceutical composition comprising the engineered polynucleotide is administered at a concentration of at least 1 μM. In some embodiments, the engineered polynucleotide, or a pharmaceutical composition comprising the engineered polynucleotide, has a cytotoxic activity of at least 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1 μM, 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM, 1.6 μM, 1.7 μM, 1.8 μM, 1.9 μM, 2.0 μM, 2.1 μM, 2.2 μM, 2.3 μM, 2.4 μM, 2.5 μM, 2.6 μM, 2.7 μM, 2.8 μM, 2.9 ... and / or administered at a concentration of 0 μM, 2.1 μM, 2.2 μM, 2.3 μM, 2.4 μM, 2.5 μM, 2.6 μM, 2.7 μM, 2.8 μM, 2.9 μM, 3.0 μM, 4.0 μM, 5.0 μM, 6.0 μM, 7.0 μM, 8.0 μM, 9.0 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, or more. In some embodiments, the engineered polynucleotide, or a pharmaceutical composition comprising the engineered polynucleotide, has a cytotoxicity of at least 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1 μM, 1.1 μM, 1.2 μM, 1.3 μM, 1.4 μM, 1.5 μM, 1.6 μM, 1.7 μM, 1.8 μM, 1.9 μM, 2.0 μM, 2.1 μM, 2.2 μM, 2.3 μM, 2.4 μM, 2.5 μM, 2.6 μM, 2.7 μM, 2.8 μM, 2.9 μM, 3.0 μM, 3.1 μM, 3.2 μM, 3.3 μM, 3.4 μM, 3.5 μM, 3.6 μM, 3.7 μM, 3.8 μM, 3.9 μM, 4.0 μM, 4.1 μM, 4.2 μM, 4.3 μM, 4.4 μM, 4.5 μM, 4.6 μM, 4.7 μM, 4.8 μM, 4.9 μM, 5.0 μM, 5.1 μM, 5.2 μM, 5.3 μM, 5.4 μM, 5.5 μM, 5.6 μM, 5.7 μM, 5.8 μM, 5.9 μM, 6.0 μM, 6.1 μM, 6.2 μM, 6.3 μM, 6.4 μM, 6.5 μM, 6.6 μM, 6.7 μM, 6.8 μM, 6.9 μM

[0033] The compound may be administered at a concentration of 2.1 μM, 2.2 μM, 2.3 μM, 2.4 μM, 2.5 μM, 2.6 μM, 2.7 μM, 2.8 μM, 2.9 μM, 3.0 μM, 4.0 μM, 5.0 μM, 6.0 μM, 7.0 μM, 8.0 μM, 9.0 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM or less.In some embodiments, the engineered polynucleotide, or pharmaceutical composition comprising the engineered polynucleotide, is administered at a concentration of about 0.25 μM to 1 μM.

[0194] 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 oral administration. However, in some instances, the methods involve administering the engineered polynucleotide, or pharmaceutical composition comprising the engineered polynucleotide, by intraperitoneal injection. In some instances, the methods involve administering the engineered polynucleotide, or pharmaceutical composition comprising the engineered polynucleotide, by intravenous ("iv") administration. In some instances, the methods involve administering the engineered polynucleotide, or pharmaceutical composition comprising the engineered polynucleotide, by intratumoral administration. It is contemplated that the engineered polynucleotides disclosed herein, or pharmaceutical compositions comprising engineered polynucleotides, may also be administered by other routes, such as subcutaneous injection, intramuscular injection, intradermal injection, transdermal injection, transdermal administration, intranasal administration, intralymphatic injection, intrathecal administration, pulmonary administration, 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 a disease or disorder.

[0195] The appropriate dose and dosage 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, comorbidities, 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.

[0196] This specification describes a method for treating cancer in a subject in need of cancer treatment using an engineered polynucleotide or a vector encoding the engineered polynucleotide.The engineered polynucleotide can be the engineered polynucleotide described in International Patent Application PCT / US2022 / 037391, which is incorporated by reference in its entirety.

[0197] Described herein are methods for treating cancer in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising an engineered polynucleotide. In some embodiments, the engineered polynucleotide comprises (i) one or more targeting moieties configured to specifically bind to a pre-messenger ribonucleic acid (pre-mRNA) at a target sequence (e.g., a conserved splice site) 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.

[0198] In some embodiments, the cancer is selected from the group consisting of brain cancer, prostate cancer, breast cancer, kidney cancer, lung cancer, and liver cancer. In some embodiments, the cancer is glioblastoma. In some embodiments, the cancer is breast adenocarcinoma. In some embodiments, the cancer is neuroblastoma. In some embodiments, the cancer is prostate cancer. In some embodiments, the cancer is renal carcinoma. In some embodiments, the cancer is hepatocellular carcinoma. In some embodiments, the cancer is renal adenocarcinoma.

[0199] In some embodiments, provided herein are methods comprising contacting a cell with an engineered polynucleotide or a vector encoding an engineered polynucleotide as described herein. In some embodiments, contacting a cell with the engineered polynucleotide or the vector encoding the engineered polynucleotide results in a cytotoxic or cytostatic effect. In some embodiments, contacting a cell with the engineered polynucleotide or the vector encoding the engineered polynucleotide results in a change in cell viability. In some embodiments, contacting a cell with the engineered polynucleotide or the vector encoding the engineered polynucleotide results in internalization of the engineered polynucleotide by the cell. In some embodiments, contacting a cell with the engineered polynucleotide or the vector encoding the engineered polynucleotide results in a decrease in mitochondrial activity. In some embodiments, contacting a cell with the engineered polynucleotide or the vector encoding the engineered polynucleotide results in a decrease in cell proliferation. In some embodiments, contacting cells with the engineered polynucleotide or a vector encoding the engineered polynucleotide alters the distribution of cell cycle phases, consisting of Sub / G1: non-proliferative state (quiescence), G1 / G0: cell growth, S: DNA replication, G2 / M: DNA segregation, and mitosis. In some embodiments, contacting cells with the engineered polynucleotide or a vector encoding the engineered polynucleotide increases the propensity of the cells to be in G2 / M phase.

[0200] In some embodiments, contacting cells with the engineered polynucleotide or the vector encoding the engineered polynucleotide increases apoptosis or necrosis. In some embodiments, contacting cells with the engineered polynucleotide or the vector encoding the engineered polynucleotide decreases MAPT expression. In some embodiments, contacting cells with the engineered polynucleotide or the vector encoding the engineered polynucleotide decreases the amount of TAU. In some embodiments, contacting cells with the engineered polynucleotide or the vector encoding the engineered polynucleotide changes the mitochondrial membrane potential.

[0201] Some embodiments provided herein are methods comprising contacting a plurality of cells with an engineered polynucleotide described herein or a vector encoding the engineered polynucleotide. In some embodiments, contacting a plurality of cells with the engineered polynucleotide or a vector encoding the engineered polynucleotide alters the distribution of cell cycle phases, with the cell cycle phases consisting of Sub / G1: non-proliferative state (quiescence), G1 / G0: cell growth, S: DNA replication, G2 / M: DNA segregation, and mitosis. In some embodiments, contacting a plurality of cells with the engineered polynucleotide or a vector encoding the engineered polynucleotide increases the number of cells in the G2 / M phase. In some embodiments, contacting a cell with the engineered polynucleotide or a vector encoding the engineered polynucleotide increases the number of cells in apoptosis or necrosis.

[0202] Some embodiments provided herein are methods for treating a subject having or suspected of having cancer. Some embodiments provided herein are methods for treating a tumor in a subject. In some embodiments, contacting cells of a subject with an engineered polynucleotide or a vector encoding an engineered polynucleotide reduces tumor progression. In some embodiments, the tumor is a glioblastoma. In some embodiments, the engineered polynucleotide or the vector encoding an engineered polynucleotide is administered intravenously. In some embodiments, the engineered polynucleotide or the vector encoding an engineered polynucleotide is administered intratumorally. In some embodiments, the engineered polynucleotide or the vector encoding an engineered polynucleotide is administered subcutaneously. In some embodiments, the engineered polynucleotide or the vector encoding an engineered polynucleotide is administered once. In some embodiments, the engineered polynucleotide or the vector encoding an engineered polynucleotide is administered daily. In some embodiments, the engineered polynucleotide or the vector encoding an engineered polynucleotide is administered daily for 1 to 21 days. In some embodiments, administering the engineered polynucleotide or the vector encoding the engineered polynucleotide to a subject in need thereof reduces tumor volume. In some embodiments, administering the engineered polynucleotide, or a vector encoding the engineered polynucleotide, to a subject in need thereof reduces tumor volume ratio.

[0203] In some embodiments, the cells are glioblastoma. In some embodiments, the cells are breast adenocarcinoma. In some embodiments, the cells are neuroblastoma. In some embodiments, the cells are prostate cancer. In some embodiments, the cells are renal carcinoma. In some embodiments, the cells are hepatocellular carcinoma. In some embodiments, the cells are renal adenocarcinoma. In some embodiments, the cells are neurons. In some embodiments, the cells are from a subject diagnosed with cancer. In some embodiments, the cells are neurons from a subject diagnosed with cancer.

[0204] In some embodiments, the engineered polynucleotide is ASMO1. In some embodiments, ASMO1 directly or indirectly interacts with U1-C and U1-70K. In some embodiments, ASMO1 directly or indirectly interacts with U1 complex and MAPT pre-mRNA.

[0205] In various embodiments, the subject is administered a taxane as if it had been administered previously, or is co-administered. The taxane can include a taxane effective in treating cancer. Administration of the engineered polynucleotide can improve the subject's prognosis, for example, compared to before administration of the engineered polynucleotide, or compared to another subject administered a taxane but not the engineered polypeptide. The engineered polypeptide can alter the amount of TAU present in the subject. The taxane can bind to TAU, resulting in a reduction in the taxane's ability to bind to other targets in the cell. By reducing the amount of TAU, the taxane will have less "off-target" binding, which can increase efficacy if present. In various embodiments, the subject is not administered a taxane as if it had been administered previously, or is not co-administered.

[0206] In various embodiments, the subject is administered the engineered polynucleotide without another anti-cancer or cancer therapeutic agent.

[0207] In various embodiments, the subject is administered another therapeutic agent for cancer (e.g., a drug, neoadjuvant, adjuvant) as previously administered, and co-administration of the engineered polynucleotide may improve the subject's prognosis or improve the efficacy of treatment compared to administration of the other therapeutic agent alone.

[0208] 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.

[0209] 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 inclusive, as is the term "comprising."

[0210] 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.

[0211] 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.

[0212] 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.

[0213] 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.

[0214] The terms "increased," "increasing," or "increase" are used herein generally to mean a statically significant increase. In some embodiments, the term "increased" or "increase" refers to an increase of at least 10% compared to a baseline level, e.g., an increase of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or up to a 100% increase, or any increase between 10 and 100%, compared to a baseline 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 baseline level.

[0215] The terms "decreased," "decreasing," or "decrease" are generally used herein to mean a decrease by a statistically significant amount. In some embodiments, "decreased" or "decreased" refers to 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. The 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.

[0216] 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 also encompasses 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.

[0217] 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 a target sequence therein, 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.

[0218] Embodiment 2. The engineered polynucleotide of embodiment 1, wherein the 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.

[0219] 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.

[0220] Embodiment 4. The engineered polynucleotide of embodiment 3, wherein the first targeting sequence comprises a consensus sequence in the target sequence.

[0221] Embodiment 5. The engineered polynucleotide of embodiment 3 or 4, wherein the second targeting sequence comprises a consensus sequence in the target sequence.

[0222] Embodiment 6. The engineered polynucleotide of any one of embodiments 3 to 5, wherein the first target sequence and the second target sequence are separated in the target sequence by a spacing sequence of 5 nucleotides or less (e.g., 1 or 2 nucleotides).

[0223] 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.

[0224] Embodiment 8. The engineered polynucleotide of embodiment 7, wherein the first targeting sequence and the second targeting sequence are both 5' or 3' to an exon-intron boundary.

[0225] Embodiment 9. The engineered polynucleotide of embodiment 7, wherein one of the first targeting sequence and the second targeting sequence is 5' to an exon-intron boundary, and the other of the first targeting sequence and the second targeting sequence is 3' to an exon-intron boundary.

[0226] 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.

[0227] Embodiment 11. The engineered polynucleotide of embodiment 10, wherein the first targeting sequence or the second targeting sequence comprises a splice site (e.g., 5'ss) in the pre-mRNA.

[0228] 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.

[0229] 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.

[0230] Embodiment 14. The engineered polynucleotide of embodiment 12, wherein the first targeting moiety comprises a sequence identical or complementary to a sequence selected from the exon sequence column of Table 1, and the second targeting moiety comprises a sequence identical or complementary to a sequence set forth in the intron sequence column of Table 1.

[0231] Embodiment 15. The engineered polynucleotide of embodiment 12, wherein the first targeting moiety comprises a sequence identical or complementary to a sequence set forth in the intron sequence column of Table 1, and the second targeting moiety comprises a sequence identical or complementary to a sequence set forth in the exon sequence column of Table 1.

[0232] 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).

[0233] 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.

[0234] 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.

[0235] 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.

[0236] Embodiment 20 The engineered polynucleotide of embodiment 19, wherein the spliceosome portion comprises U1 snRNA and spliceosome proteins.

[0237] 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.

[0238] 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.

[0239] Embodiment 23. The engineered polynucleotide of any one of embodiments 1 to 22, wherein the recruitment portion comprises a nucleotide sequence at least 70%, 80%, 85%, or 90% identical to or complementary to any one of SEQ ID NOs: 1-2.

[0240] 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.

[0241] Embodiment 25. The engineered polynucleotide of any one of embodiments 1 to 24, wherein the engineered polynucleotide comprises a (e.g., secondary) structural feature.

[0242] Embodiment 26 The engineered polynucleotide of embodiment 25, wherein the engineered polynucleotide comprises an apical loop, an upper stem, an internal loop, a lower stem, or a combination thereof.

[0243] 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.

[0244] Embodiment 28. The engineered polynucleotide of embodiment 27, wherein the stem sequence of the stem (e.g., the lower stem or the upper stem) comprises no more than about 5, 4, or 3 nucleotides.

[0245] Embodiment 29. The engineered polynucleotide of embodiment 27, 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.

[0246] Embodiment 30. The engineered polynucleotide of embodiment 29, wherein the internal loop comprises a nucleic acid sequence of 10, 9, or 8 nucleotides or less.

[0247] Embodiment 31. The engineered polynucleotide of embodiment 29 or 30, wherein the stem sequence of the additional stem (e.g., the upper stem) comprises no more than about 5, 4, or 3 nucleotides.

[0248] Embodiment 32. The engineered polynucleotide of any one of embodiments 29 to 31, wherein the engineered polynucleotide further comprises an apical loop.

[0249] Embodiment 33. The engineered polynucleotide of embodiment 32, wherein the apical loop comprises a nucleic acid sequence of 10, 9, 8, 7, 6, or 5 nucleotides or less.

[0250] Embodiment 34. The engineered polynucleotide of any one of embodiments 1 to 33, wherein the engineered polynucleotide does not contain any intramolecular disulfide bonds.

[0251] Embodiment 35. The engineered polynucleotide of any one of embodiments 1 to 34, wherein upon association with the engineered polynucleotide and the spliceosome moiety, the pre-mRNA exhibits substantially no base pairing with the RNA binding domain (RBD) of U1 snRNA.

[0252] Embodiment 36. The engineered polynucleotide of any one of embodiments 1 to 35, wherein upon association with the engineered polynucleotide and the spliceosome moiety, the pre-mRNA exhibits substantially no base-specific interaction with U1-C protein.

[0253] 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.

[0254] Embodiment 38. The engineered polynucleotide of embodiment 37, wherein the 5'-targeting portion of the engineered polynucleotide is configured to specifically interact with a zinc finger of a U1-C protein.

[0255] Embodiment 39. The engineered polynucleotide of embodiment 37, 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).

[0256] Embodiment 40. The engineered polynucleotide of any one of embodiments 37 to 39, 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 the U1-C protein.

[0257] 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.

[0258] 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.

[0259] 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.

[0260] Embodiment 44. The engineered polynucleotide of any one of embodiments 41 to 43, wherein the subsequence does not include a sequence corresponding to 5'-CACGUUA-3' of SL2 of U1 snRNA.

[0261] Embodiment 45. The engineered polynucleotide of any one of embodiments 1 to 44, wherein the engineered polynucleotide exhibits substantially no base pairing with the anchor sequence of SL2 of U1 snRNA.

[0262] Embodiment 46. The engineered polynucleotide of embodiment 45, wherein the internal loop of the engineered polynucleotide exhibits substantially no base pairing with the anchor sequence of SL2 of U1 snRNA.

[0263] Embodiment 47. The engineered polynucleotide of embodiment 45 or 46, wherein the lower stem of the engineered polynucleotide exhibits substantially no base pairing with the anchor sequence of SL2 of U1 snRNA.

[0264] 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'.

[0265] Embodiment 49. The engineered polynucleotide of any one of embodiments 1 to 48, wherein the engineered polynucleotide exhibits substantially no base pairing with the H helix of U1 snRNA.

[0266] Embodiment 50. The engineered polynucleotide of any one of embodiments 1 to 49, wherein the engineered polynucleotide comprises at least one chemical modification.

[0267] Embodiment 51. The engineered polynucleotide of embodiment 50, wherein the engineered polynucleotide comprises at least one 2'-modified (e.g., 2'-methoxy, 2'-methoxymethyl, or 2'-methoxyethyl) nucleotide.

[0268] Embodiment 52. The engineered polynucleotide of embodiment 50, wherein at least about 50%, 60%, 70%, 80%, or 90% of the nucleotides of the engineered polynucleotide are chemically modified nucleotides.

[0269] Embodiment 53. The engineered polynucleotide of embodiment 50, wherein at least about 50%, 60%, 70%, 80%, or 90% of the nucleotides of the engineered polynucleotide are 2'-modified (e.g., 2'-methoxy, 2'-methoxymethyl, or 2'-methoxyethyl) nucleotides.

[0270] Embodiment 54. The engineered polynucleotide of any one of embodiments 50 to 53, wherein the engineered polynucleotide comprises at least one phosphorothioate internucleotide linkage.

[0271] 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 the engineered polynucleotide are chemically modified.

[0272] 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 the engineered polynucleotide are phosphorothioate.

[0273] 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.

[0274] 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.

[0275] 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.

[0276] Embodiment 60. The engineered polynucleotide of any one of embodiments 1 to 59, 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.

[0277] 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.

[0278] 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.

[0279] Embodiment 63. An engineered polynucleotide comprising a nucleotide sequence at least 7%, 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.

[0280] Embodiment 64. The engineered polynucleotide of embodiment 63, wherein the nucleotide sequence comprises a nucleotide sequence identical to or complementary to any one of SEQ ID NOs: 1-4.

[0281] Embodiment 65. The engineered polynucleotide of embodiment 63, wherein the structural feature comprises one or more stem-loop structures.

[0282] Embodiment 66 The engineered polynucleotide of embodiment 63, wherein the structural feature comprises an apical loop, an upper stem, an internal loop, a lower stem, or a combination thereof.

[0283] 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.

[0284] Embodiment 68. The engineered polynucleotide of embodiment 67, wherein the stem sequence of the stem (e.g., the lower stem or the upper stem) comprises no more than about 5, 4, or 3 nucleotides.

[0285] Embodiment 69. The engineered polynucleotide of any one of embodiments 63 to 68, 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.

[0286] Embodiment 70. The engineered polynucleotide of embodiment 69, wherein the internal loop comprises a nucleic acid sequence of 10, 9, or 8 nucleotides or less.

[0287] 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, 4, or 3 nucleotides.

[0288] Embodiment 72. The engineered polynucleotide of any one of embodiments 69 to 71, wherein the engineered polynucleotide further comprises an apical loop.

[0289] Embodiment 73. The engineered polynucleotide of embodiment 72, wherein the apical loop comprises a nucleic acid sequence of 10, 9, 8, 7, 6, or 5 nucleotides or less.

[0290] 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 the target gene.

[0291] Embodiment 75. The engineered polynucleotide of embodiment 74, wherein the 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.

[0292] Embodiment 76. The engineered polynucleotide of embodiment 74, wherein the target gene is microtubule-associated protein TAU (MAPT).

[0293] Embodiment 77. The engineered polynucleotide of any one of embodiments 63 to 75, wherein the engineered polynucleotide comprises at least one chemical modification.

[0294] Embodiment 78. The engineered polynucleotide of embodiment 77, wherein the engineered polynucleotide comprises at least one phosphorothioate internucleotide linkage.

[0295] Embodiment 79. The engineered polynucleotide of embodiment 77, wherein at least about 50%, 60%, 70%, 80%, or 90% of the internucleotide linkages of the engineered polynucleotide are chemically modified.

[0296] Embodiment 80. The engineered polynucleotide of embodiment 77, wherein at least about 50%, 60%, 70%, 80%, or 90% of the internucleotide linkages of the engineered polynucleotide are phosphorothioate.

[0297] 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.

[0298] 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 of the engineered polynucleotide are chemically modified nucleotides.

[0299] 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 of the engineered polynucleotide are 2'-modified (e.g., 2'-methoxy, 2'-methoxymethyl, or 2'-methoxyethyl) nucleotides.

[0300] 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.

[0301] Embodiment 85. A method for altering a pre-messenger ribonucleic acid (pre-mRNA) in a cell, comprising the step of 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 a target sequence therein, 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.

[0302] Embodiment 86. The method of embodiment 85, wherein the 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.

[0303] Embodiment 87. The method of embodiment 85 or 86, wherein the pre-mRNA corresponds to a target gene.

[0304] Embodiment 88. The method of embodiment 87, wherein the target gene is microtubule-associated protein TAU (MAPT).

[0305] Embodiment 89. The method of any one of embodiments 85 to 88, wherein the expression or activity of a target gene is altered.

[0306] Embodiment 90. The method of any one of embodiments 85 to 89, wherein prior to the contacting step, the cell exhibits aberrant messenger ribonucleic acid (mRNA) or protein corresponding to the target gene.

[0307] 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 pre-mRNA at a plurality of target sequences, including the target sequence. [Example]

[0308] The following illustrative examples represent embodiments of the stimuli, systems, and methods described herein and are not intended to be limiting in any way.

[0309] 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 plaques encoded by the MAPT target gene).

[0310] Example 2. In vitro evaluation of the cytotoxic effects of ASMO1 (also known as "APT20TTMG" or "ASMO AP20TTMG") on a panel of human cancer cell lines Cell lines were maintained in growth medium containing 10% fetal bovine serum (FBS) until the time of experimentation. Cells were subcultured by trypsinization, dispensing the cell suspension into a fresh flask, and mobilizing fresh culture medium. To perform the assay, cells were trypsinized, neutralized with complete medium (10% FBS), centrifuged, counted using a hemocytometer, and seeded with the corresponding medium containing 10% FBS. 2.5 × 10 4 Cell lines other than the SH-SY5Y cell line were seeded at a density of 1 × 10 per well. 4 Cells were seeded at a density of 0.1% FBS (U87MG, MCF-7, SH-SY5Y, and 786-0) and 1% FBS (PC-3) for different experimental groups. Untreated cells were used as a negative control (medium only). After 24, 48, 72, and 96 hours of incubation, the effects of ASMO1 and the control on cell cytotoxicity were determined by MTT assay.

[0311] Briefly, plates were removed, and 20 μl of 5 mg / ml MTT3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide solution was added to all wells. Cells were incubated at 37°C for 3 hours. After this period, the supernatant was aspirated, and 100 μl of DMSO was added to each well to dissolve the formazan crystals. The absorbance of each well was then read at 540 nm using a Synergy HT microplate reader.

[0312] GraphPad Prism 9 software (GraphPad Software Inc., San Diego, CA, USA) was used for graphics generation.

[0313] Figure 11, panels A–D, demonstrate the effect of incubation of ASMO1 with APT20TTMG in the U-87MG cell line. At 24 hours, cell viability tended to decrease compared to the negative control and vehicle groups, suggesting a cytotoxic effect. A decrease in absorbance values ​​was observed within 48 hours, suggesting a cytotoxic and / or cytostatic effect. However, values ​​at 72 and 96 hours were similar to those in the vehicle group, suggesting that the viability-reducing effect had ceased. Cells were seeded and treated with seven different concentrations of APT20TTMG (0.03, 0.1, 0.3, 1, 3, 10, and 30 μM) containing medium (0.1% FBS). MTT assays were performed at four different time points, and the graphs are presented as absorbance (540 nm). (A) 24-hour incubation; (B) 48-hour incubation; (C) 72-hour incubation. (D) 96 h incubation.

[0314] As shown in Figure 12A-D, ASMO1 APT20TTMG does not appear to exert cytotoxic or cytostatic effects on MCF-7 cell line. Cells were seeded and treated with seven different concentrations of APT20TTMG (0.03, 0.1, 0.3, 1, 3, 10, and 30 μM) containing medium (0.1% FBS). MTT assays were performed at four different time points, and the graphs are expressed as absorbance (540 nm). (A) 24-hour incubation; (B) 48-hour incubation; (C) 72-hour incubation; (D) 96-hour incubation.

[0315] As shown in Figure 13A-D, ASMO1 APT20TTMG appears to have a cytotoxic effect at concentrations of 1 μM for up to 72 hours. The profile at 96 hours is slightly different, with some effects beginning at 0.3 μM. Cells were seeded and treated with seven different concentrations of APT20TTMG (0.03, 0.1, 0.3, 1, 3, 10, and 30 μM) containing medium (0.1% FBS). MTT assays were performed at four different time points, and graphs are presented as absorbance (540 nm). (A) 24-hour incubation. (B) 48-hour incubation. (C) 72-hour incubation. (D) 96-hour incubation.

[0316] As shown in Figure 14A-C, ASMO1 APT20TTMG does not appear to exert cytotoxic or cytostatic effects on the PC-3 cell line. Cells were seeded and treated with seven different concentrations of APT20TTMG (0.03, 0.1, 0.3, 1, 3, 10, and 30 μM) in medium (1% FBS). MTT assays were performed at three different times, and the graphs are expressed as absorbance (540 nm). (A) 24-hour incubation. (B) 48-hour incubation. (C) 72-hour incubation.

[0317] As shown in Figure 15A-C, ASMO1 APT20TTMG appears to improve cell viability of the 786-O strain at 24 hours. However, in the remaining two times, no significant difference was observed compared to the control (vehicle group) except at the highest concentration (30 pM). Cells were seeded and treated with seven different concentrations of APT20TTMG (0.03, 0.1, 0.3, 1, 3, 10, and 30 μM) containing medium (0.1% FBS). MTT assays were performed at three different times, and the graphs are expressed as absorbance (540 nm). (A) 24-hour incubation. (B) 48-hour incubation. (C) 72-hour incubation.

[0318] The results of this study demonstrated that the effect of ASMO1 was dependent on the type of cell line, time, and concentration used, with improved efficacy against glioblastoma and neuroblastoma.

[0319] Example 3. Evaluation of the antitumor activity of ASMO1 in breast cancer cell line (MCF-7) MCF-7 cells were maintained in Roswell Park Memorial Institute 1640 medium (RPMI 1640) supplemented with 10% fetal bovine serum (FBS) and a 1% solution of penicillin / streptomycin. Cells were plated at 75 cm 2 Cells were cultured in cell culture flasks and incubated at 37°C, 5% CO2, and controlled humidity. After reaching 80-90% confluency, cells were washed with 10 mL of phosphate-buffered saline (PBS) and detached from the flask with trypsin-EDTA solution (0.25% / 0.03%), followed by neutralization with complete medium. Cells were then transferred to conical tubes and centrifuged at 1000 rpm for 5 minutes. The pellet was resuspended in culture medium, and cells were stained with 0.4% trypan blue dye. Cell viability was then determined by counting using a TC20™ automated counter (BioRad, Hercules, CA, USA). A cell viability value greater than 90% was expected.

[0320] Because the molecular target of ASMO APT20TTMG is located in the cell nucleus, APT20TTMG is internalized by cells to initiate its proposed action. Cellular uptake of oligonucleotides is not fully understood but is expected to occur in two stages: adsorption and internalization. Adsorption of phosphorothioate-modified antisense oligonucleotides to the cell surface is thought to be rapid and does not require energy. After adsorption, different endocytic pathways, including micropinocytosis, can internalize this type of oligonucleotide. Cell surface proteins can direct oligonucleotide internalization via nonconventional endocytic pathways or via clathrin- or caveolin-dependent endocytic pathways. Furthermore, different cell types have different oligonucleotide binding capacities, and rapidly growing cells, including malignant cells, internalize oligonucleotides more efficiently than slow-growing cells.

[0321] To investigate the internalization of ASMO APT20TTMG by MCF-7 cell lines, quantification of intracellular fluorescence was measured by flow cytometry. For this purpose, ASMO APT20TTMG was conjugated to the fluorescent dye carboxyfluorescein. We hypothesize that the percentage of internalized ASMO molecules is likely proportional to the quantified fluorescent signal detected inside the cells. Cell lines were cultured in 24-well plates for 24 hours at 5 × 10 per well. 4Cells were cultured with ASMO1. The cells were then incubated with ASMO1, prepared in FBS-free culture medium, at a concentration of 1 μM for 0.5, 1, 2, 4, and 6 hours. Untreated cells were used as a negative control. After incubation, the cells were washed twice with 1 mL of PBS per well and then incubated with 200 μL of trypsin / EDTA solution per well for approximately 3 minutes to disaggregate the adherent cells. The trypsin was neutralized by the addition of complete medium, and the cells were centrifuged at 1500 rpm for 5 minutes. Subsequently, the cells were washed with 2 mL of PBS per sample and centrifuged again at 1500 rpm for 5 minutes. Finally, the supernatant was discarded, and the cells were resuspended in 200 μL of PBS. Cellular fluorescence, corresponding to cellular uptake of carboxycarboxyfluorescein-labeled APT20TTMG, was analyzed using a BD FACSCANTO II Flow cytometer (BD Biosciences, NJ, USA) by acquiring 10,000 gated events for each sample.

[0322] An MTT assay was performed to assess cell viability in MCF-7 cell lines after treatment with ASMO APT20TTMG, thereby inferring ASMO1 cytotoxicity. The tetrazolium salt reduction (MTT) method evaluates cell viability by assessing mitochondrial integrity. Viable cells can reduce the yellow, water-soluble MTT reagent to an insoluble purple formazan product. Formazan quantification could be measured by spectrophotometric absorbance at 560 nm. The amount of formazan is directly proportional to the number of viable cells in culture.

[0323] In this assay, MCF-7 cells were plated in 96-well plates at 1 × 10 per well. 4Cells were seeded at a density of 100 μM and cultured for 24 hours for adhesion. Subsequently, cells were incubated with eight concentrations (0.0078–1 μM) of APT20TTMG (prepared in culture medium containing 2% FBS) for a 48-hour period. Cells were then washed with 150 μL of PBS per well and incubated with 100 μL of MTT solution (prepared in culture medium at a concentration of 0.5 mg / mL) per well in an incubator for 3 hours. The MTT solution was then removed from the plate, and the resulting formazan crystals were solubilized with 100 μL of dimethyl sulfoxide (DMSO) per well and mixed at 300 rpm in an orbital shaker for 20 minutes. Finally, the absorbance of the samples was measured at a wavelength of 560 nm in a plate reader spectrophotometer (Multiskan Spectrum, Thermo Scientific, Waltham, MA, USA). The absorbance of each sample was used to determine cell viability after each treatment condition.

[0324] The cell cycle consists of DNA replication (S phase), mitosis (M), and cytokinesis, separated by two gaps (G1 and G2). A non-proliferative state known as G0 (or quiescence) can also occur during the G1 phase. These gap phases are important for cell cycle regulation and decisions related to entering the cell cycle in G1 or initiating the process for DNA segregation in G2. In this way, cell cycle progression regulates cell proliferation, and its dysfunction plays a key role in cancer progression, primarily in malignant tumors. Controlling cell cycle progression by inducing cell cycle arrest may be a key feature of cancer therapy.

[0325] We performed cell cycle assays to understand how ASMO APT20TTMG affects the cell cycle of MCF-7 cell line and its possible mechanism of action. First, cells were cultured at 5 × 10 per well in 12-well plates. 4Cells were seeded at a density of 1000 μg / mL and cultured for 24 hours for adhesion. Then, they were treated with three different concentrations of APT20TTMG (0.25, 0.5, and 1 μM) for 48 hours. Afterwards, cells were harvested using trypsin / EDTA solution, washed with 2.0 mL of PBS buffer, and centrifuged at 1500 rpm for 5 minutes. Cells were then fixed with ice-cold 70% ethanol and incubated overnight at 4°C. Afterwards, cells were washed again with 2.0 mL of PBS and incubated with propidium iodide solution (50 μg / mL) and RNAse (200 μg / mL) at room temperature for 1 hour, protected from light. Finally, cells were analyzed in a flow cytometer (BD FACSCanto II, BD Biosciences) by acquiring 10,000 gated events for each sample.

[0326] Although several forms of cell death exist and follow distinct pathways, two distinct processes remain predominant: apoptosis (also known as programmed cell death) and necrosis (unregulated cell death). Apoptosis is characterized by several characteristic morphological changes in the cell's structure, along with several enzyme-dependent biochemical processes. This results in the clearance of the cell from the body with minimal damage to surrounding tissues. Cancer cells often evade apoptosis by expressing proteins that inhibit the apoptotic process or by activating signaling pathways that promote cell survival. Furthermore, cancer cells often exhibit abnormalities in signaling pathways that control the cell cycle and cell proliferation, which can lead to uncontrolled growth and proliferation of cancer cells. While dysregulation of the apoptotic process can contribute to the development and progression of cancer, inducing apoptosis in cancer cells can be an effective strategy for cancer treatment.

[0327] The apoptotic potential of ASMO APT20TTMG was assessed using a method of double-labeling cells with Annexin V and propidium iodide. Annexin V is a peptide with a high affinity for phosphatidylserine, a phospholipid that is asymmetrically distributed in the inner leaflet of the plasma membrane lipid bilayer under physiological conditions. During the process of apoptotic cell death, the asymmetric distribution of phospholipids is lost, and phosphatidylserine translocates to the outer leaflet of the plasma membrane through a process called exosomal translocation. The exposure of phosphatidylserine could be measured using Annexin V conjugated to a fluorescent dye. On the other hand, propidium iodide is a DNA intercalator that can only penetrate the interior of cells if the selective permeability of the plasma membrane is impaired, which apparently occurs during some processes of cell death.

[0328] The pro-apoptotic potential of APT20TTMG was evaluated in MCF-7 cells using apoptosis detection with Annexin V / propidium iodide kit (eBioScience). Cells were first plated at 1 × 10 per well in 24-well plates. 5 The cells were cultured at a density of 1000 x 1000 for 24 hours to allow adhesion. The cells were then treated with three concentrations of APT20TTMG (0.25, 0.5, and 1 μM) for 24 hours and then harvested with 0.05% trypsin solution. After treatment, the cells were washed once with 2 mL of ice-cold PBS and again with the binding buffer provided by the kit. The samples were labeled with Annexin V conjugated to FITC for 15 minutes and washed again with the binding buffer. Finally, the samples were labeled with propidium iodide and analyzed in a flow cytometer (BD FACSCanto II, BD Biosciences) by acquiring 10,000 gated events per sample.

[0329] Mitochondria are essential for cell survival and can contribute to the uncontrolled growth and proliferation of these cells in cancer. Mitochondria in cancer cells have different functions and structures than mitochondria in normal cells. One key difference is that cancer cells have an altered membrane potential compared to healthy cells, which leads to different forms of cellular energy production. The mitochondrial membrane potential represents the difference in charge across the inner mitochondrial membrane and is essential for ATP synthesis by oxidative phosphorylation. Loss of mitochondrial membrane potential is an early event during apoptosis, a common form of tumor cell death in response to treatment.

[0330] The effect of ASMO 1 APT20TTMG on mitochondrial membrane potential in MCF-7 cell lines was assessed using MitoTracker Red dye, a cationic fluorophore that accumulates in the mitochondrial matrix, providing an indication of the viability and integrity of the organelle.

[0331] For this purpose, cells were plated at 1 × 10 per well in a 24-well plate for cell attachment. 5 Cells were cultured overnight at a density of 1000 μM. Then, cells were treated with three concentrations of APT20TTMG (0.25, 0.5, and 1 μM) for 24 hours. After ASMO1 treatment, cells were harvested using 0.05% trypsin solution and washed twice with 2.0 mL of PBS buffer. Finally, the samples were incubated with 500 μL of MitoTracker Red fluorescent dye solution at a concentration of 50 nM in an incubator for 15 minutes. The cells were then washed twice with 2 mL of PBS and subsequently analyzed in a flow cytometer (BD FACSCanto II, BD Biosciences) by acquiring 10,000 gated events per sample.

[0332] Values ​​were expressed as mean ± standard error of the mean. To assess statistical differences between groups, one-way ANOVA test was performed, followed by Dunnett's multiple comparison test. A P value of less than 0.05 was considered to indicate significance. GraphPad Prism 9 software (GraphPad Software Inc., San Diego, CA, USA) was used for statistical analysis.

[0333] As shown in Figure 16, the internalization value of ASMO APT20TTMG was very high, showing 75.3±1.4% in the first 0.5 hours after incubation with MCF-7 cell line. After 1 hour of incubation, the degree of internalization remained constant for the rest of the evaluation period, with an approximate value of approximately 80%.

[0334] As shown in Figure 17, after 48 hours of incubation, cell viability decreased at concentrations of 0.5 and 1 μM, reaching 86.4 ± 1.6% (p = 0.0235) and 85 ± 2.7% (p = 0.0120), respectively. After determining the concentration range that could have an antitumor effect, new tests were performed at concentrations of 0.25, 0.5, and 1 μM. This assessment of cell viability may represent mitochondrial activity, but may also indirectly represent cell proliferation. A reduction in cell number was observed at the two highest concentrations tested, likely due to the presence of ATP.

[0335] As shown in Figure 18, the only statistically significant alteration was in the S phase, i.e., the phase in which DNA synthesis occurs; lower concentrations of cells were present and no cells were retained in this phase after incubation at 0.25 and 0.5 μM.

[0336] As shown in Figure 19, no changes were observed in any of the parameters evaluated, i.e., all treatments showed a similar profile to the negative control (untreated cells) in terms of the percentage of viable cells in immediate / late apoptosis or necrosis.

[0337] As shown in Figure 20, no changes in mitochondrial membrane potential were observed, ie, all treatments showed a profile similar to that of the negative control (untreated cells).

[0338] Upon completion of the study, we found that ASMO1 was efficiently internalized by the carcinoma breast cell line MCF-7. Furthermore, at higher concentrations, we observed a decrease in cell viability, which may be related to a decrease in cell proliferation. Results showed that APT20TTMG reduced the number of cells in the S phase of the cell cycle, although no changes in mitochondrial membrane potential or apoptosis were observed.

[0339] Example 4. Evaluation of the antitumor potential of ASMO1 APT20TTMG in neuroblastoma cell lines (SH-SY5Y and SK-N-SH) Under physiological conditions, the TAU protein is a phosphoprotein that promotes microtubule assembly and stabilization in cells. TAU also plays important roles in chromatin structure, signal transduction, and nucleic acid protection. Indeed, this protein has been described to have extensive interactions, including with cancer-associated kinase proteins, PI3K / AKT (associated with cell survival and proliferation in many cancers), and Rho-ROCK signaling (involved in cell migration and invasive phenotypes). Consequently, despite its well-known role in neurodegenerative diseases, recent studies have recognized that TAU ​​may also be involved in the progression of many cancers, as well as cell migration and invasiveness. Furthermore, as a microtubule-binding protein, TAU may interfere with the binding of taxanes (microtubule-stabilizing drugs) to tubulin, thereby contributing to resistance to taxanes used in cancer treatment. Thus, anti-TAU molecules may also be a strategy for improving the efficacy of taxane-based chemotherapy. Therefore, modulation of TAU expression at both the mRNA and protein levels can be used for cancer chemotherapy.

[0340] Cells were harvested by rinsing once with ice-cold PBS and pelleted by centrifugation at 2,500 g for 5 minutes at 4°C. Each sample was resuspended and lysed in 50 μl of ice-cold radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific, USA) containing EDTA-free Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, USA) 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 15 minutes at 4°C. The supernatant was collected, and total protein concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific, USA) according to the manufacturer's instructions. Fifteen micrograms of total protein in the cell lysate was denatured in NuPAGE LDS sample buffer (Thermo Fisher Scientific, USA) containing NuPAGE sample reducing agent (DTT, Thermo Fisher Scientific, USA) and boiled at 90°C for 5 minutes. Using an XCell SureLock Mini-cell system (Thermo Fisher Scientific, USA), samples were separated on NuPAGE 4-12% Bis-Tris gels (Thermo Fisher Scientific, USA) and electrophoresed at 120V in NuPAGE MES SDS running buffer (Thermo Fisher Scientific, USA). The resolved proteins were transferred to a polyvinylidene difluoride (PVDF) 0.45-μm membrane (Thermo Fisher Scientific, USA) in NuPAGE transfer buffer (Thermo Fisher Scientific, USA) containing 20% ​​methanol at 120V for 75 minutes at 4°C.PVDF membranes were blocked in TBS-T containing 5% (w / v) dry milk powder for 1 hour at room temperature and then incubated overnight at 4°C with whole TAU primary antibody (Cell Signaling Technology, catalog no. 46687 or Abeam, catalog no. ab80579) diluted in TBS-T containing 2% (w / v) BSA. The membranes were then washed in TBS-T and incubated with HRP-conjugated secondary antibody at a dilution of 1:5000 in TBS-T for 1 hour at room temperature. The membranes were washed again with TBS-T, and proteins were visualized with enhanced chemiluminescence (ECL) reagent (Thermo Fisher Scientific, USA). Images were captured using the Jess Simple Western Imaging suite. Densitometry was performed using Image J (v1.48k, NIH), and all quantifications were normalized to vinculin levels.

[0341] At all time points, cells were rinsed once with PBS before RNA extraction using the RNeasy Plus Kit (Qiagen). RNA (0.1–1 μg) was converted to cDNA using the SuperScript IV First-Strand Synthesis System (Thermo Fisher) according to the manufacturer's instructions. After RNA removal, 5–10 ng of cDNA was amplified by qPCR. Taqman probes (Thermo Fisher) were used: total TAU (MAPT, Assay ID: Hs00902193_ml) and GAPDH (GAPDH, Assay ID: Hs99999905_ml). Gene expression was analyzed using the Applied Biosystems QuantStudio™ 12K Flex Real-Time PCR System. Gene expression was quantified using the comparative threshold cycle (2^-ΔΔCT) Livak method, compared to cells treated with Lipofectamine 3000 alone. Gene expression was normalized to the reference gene GAPDH.

[0342] Values ​​were expressed as mean ± standard error of the mean. To evaluate statistical differences between groups, one-way ANOVA test was performed, followed by Dunnett's multiple comparison test. Western blot and qPCR analysis of the final experiment were performed with two-tailed t-tests, and a p value of less than 0.05 was considered statistically significant compared to MOCK (cells treated with Lipofectamine 3000 only). GraphPad Prism 9 software (GraphPad Software Inc., San Diego, CA, USA) was used to generate graphics.

[0343] As shown in Figure 21, internalization of ASMO1 APT20TTMG was observed, representing approximately 50% at all time points evaluated. As shown in Figures 22A-B, no change in cell viability was observed at any of the concentrations and time points evaluated. As shown in Figure 23, no change in cell cycle phase was observed at any of the concentrations evaluated. As shown in Figure 24, an increase in necrotic cells was observed after 24 hours of incubation with ASMO1 APT20TTMG (0.25 μM). As shown in Figure 25, no change in mitochondrial membrane potential was observed, i.e., all treatments showed a profile similar to that of the negative control (untreated cells). As shown in Figure 26, after 0.5 μM APT20TTMG ASMO1, the number of viable cells remained approximately the same across four assays, demonstrating a cytostatic effect. As shown in Figure 27A-B, except for the transient increase observed at 24 hours, the regulatory profile of MAPT expression at the two longest time points was observed, with a decrease of 82±1.2% at 96 hours and 74±4.3% at 144 hours. Similar profiles were observed at the two highest concentrations in protein quantification, with a decrease of 40±13.3% and 46±14.3%.

[0344] ASMO1 is internalized by the neuroblastoma cell line SH-SY5Y. However, no changes in cell viability or proliferation, cell cycle, or mitochondrial membrane potential were observed. Cytotoxicity or cytostatic potential may be demonstrated based on subtle changes in necrotic cells.

[0345] For the SK-N-SH neuroblastoma cell line, APT20TTMG could reduce TAU protein levels and MAPT gene expression in a time-dependent manner, with the number of viable cells remaining roughly the same across four analyses (cytostatic effect).

[0346] Example 5. Evaluation of the antitumor potential of ASMO APT20TTMG in glioblastoma cell line (U87-MG) The methodology in this example is the same as that described in Example 4. Preliminary studies with five different human tumor lines showed antitumor potential after incubation with ASMO1, and three of the most promising lines, including glioblastoma, were further evaluated.

[0347] The in vitro effects of APT20TTMG on the viability and proliferation of the human cancer cell line U87MG (human glioblastoma) were also assessed by MTT and BrdU assays, respectively. Cells were treated with various concentrations of APT20TTMG and the reference in technical triplicates (experiments were performed in three separate sets) for 48, 96, and 144 hours. APT20TTMG and the reference were read every 48 hours.

[0348] Values ​​were expressed as mean ± standard error of the mean. To evaluate statistical differences between groups, one-way ANOVA test was performed, followed by Dunnett's multiple comparison test. Graphics were generated using GraphPad Prism 9 software (GraphPad Software Inc., San Diego, CA, USA).

[0349] As shown in Figure 28, ASMO1 internalization increases at the longest evaluation time. The values ​​obtained were 1 hour (47.45 ± 4.7%), 2 hours (45.45 ± 3.9%), 4 hours (58 ± 4.8%), and 6 hours (63.65 ± 3.2%). Cells were exposed to the fluorescent dye-conjugated compound FAM at a concentration of 1 μM, and the resulting intracellular fluorescence was quantified at 1, 2, 4, and 6 hours using flow cytometry techniques by acquiring 10,000 gated events. Data from two independent experiments performed with two technical replicates.

[0350] As shown in Figure 29, APT20TTMG at a concentration of 0.25 μM significantly altered two cell cycle phases, G1 / G0 and G2 / M. First, we observed a decrease in retention (p=0.0036) and an increase in retention (p=0.0425) in G2 / M, suggesting that ASMO1 prevented cell proliferation from this cell line. Cells were seeded and treated with three different concentrations of APT20TTMG (0.25, 0.5, and 1 μM), and the cell cycle phase was assessed after 48 hours of incubation. Cell proliferation is regulated by cell cycle progression. Phases: Sub / G1: non-proliferative state (quiescence), G1 / G0: cell growth, S: DNA replication, G2 / M: DNA segregation and mitosis. Results are expressed as the mean ± standard error of three independent experiments.

[0351] As shown in Figure 30, apoptosis assays after incubation with APT20TTMG revealed that the two highest concentrations reduced cell viability, significantly increasing late apoptosis, while the 0.25 μM concentration only showed a trend toward this state. In addition to the results obtained on the cell cycle and apoptosis, ASMO1 also altered mitochondrial membrane potential and reduced labeling at concentrations of 0.25 and 0.5 μM, as shown in Figure 31. Cells were seeded and treated with three different concentrations of APT20TTMG (0.25, 0.5, and 1 μM), and cell cycle phases were assessed after 48 hours of incubation. Cell proliferation is regulated by cell cycle progression: Sub / G1: non-proliferative state (quiescence), G1 / G0: cell growth, S: DNA replication, G2 / M: DNA segregation and mitosis. Results are expressed as the mean ± SEM of three independent experiments.

[0352] As shown in Figures 32-33, an MTT assay (Figure 32) was also performed using five different concentrations of APT20TTMG (0.003, 0.03, 0.3, 3, and 30 μM) at 48, 96, and 144 h of incubation. After 48 h of incubation, only the 30 μM concentration of APT20TTMG was observed to reduce cell viability. However, after 96 and 144 h of incubation, the three highest concentrations of APT20TTMG (0.3, 3, and 30 μM) also exhibited this ability, suggesting possible cytotoxicity and / or cytostatic potential. Finally, to confirm whether APT20TTMG is a cytotoxic or cytostatic molecule, given its ability to reduce cell viability at certain concentrations, a cell proliferation assay (Figure 33) was also performed using the U87-MG cell line under the same conditions as the MTT assay. At the lowest concentrations of APT20TTMG (0.003 and 0.03 μM), we observed a statistically significant decrease in proliferation over the final 144 hours of the assay, despite an increase in cell proliferation over the first 48 hours of incubation at the 0.03 μM concentration. This decrease in profile, without a change in cell viability, indicates that APT20TTMG is cytostatic at its lowest concentrations (0.003 and 0.03 μM). At the highest concentrations of APT20TTMG (0.3, 3, and 30 μM), in addition to a transient increase in cell proliferation over the first 48 hours of the assay, we observed a significant decrease in cell proliferation by 144 hours of incubation. At the highest concentrations of APT20TTMG (0.3, 3, and 30 μM), in addition to observing a transient increase in cell proliferation during the first 48 hours of the assay (which may be related to a beneficial increase in astrocyte numbers in the astrogliosis activation pathway), we observed a significant decrease in cell proliferation at 144 hours of incubation. This data (Figure 33), along with the MTT results (Figure 32), indicates that APT20TTMG is cytotoxic at the highest tested concentration. Cells were seeded and treated with five different concentrations (0.003, 0.03, 0.33, and 30 μM). BrdU assays were performed after 48, 96, or 144 hours of incubation.Data are plotted as the percentage (%) of cell viability compared to the negative control (vehicle). Results are expressed as the mean ± SEM of three independent experiments performed with three technical replicates. Statistical analysis was performed using one-way ANOVA followed by Dunnett's multiple comparison test. *p<0.05, **p<0.01, ***p<0.001, ****p<0...

Claims

1. 1. A method of treating cancer 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 a target sequence in (ii) a recruitment moiety configured to recruit a spliceosome moiety; The method wherein, upon association with the pre-mRNA and the engineered polynucleotide, the spliceosome portion alters the pre-mRNA at or near the target sequence.

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

3. 3. The method of claim 2, wherein the prognosis of the subject is improved compared to a subject receiving the taxane but not the engineered polynucleotide.

4. 3. The method of claim 2, wherein the total level of tau in the subject is reduced, thereby reducing the amount of tau that binds to the taxane.

5. 1. A method of improving the prognosis of a subject suffering from cancer and who has been administered a taxane, 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 a target sequence in (ii) a recruitment moiety configured to recruit a spliceosome moiety; The method wherein, upon association with the pre-mRNA and the engineered polynucleotide, the spliceosome portion alters the pre-mRNA at or near the target sequence.

6. 6. The method of claim 5, wherein the total level of tau in the subject is reduced, thereby reducing the amount of tau bound to the taxane in the subject.

7. The method of any one of claims 1 to 6, wherein the cancer is selected from the group consisting of brain cancer, prostate cancer, breast cancer, renal cancer, kidney cancer, lung cancer, and liver cancer.

8. 7. The method of any one of claims 1 to 6, wherein the cancer is selected from the group consisting of glioblastoma, neuroblastoma, hepatocellular carcinoma, lung carcinoma, breast adenocarcinoma, human prostate cancer, renal cell carcinoma, and renal adenocarcinoma.

9. The method of any one of claims 1 to 6, wherein the cancer is glioblastoma.

10. The method according to any one of claims 1 to 9, wherein the tumor volume ratio is reduced.

11. The method of any one of claims 1 to 10, wherein the method reduces tumor progression.

12. The method of any one of claims 1 to 11, wherein the expression of tau is altered.

13. The method according to any one of claims 1 to 12, wherein the expression of TAU is reduced.

14. The method of any one of claims 1 to 13, wherein the total amount of TAU in the subject is reduced.

15. The method of any one of claims 1 to 14, wherein the expression of AKT is reduced.

16. The method of any one of claims 1 to 15, wherein the expression of glial fibrillary acidic protein (GFAP) is reduced.

17. The method of any one of claims 1 to 16, wherein the engineered polynucleotide is administered intratumorally.

18. 17. The method of any one of claims 1 to 16, wherein the engineered polynucleotide is administered intravenously.

19. 17. The method of any one of claims 1 to 16, wherein the engineered polynucleotide is administered intrathecally.

20. 17. The method of any one of claims 1 to 16, 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.

21. The method of any one of claims 1 to 20, wherein premature polyadenylation of one or more transcripts of said subject is reduced.

22. 22. The method of any one of claims 1 to 21, wherein cryptic splicing of one or more transcripts of said subject is reduced.

23. The method of any one of claims 1 to 22, which improves a score associated with a histopathological finding.

24. 24. The method of claim 23, wherein the histopathological findings include tumor grade, lipid content, necrosis, or nuclear to cytoplasmic (N:C) ratio.

25. 1. A method of reducing the viability of a cell, comprising administering to the cell an engineered polynucleotide, wherein the engineered polynucleotide: (i) one or more targeting moieties configured to specifically bind to pre-messenger ribonucleic acid (pre-mRNA) at a target sequence in (ii) a recruitment moiety configured to recruit a spliceosome moiety; The method wherein, upon association with the pre-mRNA and the engineered polynucleotide, the spliceosome portion alters the pre-mRNA at or near the target sequence.

26. 1. A method of reducing the proliferation rate of a cell, comprising administering to the cell an engineered polynucleotide, wherein the engineered polynucleotide: (i) one or more targeting moieties configured to specifically bind to pre-messenger ribonucleic acid (pre-mRNA) at the target, which may be a conserved splice site sequence present in (ii) a recruitment moiety configured to recruit a spliceosome moiety; The method wherein, upon association with the pre-mRNA and the engineered polynucleotide, the spliceosome portion alters the pre-mRNA at or near a target sequence.

27. The method according to any one of claims 25 to 26, wherein cell necrosis or cell apoptosis is increased.

28. The method of any one of claims 25 to 27, wherein the method increases the tendency of the cells to be in G2 / M phase.

29. The method of any one of claims 25 to 28, wherein the cells are tumor cells.

30. 30. The method of claim 29, wherein the tumor cells comprise a glioma, a neuroblastoma, or a carcinoma.

31. 1. A method of altering cell cycle phase distribution in a plurality of cells, comprising administering to said plurality of cells 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 a target sequence in (ii) a recruitment moiety configured to recruit a spliceosome moiety; The method wherein, upon association with the pre-mRNA and the engineered polynucleotide, the spliceosome portion alters the pre-mRNA at or near the target sequence.

32. 32. The method of claim 31, wherein the number of cells in the G2 / M phase is increased.

33. 32. The method of claim 31, wherein the number of cells in a necrotic or apoptotic stage is increased.

34. 34. The method of any one of claims 31 to 33, wherein the plurality of cells comprises tumor cells.

35. 35. The method of claim 34, wherein the tumor cells comprise a glioma, a neuroblastoma, or a carcinoma.

36. 1. A method of reducing tau expression by a neuron, comprising administering to said neuron an engineered polynucleotide, wherein said engineered polynucleotide: (ii) one or more targeting moieties configured to specifically bind to pre-messenger ribonucleic acid (pre-mRNA) at a target sequence in the (ii) a recruitment moiety configured to recruit a spliceosome moiety; The method wherein, upon association with the pre-mRNA and the engineered polynucleotide, the spliceosome portion alters the pre-mRNA at or near the target sequence.

37. 37. The method of claim 36, wherein the neuron is derived from an individual suffering from cancer.

38. 38. The method of claim 36 or 37, wherein the method reduces cryptic splicing of one or more transcripts in the neuron.

39. A method according to any one of claims 1 to 38, wherein the formation of the U1 snRNP complex is modulated.

40. 40. The method of any one of claims 1 to 39, wherein the engineered polynucleotide is administered at a concentration of at least 0.25 μM.

41. 40. The method of any one of claims 1 to 39, wherein the engineered polynucleotide is administered at a concentration of at least 0.5 μM.

42. 40. The method of any one of claims 1 to 39, wherein the engineered polynucleotide is administered at a concentration of about 0.25 μM to about 1 μM.

43. 43. The method of any one of claims 1 to 42, wherein a targeting moiety of the one or more targeting moieties is sufficiently identical to or complementary to a consensus sequence in the target sequence of a target gene.

44. 44. The method of any one of claims 1 to 43, wherein the targeting moiety is complementary to and / or hybridizes with said target sequence.

45. 45. The method of any one of claims 1 to 44, wherein the targeting moiety is complementary to and / or hybridizes with a consensus sequence of the target sequence.

46. 46. ​​The method of any one of claims 1 to 45, wherein the target sequence comprises a splice site.

47. 47. The method of claim 46, wherein the splice site is a conserved splice site.

48. 48. The method of claim 47, wherein the splice site comprises 5'-GU-3'.

49. The method of any one of claims 43 to 48, wherein the pre-mRNA is encoded by the target gene.

50. 50. The method of claim 49, wherein the expression or activity of the target gene is altered.

51. 53. The method of any one of claims 1-52, 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.

52. 52. The method of claim 51 , wherein the first targeting sequence comprises a consensus sequence in the target sequence.

53. 53. The method of claim 51 or 52, wherein the second targeting sequence comprises a consensus sequence in the target sequence.

54. 54. The method of any one of claims 51 to 53, 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).

55. 55. The method of any one of claims 1 to 54, wherein the target sequence comprises an exon-intron boundary in the pre-mRNA.

56. 56. The method of claim 55, wherein the first targeting sequence and the second targeting sequence are both 5' or 3' to the exon-intron boundary.

57. 56. The method of claim 55, 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.

58. 58. The method of any one of claims 1 to 57, wherein the target sequence comprises a splice site in the pre-mRNA.

59. 59. The method of claim 58, wherein the first targeting sequence or the second targeting sequence comprises a splice site (e.g., 5'ss) in the pre-mRNA.

60. 60. The method of any one of claims 1-59, 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.

61. 61. The method of claim 60, wherein the first targeting moiety or the second targeting moiety comprises a sequence identical to or complementary to a sequence shown in Table 1.

62. 61. The method of Claim 60, wherein said first targeting moiety comprises a sequence identical or complementary to a sequence selected from the exon sequence column of Table 1, and said second targeting moiety comprises a sequence identical or complementary to a sequence shown in the intron sequence column of Table 1.

63. 61. The method of Claim 60, wherein said first targeting moiety comprises a sequence identical or complementary to a sequence set forth in the intron sequence column of Table 1, and said second targeting moiety comprises a sequence identical or complementary to a sequence set forth in the exon sequence column of Table 1.

64. 61. The method of claim 60, 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).

65. 61. The method of Claim 60, 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).

66. 61. The method of claim 60, 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.

67. 67. The method of any one of claims 1 to 66, 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, e.g., U1 snRNA.

68. 68. The method of any one of claims 1 to 67, 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, such as U1 snRNA.

69. 69. The method of any one of claims 67 or 68, 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.

70. 70. The method of any one of claims 1 to 69, 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.

71. 68. The method of claim 67, wherein the spliceosome portion comprises U1 snRNA and spliceosome proteins.

72. 72. The method of any one of claims 67 to 71, wherein the spliceosomal snRNA is selected from U1, U2, U4, U5, U6, U11, U12, U14atac, U6atac, and combinations thereof.

73. 73. The method of any one of claims 67 to 72, wherein the spliceosome protein is selected from Sm, U1-70k, U1A, U1C, and combinations thereof.

74. 74. The method of any one of claims 1 to 73, 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 or 2.

75. 75. The method of claim 74, wherein the recruitment moiety comprises a nucleotide sequence identical to or complementary to any one of SEQ ID NOs: 1 or 2.

76. 76. The method of any one of claims 1 to 75, wherein the engineered polynucleotide comprises a secondary structural feature.

77. 77. The method of any one of claims 1-76, wherein said engineered polynucleotide comprises an apical loop, an upper stem, an internal loop, a lower stem, or a combination thereof.

78. 78. The method of any one of claims 1-77, 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.

79. 79. The method of claim 78, wherein the stem sequence of the stem (e.g., the lower stem or the upper stem) comprises no more than about 5, 4, or 3 nucleotides.

80. 79. The method of claim 77 or 78, 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.

81. 81. The method of claim 80, wherein the internal loop comprises a nucleic acid sequence of 10, 9, or 8 nucleotides or less.

82. 82. The method of claim 80 or 81, wherein the stem sequence of the additional stem (e.g., the upper stem) comprises no more than about 5, 4, or 3 nucleotides.

83. 83. The method of any one of claims 80-82, wherein the engineered polynucleotide further comprises an apical loop.

84. 84. The method of Claim 83, wherein the apical loop comprises a nucleic acid sequence of no more than 10, 9, 8, 7, 6, or 5 nucleotides.

85. 85. The method of any one of claims 1 to 84, wherein the engineered polynucleotide does not contain any intramolecular disulfide bonds.

86. 86. The method of any one of claims 1-85, wherein the pre-mRNA, when associated with the engineered polynucleotide and the spliceosome moiety, exhibits substantially no base pairing with the RNA binding domain (RBD) of U1 snRNA.

87. 87. The method of any one of claims 1 to 86, wherein the pre-mRNA, when associated with the engineered polynucleotide and the spliceosome portion, exhibits substantially no base-specific interactions with U1-C protein.

88. 88. The method of any one of claims 1 to 87, wherein the engineered polynucleotide is configured to specifically interact with a zinc finger of a U1-C protein.

89. 89. The method of claim 88, wherein the 5'-targeting portion of the engineered polynucleotide is configured to specifically interact with a zinc finger of a U1-C protein.

90. 90. The method of claim 88 or 89, 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).

91. 91. The method of any one of claims 88-90, 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.

92. 92. The method of any one of claims 88-91, wherein the aid recruiting moiety comprises a nucleotide sequence comprising a phosphorothioate internucleotide linkage that binds to the U1-C zinc finger.

93. 93. The method of any one of claims 1 to 92, wherein the engineered polynucleotide comprises a nucleotide sequence complementary to a subsequence of stem loop II (SU2) of U1 snRNA.

94. 94. The method of Claim 93, 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 (SU2) of U1 snRNA.

95. 95. The method of claim 93 or 94, wherein the partial sequence comprises a sequence corresponding to 5'-GGCCU-3' of SL2 of U1 snRNA.

96. The method of any one of claims 93 to 95, wherein the partial sequence does not include a sequence corresponding to 5'-CACGUUA-3' of SL2 of U1 snRNA.

97. 97. The method of any one of claims 1 to 96, wherein the recruitment moiety is complementary to the stem-loop II region of snRNA, such as U1 snRNA.

98. 98. The method of any one of claims 1 to 97, wherein the recruitment moiety hybridizes with the stem-loop II region of snRNA, such as U1 snRNA.

99. 99. The method of any one of claims 1-98, wherein the recruitment moiety comprises AGGCC.

100. 100. The method of any one of claims 1-99, 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.

101. 101. The method of any one of claims 1-100, 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 shown in Tables 2-3.

102. 102. The method of any one of claims 1 to 101, wherein the recruitment nucleotide sequence comprises (i) a nucleotide sequence complementary to at least 4 nucleotides of stem loop II (SU2) of U1 snRNA.

103. 103. The method of any one of claims 1 to 102, wherein the engineered polynucleotide exhibits substantially no base pairing with the anchor sequence of SL2 of U1 snRNA.

104. 104. The method of Claim 103, wherein the internal loop of the engineered polynucleotide exhibits substantially no base pairing with the anchor sequence of the SL2 of U1 snRNA.

105. 105. The method of claim 103 or 104, wherein the lower stem of the engineered polynucleotide exhibits substantially no base pairing with the anchor sequence of the SL2 of U1 snRNA.

106. 106. The method of any one of claims 103 to 105, wherein the anchor sequence comprises a sequence corresponding to 5'-CACGUUA-3'.

107. 107. The method of any one of claims 1 to 106, wherein the engineered polynucleotide exhibits substantially no base pairing with the H helix of U1 snRNA.

108. 108. The method of any one of claims 1 to 107, wherein the engineered polynucleotide comprises at least one chemical modification.

109. 109. The method of Claim 108, wherein at least about 50%, 60%, 70%, 80%, or 90% of the nucleotides of said engineered polynucleotide are chemically modified nucleotides.

110. 110. The method of claim 108 or 109, wherein the engineered polynucleotide comprises at least one 2'-modified nucleotide.

111. 111. The method of claim 110, wherein at least about 50%, 60%, 70%, 80%, or 90% of the nucleotides of said engineered polynucleotide are 2'-modified nucleotides.

112. 112. The method of any one of claims 109 to 111, wherein the 2'-modified nucleotides comprise 2'-methoxy, 2'-methoxymethyl, 2'-methoxyethyl, 2'-fluoro, or 2'-aminoethyl nucleotides.

113. 113. The method of any one of Claims 108-112, wherein said engineered polynucleotide comprises nucleotides joined by internucleotide linkages, wherein at least one of said internucleotide linkages does not comprise a phosphate.

114. 114. The engineered polynucleotide of any one of Claims 108-113, wherein said engineered polynucleotide comprises nucleotides connected by internucleotide linkages, wherein at least one of said 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).

115. 115. The method of any one of claims 108 to 114, wherein the engineered polynucleotide comprises at least one phosphorothioate internucleotide linkage.

116. 113. The method of any one of claims 108-112, wherein at least about 50%, 60%, 70%, 80%, or 90% of the internucleotide linkages of the engineered polynucleotide are chemically modified.

117. 117. The method of any one of claims 108-116, wherein at least about 50%, 60%, 70%, 80%, or 90% of the internucleotide linkages are phosphorathioate.

118. 118. The method of any one of claims 108-117, 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.

119. 119. The method of any one of Claims 1-118, 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.

120. 120. The method of any one of claims 1-119, wherein the recruitment moiety comprises from about 10 to about 30 nucleotides, or from about 10 to about 20 nucleotides.

121. 121. The method of any one of claims 1-120, 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.

122. 122. The method of any one of claims 1-121, 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.

123. 123. The method of any one of claims 1-122, wherein the spliceosome portion, upon association with the engineered polynucleotide and the pre-RNA, cleaves or splices the pre-mRNA in the target sequence.

124. 124. The method of any one of claims 1 to 123, wherein the spliceosome portion further promotes modification of the cleaved pre-mRNA.

125. 125. The method of any one of claims 1 to 124, wherein the engineered polynucleotide comprises a nucleotide sequence at least 70%, 80%, 85%, or 90% identical to or complementary to any one of SEQ ID NOs: 1-4.

126. 126. The method of any one of claims 1 to 125, wherein the engineered polynucleotide comprises a nucleotide sequence identical to or complementary to any one of SEQ ID NOs: 3 or 4.

127. the engineered polynucleotide is a first targeting moiety configured to specifically bind to pre-messenger ribonucleic acid (pre-mRNA) at a first targeting sequence in (i), 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; and a second targeting moiety configured to specifically bind to the pre-mRNA at a second targeting sequence in (iii), wherein the second targeting moiety comprises a sequence identical or complementary to 5'-CG-3'.

128. 1. A method of treating a subject having cancer, comprising administering to the subject a pharmaceutical composition comprising an engineered polynucleotide having the nucleotide sequence of SEQ ID NO:

3.

129. 129. The method of Claim 128, wherein all internucleotide linkages of said engineered polynucleotide comprise phosphorothioate linkages.

130. 130. The method of any one of claims 128-129, wherein the nucleotides at positions 1-5 and 20-24 of the engineered polynucleotide comprise 2'-O-methyl moieties.

131. 131. The method of any one of claims 128 to 130, further comprising administering a taxane.

132. A method of treating a subject having cancer, wherein the subject is being administered a taxane, the method comprising the step of (i) administering to the subject a pharmaceutical composition comprising an engineered polynucleotide having the nucleotide sequence of SEQ ID NO: 3, wherein the internucleotide linkages of the engineered polynucleotide comprise phosphorothioate linkages and the nucleotides at positions 1 to 5 and 20 to 24 of the engineered polynucleotide comprise 2'-O-methyl moieties, and wherein the administration reduces TAU protein levels and TAU binding to taxanes in the subject.

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