Polynucleotide compositions and methods for treating cancer

Abstract

The present disclosure provides engineered polynucleotides that interact with mRNA precursors and spliceosomes to modulate gene expression. The engineered polynucleotides may have a stem-loop structure with recruitment spliceosomes and a targeting sequence complementary to the target sequence at the exon-intron splice point, and may include nucleotides with 2'modifications and thiophosphate linkages The engineered polynucleotides may be administered to a subject to treat cancer.

Description

[0001] Cross-referencing

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 480,466, filed January 18, 2023, the entire contents of which are incorporated herein by reference. Background Technology

[0003] Aberrant splicing is associated with many disease states. Proper splicing of precursor messenger RNA (mRNA) is crucial for correct protein translation. Splicing efficiency must be modulated to ensure proper splicing of mRNA precursors and appropriate premature polyadenylation inhibition, thereby avoiding the production of misfolded and potentially pathogenic proteins. Gene regulation efficiency 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

[0004] In some respects, this document describes a method for treating cancer in a subject of need, the method comprising administering to the subject a pharmaceutical composition comprising an engineered polynucleotide, the polynucleotide comprising: (i) one or more targeting portions configured to specifically bind the mRNA precursor at a target sequence in a messenger ribonucleic acid precursor (mRNA precursor); and (ii) a recruitment portion configured to recruit a spliceosome portion, wherein, upon binding with the mRNA precursor and the engineered polynucleotide, the spliceosome portion alters the mRNA precursor in or near the target sequence.

[0005] In some aspects, this document describes a method for treating cancer in a subject of need, the method comprising: administering a pharmaceutical composition to the subject, the pharmaceutical composition comprising (a) a taxane and (b) an engineered polynucleotide comprising: (i) one or more targeting portions configured to specifically bind the mRNA precursor at a target sequence in a messenger ribonucleic acid precursor (mRNA precursor); and (ii) a recruitment portion configured to recruit a spliceosome portion, wherein, upon binding with the mRNA precursor and the engineered polynucleotide, the spliceosome portion alters the mRNA precursor at or near the target sequence. In some embodiments, the method improves the prognosis of the subject compared to a subject given a 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.

[0006] In some aspects, this document describes a method for improving the prognosis of cancer patients who have been treated with taxanes, wherein the method comprises: administering to the subject a pharmaceutical composition comprising an engineered polynucleotide, the engineered polynucleotide comprising: (i) one or more targeting portions configured to specifically bind the mRNA precursor at a target sequence in a messenger ribonucleic acid precursor (mRNA precursor); and (ii) a recruitment portion configured to recruit a spliceosome portion, wherein, upon binding with the mRNA precursor and the engineered polynucleotide, the spliceosome portion alters the mRNA precursor 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.

[0007] In some embodiments, the cancer is selected from: brain cancer, prostate cancer, breast cancer, renal cancer, lung cancer, and liver cancer. In some embodiments, the cancer is selected from: glioblastoma (GBM), neuroblastoma, hepatocellular carcinoma, breast adenocarcinoma, human prostate adenocarcinoma, renal cell carcinoma, and renal adenocarcinoma. In some embodiments, the cancer is GBM.

[0008] In some embodiments, the methods disclosed herein reduce premature polyadenylation of one or more transcripts of the subject. In some embodiments, the methods disclosed herein reduce covert splicing of one or more transcripts of the subject. 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 nucleocytoplasmic ratio (N:C).

[0009] In some embodiments, the method reduces the tumor volume ratio. In some embodiments, the method slows 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 the 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 intrathecally. In some embodiments, the engineered polynucleotide is administered via subcutaneous injection, intramuscular injection, intradermal injection, percutaneous administration, intranasal administration, intralymphatic injection, intrathecal administration, pulmonary administration, rectal administration, gastric administration, or any other suitable parenteral administration.

[0010] In some respects, this document describes a method for reducing cell viability, the method comprising: administering an engineered polynucleotide to cells, the engineered polynucleotide comprising: (i) one or more targeting portions configured to specifically bind a messenger RNA precursor; and (ii) a recruitment portion configured to recruit a splice portion, wherein, when bound to the mRNA precursor and the engineered polynucleotide, the splice portion alters the mRNA precursor at or near the target sequence; (i) one or more targeting portions configured to specifically bind the mRNA precursor at a target sequence in the messenger RNA precursor (mRNA precursor); and (ii) a recruitment portion configured to recruit a splice portion, wherein, when bound to the mRNA precursor and the engineered polynucleotide, the splice portion alters the mRNA precursor at or near the target sequence.

[0011] In some respects, this document describes a method for reducing cell proliferation, the method comprising: administering an engineered polynucleotide to cells, the engineered polynucleotide comprising: (i) one or more targeting portions configured to specifically bind the mRNA precursor at a target sequence in a messenger ribonucleic acid precursor (mRNA precursor); and (ii) a recruitment portion configured to recruit a spliceosome portion, wherein, upon binding to the mRNA precursor and the engineered polynucleotide, the spliceosome portion alters the mRNA precursor in or near the target sequence; (i) one or more targeting portions configured to specifically bind the mRNA precursor at a target sequence in a messenger ribonucleic acid precursor (mRNA precursor); and (ii) a recruitment portion configured to recruit a spliceosome portion, wherein, upon binding to the mRNA precursor and the engineered polynucleotide, the spliceosome portion alters the mRNA precursor in or near the target sequence.

[0012] In some embodiments, the method increases cell necrosis or apoptosis. In some embodiments, the method increases the tendency of the cells to be in the G2 / M phase. In some embodiments, the cells are tumor cells. In some embodiments, the tumor cells are gliomas, neuroblastomas, or carcinomas.

[0013] In some respects, this document describes a method for altering the cell cycle phase distribution of a plurality of cells, the method comprising: administering an engineered polynucleotide to the plurality of cells, the engineered polynucleotide comprising: (i) one or more targeting portions configured to specifically bind the mRNA precursor at a target sequence in a messenger ribonucleic acid precursor (mRNA precursor); and (ii) a recruitment portion configured to recruit a spliceosome portion, wherein, upon binding to the mRNA precursor and the engineered polynucleotide, the spliceosome portion alters the mRNA precursor in or near the target sequence; (i) one or more targeting portions configured to specifically bind the mRNA precursor at a target sequence in a messenger ribonucleic acid precursor (mRNA precursor); and (ii) a recruitment portion configured to recruit a spliceosome portion, wherein, upon binding to the mRNA precursor and the engineered polynucleotide, the spliceosome portion alters the mRNA precursor in or near the target sequence.

[0014] In some embodiments, the method increases the number of cells in the G2 / M phase. In some embodiments, the method increases the number of cells in the necrosis or apoptosis phase. In some embodiments, the plurality of cells includes tumor cells. In some embodiments, the tumor cells include glioma, neuroblastoma, or carcinoma.

[0015] In some aspects, this document describes a method for reducing tau expression in neurons, the method comprising: administering an engineered polynucleotide to the neuron, the engineered polynucleotide comprising: (i) one or more targeting portions configured to specifically bind the mRNA precursor at a target sequence in a messenger ribonucleic acid precursor (mRNA precursor); and (ii) a recruitment portion configured to recruit a spliceosome portion, wherein, upon binding to the mRNA precursor and the engineered polynucleotide, the spliceosome portion alters the mRNA precursor in or near the target sequence. In some embodiments, the neuron is derived from an individual with cancer. In some embodiments, the method reduces covert splicing of one or more transcripts of the neuron.

[0016] In some respects, this document describes a method for treating a subject suffering from cancer, the method comprising administering a pharmaceutical composition to the subject, the pharmaceutical composition comprising an engineered polynucleotide having the nucleotide sequence of SEQ ID NO:3, wherein all internucleotide bonds of the engineered polynucleotide comprise phosphate thioester bonds, and wherein the nucleotides at positions 1-5 and 20-24 of the engineered polynucleotide comprise a 2'-O-methyl moiety, thereby modulating the formation of the U1 snRNP complex.

[0017] In some aspects, this document describes a method for treating a subject suffering from cancer, the method comprising administering a pharmaceutical composition to the subject, the pharmaceutical composition comprising an engineered polynucleotide having the nucleotide sequence of SEQ ID NO:3. In some embodiments, the internucleotide bonds of the engineered polynucleotide comprise phosphate thioester bonds, and in some embodiments, the nucleotides at positions 1-5 and 20-24 of the engineered polynucleotide comprise a 2'-O-methyl moiety. In some embodiments, the method further comprises administering a taxane drug to the subject.

[0018] In some respects, this document describes a method for treating a subject suffering from cancer, wherein the subject has been administered a taxane drug, the method comprising administering a pharmaceutical composition to the subject, the pharmaceutical composition comprising (i) an engineered polynucleotide having the nucleotide sequence of SEQ ID NO:3, wherein the internucleotide bonds of the engineered polynucleotide comprise phosphate thioester bonds, and wherein the nucleotides at positions 1-5 and 20-24 of the engineered polynucleotide comprise a 2'-O-methyl moiety, thereby reducing the level of tau protein in the subject and the binding of tau to the taxane drug.

[0019] In some embodiments, the methods disclosed herein regulate the formation of the U1 snRNP complex.

[0020] In some aspects, this document describes engineered polynucleotides used in the methods disclosed throughout this disclosure. In some embodiments, the engineered polynucleotide comprises at least 70%, 80%, 85%, or 90% identical or complementary nucleotide sequences to any one of SEQ ID NO:1-4. In some embodiments, the engineered polynucleotide comprises identical or complementary nucleotide sequences to any one of SEQ ID NO:3 or 4.

[0021] In some embodiments, the engineered polynucleotide comprises: one or more targeting portions configured to specifically bind the mRNA precursor at a target sequence in a messenger ribonucleic acid precursor (mRNA precursor); and a recruitment portion configured to recruit a post-transcriptional regulatory portion (e.g., a spliceosome portion), wherein, upon binding to the mRNA precursor and the engineered polynucleotide, the post-transcriptional regulatory portion alters the mRNA precursor in or near the target sequence. In some embodiments, the targeting portion of the one or more targeting portions is sufficiently identical or complementary to a common sequence in the target sequence of the target gene. In some embodiments, the targeting portion is complementary to and / or hybridizes with the target sequence. In some embodiments, the targeting portion is complementary to and / or hybridizes with a common sequence of the target sequence. In some embodiments, the target sequence contains a splice site. In some embodiments, the splice site is a conserved splice site. In some embodiments, the splice site contains a 5'-GU-3'. In some embodiments, the mRNA precursor is encoded by the target gene. In some embodiments, the method alters the expression or activity of the target gene.

[0022] In some embodiments, the one or more targeting portions comprise: a first targeting portion configured to specifically bind a first target sequence in the target sequence of the mRNA precursor; and a second targeting portion configured to specifically bind a second target sequence in the target sequence of the mRNA precursor. In some embodiments, the first target sequence comprises a common sequence in the target sequence. In some embodiments, the second target sequence comprises a common sequence in the target sequence. In some embodiments, the first and second target sequences are spacer sequences separated by no more than five nucleotides (e.g., one or two nucleotides) in the target sequence. In some embodiments, the target sequence comprises an exon-intron boundary in the mRNA precursor. In some embodiments, both the first and second target sequences are located at the 5' or 3' of the exon-intron boundary. In some embodiments, one of the first and second target sequences is located at the 5' of the exon-intron boundary; and the other of the first and second target sequences is located at the 3' of the exon-intron boundary. In some embodiments, the target sequence comprises a splicing site in the mRNA precursor. In some embodiments, the first or second target sequence contains a splicing site (e.g., 5'ss) in the mRNA precursor. In some embodiments, one of the first and second target portions is located at the 5' of the recruitment portion, and the other of the first and second target portions is located at the 3' of the recruitment portion. In some embodiments, the target portion contains at least 80%, 90%, or the same sequence as the ribosome binding site of the spliceosome snRNA (e.g., U1 snRNA). In some embodiments, the second target portion contains at least 80%, 90%, or the same sequence as the ribosome binding site of the spliceosome snRNA (e.g., U1 snRNA). The sequence that is at least 80%, 90%, or the same as the ribosome binding site can be from about 2 nucleotides to about 10 nucleotides. In some embodiments, the recruitment nucleotide sequence comprises: (i) a nucleotide sequence that is complementary to at least four nucleotides of the stem-loop II (SL2) of the U1 snRNA.

[0023] In some embodiments, the first or second targeting portion comprises a sequence that is identical or complementary to the sequence shown in Table 1. In some embodiments, the first targeting portion comprises a sequence that is identical or complementary to the sequence selected from the 5'-targeting portion sequence column of Table 1; and wherein the second targeting portion comprises a sequence that is identical or complementary to the sequence shown in the 3'-targeting portion sequence column of Table 1. In some embodiments, the first targeting portion comprises a sequence that is identical or complementary to the sequence shown in the 3'-targeting portion sequence column of Table 1; and wherein the second targeting portion comprises a sequence that is identical or complementary to the sequence shown in the 5'-targeting portion sequence column of Table 1. In some embodiments, the first or second targeting portion comprises a sequence that is identical or complementary to a common sequence of the intron donor site (e.g., selected from GU, GT, GC, and CA). In some embodiments, the first or second targeting portion comprises a sequence that is identical or complementary to a common sequence of the exon donor site (e.g., G). In some embodiments, the first or second targeting portion comprises a sequence that is identical or complementary to a common sequence selected from GU, GC, G, and CA. In some embodiments, the spliceosome moiety is selected from the spliceosome ribonucleoprotein complex, spliceosome small nucleoribonucleic acid (snRNA), spliceosome protein, functional variants thereof, or functional fragments thereof. In some embodiments, the spliceosome moiety comprises U1 snRNA and spliceosome protein. In some embodiments, the spliceosome snRNA is selected from U1, U2, U4, U5, U6, U11, U12, U14atac, U6atac, and combinations thereof. In some embodiments, the spliceosome protein is selected from Sm, U1-70k, U1A, U1C, and combinations thereof. In some embodiments, the recruitment moiety comprises a nucleotide sequence that is at least 70%, 80%, 85%, or 90% identical or complementary to the sequences shown in Tables 2-3. In some embodiments, the recruitment moiety is complementary to the stem-loop-II region of the snRNA (e.g., U1 snRNA). In some embodiments, the recruitment moiety hybridizes to the stem-loop-II region of the snRNA (e.g., U1 snRNA). In some embodiments, the recruitment portion comprises AGGCC. In some embodiments, the recruitment portion comprises at least 80%, 90%, or the same nucleotide sequence as at least five consecutive nucleotides shown in Tables 2-3. In some embodiments, the recruitment portion comprises at least 80%, 90%, or the same nucleotide sequence as about 5 to about 10 consecutive nucleotides shown in Tables 2-3. In some embodiments, the recruitment portion comprises the same or complementary nucleotide sequence as the sequence shown in Tables 2-3. In some embodiments, the engineered polynucleotide comprises (e.g., secondary) structural features. In some embodiments, the engineered polynucleotide comprises a top loop, an upper stem, an inner loop, a lower stem, or a combination thereof.In some embodiments, the engineered polynucleotide comprises a loop (e.g., an inner loop) adjacent to a stem (e.g., a lower stem or an upper stem), the stem comprising two complementary stem sequences. In some embodiments, the stem sequence of the stem (e.g., the lower stem or the upper stem) comprises no more than about five, four, or three nucleotides. In some embodiments, the loop is an inner loop adjacent to the stem (e.g., the lower stem) comprising two complementary stem sequences and another stem (e.g., the upper stem). In some embodiments, the inner loop comprises a nucleic acid sequence of no more than 10, 9, or 8 nucleotides. In some embodiments, the stem sequence of the other stem (e.g., the upper stem) comprises no more than about five, four, or three nucleotides. In some embodiments, the engineered polynucleotide also comprises a apical loop. In some embodiments, the apical loop comprises a nucleic acid sequence of no more than 10, 9, 8, 7, 6, or 5 nucleotides. In some embodiments, the engineered polynucleotide does not contain any intramolecular disulfide bonds. In some embodiments, when the engineered polynucleotide binds to the spliceosome portion, the mRNA precursor substantially does not exhibit base pairing with the RNA-binding domain (RBD) of the U1 snRNA. In some embodiments, when the engineered polynucleotide binds to the spliceosome portion, the mRNA precursor substantially does not exhibit base-specific interactions with the U1-C protein. In some embodiments, the engineered polynucleotide is configured to interact specifically with the zinc finger of the U1-C protein. In some embodiments, the 5'-targeting portion of the engineered polynucleotide is configured to interact specifically 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 disulfide bonds). In some embodiments, the engineered polynucleotide is configured to non-covalently interact with the zinc finger of the U1-C protein (e.g., via hydrogen bonds). In some embodiments, the engineered polynucleotide comprises a nucleotide sequence complementary to a partial sequence of the stem-loop II (SL2) of the U1 snRNA. In some embodiments, one side of the stem-loop structure of the engineered polynucleotide comprises a nucleotide sequence complementary to a partial sequence of the stem-loop II (SL2) of the U1 snRNA. In some embodiments, the partial sequence comprises a sequence corresponding to the 5'-GGCCU-3' of the SL2 of the U1 snRNA. In some embodiments, the partial sequence does not comprise a sequence corresponding to the 5'-CACGUUA-3' of the SL2 of the U1 snRNA. In some embodiments, the engineered polynucleotide substantially does not exhibit interaction with the U1-C protein. The anchoring sequence base pairing of the SL2 of the snRNA. In some embodiments, the inner loop of the engineered polynucleotide does not substantially exhibit base pairing with the anchoring sequence base pairing of the SL2 of the U1 snRNA.In some embodiments, the lower stem of the engineered polynucleotide does not substantially exhibit base pairing with the anchoring sequence of the SL2 of the U1 snRNA. In some embodiments, the anchoring sequence comprises a sequence corresponding to 5'-CACGUUA-3'. In some embodiments, the engineered polynucleotide does not substantially exhibit base pairing with the H-helix of the U1 snRNA. In some embodiments, the engineered polynucleotide comprises at least one chemical modification. In some embodiments, the engineered polynucleotide comprises at least one 2'-modified (e.g., 2'-methoxy, 2'-methoxymethyl, or 2'-methoxyethyl) nucleotide. In some embodiments, at least about 50%, 60%, 70%, 80%, or 90% of the nucleotides of the engineered polynucleotide are chemically modified nucleotides. In some embodiments, at least about 50%, 60%, 70%, 80%, or 90% of the nucleotides of the engineered polynucleotide are 2'-modified (e.g., 2'-methoxy, 2'-methoxymethyl, or 2'-methoxyethyl) nucleotides. In some embodiments, the 2'-modified nucleotide comprises 2'-methoxy, 2'-methoxymethyl, 2'-methoxyethyl, 2'-fluorine, or 2'-aminoethyl nucleotides. In some embodiments, the engineered polynucleotide comprises nucleotides linked by internucleotide bonds, and at least one of the internucleotide bonds does not contain a phosphate ester. In some embodiments, the engineered polynucleotide comprises nucleotides linked by internucleotide bonds, and at least one of the internucleotide bonds comprises sulfur (S); selenium (Se); BR3, wherein each R is independently selected from hydrogen, alkyl, and aryl; carbon (C); or NR2, wherein each R is independently selected from hydrogen, alkyl, and aryl.

[0024] In some embodiments, the engineered polynucleotide comprises at least one thiophosphate nucleotide internucleotide bond. In some embodiments, at least about 50%, 60%, 70%, 80%, or 90% of the nucleotide internucleotide bonds of the engineered polynucleotide are chemically modified. In some embodiments, at least about 50%, 60%, 70%, 80%, or 90% of the nucleotide internucleotide bonds are thiophosphates. In some embodiments, the nucleotide internucleotide bonds comprise methylphosphonate, hydroxyamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thiomethyl acetal, methyl acetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazine, methylenedimethylhydrazine, 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, each of the one or more targeting moieties independently comprises 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 and second targeting moieties comprises about 2 nucleotides, and the other of the first and second targeting moieties comprises about 5 or 6 nucleotides. In some embodiments, the spliceosome moiety cleaves or splices the mRNA precursor in the target sequence when bound to the engineered polynucleotide and the mRNA precursor. In some embodiments, the spliceosome moiety further facilitates modification of the cleaved mRNA precursor.

[0025] In some aspects, this document describes an engineered polynucleotide comprising at least 70%, 80%, 85%, or 90% identical or complementary nucleotide sequences to those shown in Tables 2-3, characterized by (e.g., secondary) structural features. In some embodiments, the nucleotide sequences are identical or complementary to those shown in Tables 2-3. In some embodiments, the structural features comprise one or more stem-loop structures. In some embodiments, the structural features comprise a apical loop, an upper stem, an inner loop, a lower stem, or a combination thereof. In some embodiments, the engineered polynucleotide comprises a loop (e.g., an inner loop) adjacent to a stem (e.g., a lower or upper stem), the stem comprising two complementary stem sequences. In some embodiments, the stem sequence of the stem (e.g., the lower or upper stem) comprises no more than about five, four, or three nucleotides. In some embodiments, the loop is an inner loop adjacent to the stem (e.g., the lower stem) comprising two complementary stem sequences and another stem (e.g., the upper stem). In some embodiments, the inner loop comprises a nucleic acid sequence of no more than 10, 9, or 8 nucleotides. In some embodiments, the stem sequence of the other stem (e.g., the upper stem) contains no more than about five, four, or three nucleotides. In some embodiments, the engineered polynucleotide further comprises a apical loop. In some embodiments, the apical loop comprises a nucleic acid sequence of no more than 10, 9, 8, 7, 6, or 5 nucleotides. In some embodiments, the engineered polynucleotide further comprises one or more targeting portions sufficiently identical or complementary to the target sequence of the target gene. In some embodiments, the targeting portion of the one or more targeting portions is sufficiently identical or complementary to a common 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 phosphate-thioester nucleotide internucleotide bond. In some embodiments, at least about 50%, 60%, 70%, 80%, or 90% of the nucleotide internucleotide bonds of the engineered polynucleotide are chemically modified. In some embodiments, at least about 50%, 60%, 70%, 80%, or 90% of the nucleotides are linked by phosphate thioesters. 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 in the engineered polynucleotide are chemically modified nucleotides. In some embodiments, at least about 50%, 60%, 70%, 80%, or 90% of the nucleotides in the engineered polynucleotide are 2'-modified (e.g., 2'-methoxy, 2'-methoxymethyl, or 2'-methoxyethyl) nucleotides.In some embodiments, the engineered polynucleotide comprises about 10 to about 40 nucleotides, about 10 to about 35 nucleotides, about 10 to about 30 nucleotides, or about 10 to about 25 nucleotides.

[0026] In some aspects, this document describes a method for altering a precursor messenger ribonucleic acid (mRNA) in a cell, the method comprising contacting the cell with an engineered polynucleotide comprising one or more target moieties and a recruitment moieties, wherein the one or more target moieties bind the mRNA precursor at a target sequence in the mRNA precursor, and the recruitment moieties recruit a post-transcriptional regulatory moiety (e.g., a spliceosome portion) of the mRNA precursor in the vicinity of the target sequence to alter the mRNA precursor in the cell, thereby producing one or more altered mRNA precursors. In some embodiments, the target moieties in the one or more target moieties are sufficiently identical or complementary to a common sequence in the target sequence of a gene (e.g., a target gene). In some embodiments, the mRNA precursor 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 the contact, the cell exhibits an aberrant messenger ribonucleic acid (mRNA) or protein corresponding to the target gene.

[0027] In some embodiments, the engineered polynucleotide comprises: (i) a first targeting portion configured to specifically bind the mRNA precursor at a first target sequence in the messenger ribonucleic acid precursor (mRNA precursor), wherein the first targeting portion comprises a sequence identical or complementary to 5'-GTCCA-3'; (ii) a recruitment portion comprising a sequence at least 90% similar to or complementary to SEQ ID NO:1 and configured to recruit a spliceosome portion comprising U1snRNA and U1-C protein, wherein the recruitment portion comprises a top loop, an upper stem adjacent to the top loop, a lower stem, and an inner loop located between the upper stem and the lower stem; and (iii) a second targeting portion configured to specifically bind the mRNA precursor at a second target sequence in the mRNA precursor, wherein the second targeting portion comprises a sequence identical or complementary to 5'-CG-3'.

[0028] In some respects, this document describes a set of engineered polynucleotides, each engineered polynucleotide independently comprising: one or more targeting portions configured to bind a messenger ribonucleic acid precursor (mRNA precursor) at a target sequence; and a recruitment portion configured to recruit a posttranscriptional regulatory portion (e.g., a spliceosome portion), wherein the set of engineered polynucleotides is configured to specifically bind the mRNA precursor at multiple target sequences including the target sequence.

[0029] Another embodiment described herein is an engineered polynucleotide comprising: a first targeting portion configured to specifically bind to a first target sequence in a messenger ribonucleic acid precursor (mRNA precursor); a recruitment portion configured to recruit a spliceosome portion; and a second targeting portion configured to specifically bind to a second target sequence in the mRNA precursor. The recruitment portion includes a apical loop, an upper stem adjacent to the apical loop, a lower stem, and an inner loop located between the upper and lower stems. The spliceosome portion, upon binding to the mRNA precursor and the engineered polynucleotide, alters the mRNA precursor in a target sequence comprising the first target sequence and the second target sequence. In some embodiments, the first targeting portion is complementary to and / or hybridizes with the first target sequence. In some embodiments, the second targeting portion is complementary to and / or hybridizes with the second target sequence. In some aspects, the first target sequence and the second target sequence are separated by a spacer sequence of no more than five nucleotides in the target sequence. In some aspects, the target sequence comprises an exon-intron boundary in the mRNA precursor. In some aspects, the first target sequence is located at the 5' of the exon-intron boundary, and the second target sequence is located at the 3' of the exon-intron boundary. In some aspects, the first targeting portion comprises a sequence that is identical or complementary to the sequence shown in the exon sequence column of Table 1; and the second targeting portion comprises a sequence that is identical or complementary to the sequence shown in the intron sequence column of Table 1. In some embodiments, the first targeting portion comprises a sequence that is at least 80%, 90%, or identical to the ribosome binding site of the spliceosome snRNA (e.g., U1 snRNA). In some embodiments, the second targeting portion comprises a sequence that is at least 80%, 90%, or identical to the ribosome binding site of the spliceosome snRNA (e.g., U1 snRNA). In some embodiments, the sequence that is at least 80%, 90%, or identical to the ribosome binding site can be from about 2 nucleotides to about 10 nucleotides. In some embodiments, the recruitment moiety is complementary to the stem-loop-II region of snRNA (e.g., U1 snRNA). In some embodiments, the recruitment moiety hybridizes to the stem-loop-II region of snRNA (e.g., U1 snRNA). In some embodiments, the recruitment moiety comprises AGGCC. In some embodiments, the recruitment moiety comprises at least 80%, 90%, or the same nucleotide sequence as at least five consecutive nucleotides shown in Tables 2-3.In some embodiments, the recruitment portion comprises at least 80%, 90%, or the same nucleotide sequence of about 5 to about 10 consecutive nucleotides as shown in Tables 2-3. In some aspects, the spliceosome portion comprises U1 snRNA and U1-C protein. In some aspects, the upper stem or the lower stem comprises two complementary sequences, each of which comprises no more than 5 nucleotides; the inner loop comprises two nucleic acid sequences, each of which comprises no more than 5 nucleotides; and the apical loop comprises a nucleic acid sequence of no more than 8 nucleotides. In other aspects, the mRNA precursor substantially does not exhibit base pairing with the RNA-binding domain (RBD) of the U1 snRNA when bound to the engineered polynucleotide, and the spliceosome portion substantially does not exhibit base-specific interactions with the U1-C protein when bound to the engineered polynucleotide and the spliceosome portion. In another aspect, the 5'-targeting portion of the engineered polynucleotide is configured to specifically interact with the zinc finger of the U1-C protein. In another aspect, the recruitment portion comprises a nucleotide sequence complementary to at least four nucleotides of the stem-loop II (SL2) sequence of the U1 snRNA. In another aspect, the SL2 sequence of the U1 snRNA comprises 5'-GGCCU-3'. The engineered polynucleotide may have 2'-modified nucleotides. At least 50% of the nucleotides in the engineered polynucleotide may be 2'-modified nucleotides. The 2'-modified nucleotides may be 2'-methoxynucleotides. In another aspect, the engineered polynucleotide comprises nucleotides linked by internucleotide bonds, and at least one of the internucleotide bonds does not contain a phosphate ester. At least one or 50%, 60%, 70%, 80%, or 90% of the internucleotide bonds may be phosphate thioesters. Attached Figure Description

[0030] The patent or application documents contain at least one color drawing. The patent office will provide a copy of the patent or application with the color drawing upon request and upon receipt of the necessary fees.

[0031] Figure 1A schematic diagram is shown for identifying splice donor and splice acceptor sites. An example common sequence for messenger ribonucleic acid (mRNA) splicing in animals and plants is “GU_AG”, where “GU” is the example splice donor sequence and “AG” is the example splice acceptor sequence. A longer splice donor common sequence in mammals can be “GUrAGU”, where “r” represents “G” or “A”. Typically, the expression “GU_AG” means that only the 5’ and 3’ terminal nucleotides of the sequence remain unchanged as “GU” and “AG”, respectively, and the underlined sequence can be any sequence. However, the expression described herein indicates that the underlined sequence can be any sequence except one that does not match any other common sequence. The splice acceptor common sequence is preceded by a branch point sequence containing adenine, which is attached to the 5’ splice site ribonucleotide to form an intronic lasso; and a polypyrimidine bundle (C or U) between the branch point and the splice acceptor sequence. While the short GU_AG concordant sequence in introns is clearly insufficient to distinguish numerous alternative splicing events, it is surprising how little we know about other sequence information required for RNA splicing alternative regulation. One or two nucleotides located on either side of the intron are also generally conserved, and they are included in our supplementary table, but will not be discussed further here; thus, we can focus our analysis on the concordant sequence at the intron ends. In this sense, the rational design of engineered polynucleotides logically identifies the splicing intron concordant sequence (GU_AG). This then makes it possible to identify conserved regions at the donor site (downstream of the 5' exon and intron) and the acceptor site (downstream of the 3' exon and intron). Notably, the conserved and concordant regions are located within the same site as the constitutive splicing donor or acceptor. The identification of the concordant region determines the 5' splice site, the boundary between the exon and intron. Simultaneously, the identification of the conserved region identifies the transcript selected for regulation.

[0032] Figure 2A-2B Example engineered polynucleotides described herein are shown, comprising: (1) a 3'-targeting portion: 3'-GC-5'; (2) a lower stem: 3'-GA-5' / 5'-CT3'; (3) an inner loop: 3'-CC-5' / 5'-AA-3'; (4) an upper stem: 3'-GGA-5' / 5'-CCT-3'; (5) a top loop: 3'-CTT-5'; and (6) a 5'-targeting portion: 5'-GTCCA-3'. Figure 2C An example of the interaction between engineered polynucleotides and target mRNA precursor sequences is shown. Figure 2D The interaction of multiple components of an example engineered polynucleotide with a U1 RNP complex is illustrated.

[0033] Figures 3A-3BAnchoring occurs via an engineered polynucleotide “stem 5' / 3'” (aka, 5'-targeting portion and / or 3'-targeting portion) designed to interact with a conserved portion present in a constitutive donor site. Figure 3A Substitution of molecular sugars such as thiophosphate nucleotide bonds and 2'O-methyl (2'O-ME) in stem 5' / 3' (GTCCA and CG) increases resistance to endonucleases and increases the molecular strength of the interaction between the bases of stem 5' / 3' and conserved regions from constitutive donors. Figure 3B The engineered polynucleotides described in this article interact with the constitutive donor, and the RNA-binding domain (RBD) of U1 snRNA is spliced ​​and silenced by the constitutive donor splicing-exon splicing.

[0034] Figure 4 A schematic diagram of the human U1 snRNP is shown. The U1 snRNP consists 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 forming the H-helix are shown. Furthermore, the U1 snRNA sequences associated with RNA:protein or RNA:5'ss interactions are given. The loop portion of SL1 is drawn from the crystal structure. It 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 shown. The Sm loop formed by the Sm proteins shown as green circles binds to the framed Sm sites. U1-70K, shown as red, recognizes SL1. U1A, shown as yellow, binds SL2. U1C, shown in blue, is recruited to the U1 snRNP through protein-protein interactions with U1-70K and Sm proteins. The annotations indicate the interaction between U1C and the Sm loop.

[0035] Figures 5A-5CThis study demonstrates that the U1 snRNP binds to the 5' exon-intron junction of the mRNA precursor and thus plays a crucial role in the early stages of mRNA precursor splicing. Two crystal structures of the engineered U1 substructure are shown, collectively revealing at atomic resolution a nearly complete protein-protein and RNA-protein interaction network within the U1 snRNP and illustrating how the U1 snRNP recognizes the 5' splice site of the mRNA precursor. The zinc finger of U1-C interacts with the double-stranded structure between the mRNA precursor and the 5' end of the U1 snRNA. The binding of the RNA double-stranded structure is stabilized by hydrogen bonds and electrostatic interactions between U1-C and the RNA backbone surrounding the splice site, but U1-C does not have base-specific contact with the mRNA precursor. This structure, along with RNA binding assays, shows that the selection of nucleotides at the 5'-splicing site by the U1 snRNP is primarily achieved through base pairing with the U1 snRNA, while U1-C fine-tunes the relative affinity of mismatched 5'-splicing sites. Figure 5A U1-70K complexes with U1 snRNA stem-loop complex and U1-A RRM complexes with stem-loop 2. Figure 5B U1 snRNA stem-loop 1 and 2 (55-MER). Figure 5C U1 small nucleoprotein A and 70 kDa.

[0036] Figure 6 The U1-70K stem-loop complex with U1 snRNA and the U1-A RRM stem-loop 2 complex are shown to be stabilized by U1-C zinc finger.

[0037] Figure 7A schematic diagram illustrating the regulation of the spliceosome mechanism by the engineered polynucleotide (ASMO1, also known as APT20TTMG) described herein is shown. Anchoring of the targeting portions (“stem 5’ / 3’”) (5’-GTCCA-3’ and 5’-CG-3’) allows interaction with conserved sites of the constitutive donor via the RNA-binding portion (RBD) of the silenced U1 snRNA. Stabilization of the U1 snRNP complex can be observed via the strong ionic attraction of the U1-C zinc finger (induced by the disulfide bridge of the thiol in the ASMO1 stem 5’ / 3’). The mRNA precursor / ASMO1 duplex bond is stabilized by hydrogen bonding and electrostatic interactions between U1-C and the mRNA precursor backbone around the splice site, but U1-C does not make base-specific contacts with the mRNA precursor. This structure suggests that the selection of 5’-splicing nucleotides by U1 snRNP is primarily achieved through the interaction between the stem 5’ / 3’ and the mRNA precursor. Simultaneously, U1-C adjusts the relative affinity of the 5'-splicing incompatible sites and stabilizes the central core of the spliceosome mechanism through the interaction bridge between U1-70 kDa and the Sm loop. Stem loop II and the upper stem (also known as "hairpin-2"; see...) can be observed. Figure 2B Electrostatic interactions and hydrogen bridging of specific bases (5'-AGGCC-3') in the stem-loop (3'-GGA-5' / 5'-CCT-3') and inner loop (3'-mCC-5' / 5'-AA-3') are associated with polyadenylation signaling and acetylation regulation of U1-A. Notably, the anchoring portion of U1-A in stem-loop II (5'-CAACGUUA-3') is not silenced by the upper stem, thereby inducing regulation of gene expression and acetylation levels. Furthermore, the presence of the 2'-OME group induces changes in mediator molecular dynamics, promotes conformational changes in U1-snRNA, and the stem-loop II approximates ASMO1, where ASMO1 targets or recruits a different portion of the U1-A protein, thus reducing the likelihood of premature reading frame interruption through polyadenylation signaling dysregulation.

[0038] Figure 8The complexation of U1-70K with the stem-loop of U1 snRNA and the complexation of U1-A RRM with stem-loop 2 are shown, stabilized by the U1-C zinc finger. Electrostatic interactions and hydrogen bridging of specific bases (3'-CCGGA-5') in stem-loop II with the upper stem (3'-GGA-5' / 5'-CCT-3') and the inner loop (3'-mCC-5' / 5'-AA-3') are observed, and are associated with polyadenylation signaling and acetylation regulation of U1-A. Notably, the anchoring 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. Furthermore, the presence of the 2'-OME group induces changes in mediator molecular dynamics, promotes conformational changes in U1-snRNA, and approximates stem-loop II with the engineered polynucleotide (ASMO1) described herein.

[0039] Figure 9A U1-C located on SmD3 is shown, and its binding can be stabilized by the N-terminus of U1-70K. Figure 9B This shows that U1-C forms hydrogen bonds with the sugar-phosphate backbone atoms but does not contact RNA bases. On the 5'SS strand, the nucleotides of the exon sequences are stained blue, and the intron sequences are stained yellowish-brown. Figure 9C A diagram illustrating 5'-splicing site recognition is shown. Red dashed lines: Hydrogen bonds formed by the U1-C zinc finger amino acid side chains. Blue dashed lines: Hydrogen bonds formed by the U1-C zinc finger backbone atoms. Green dashed lines: Disulfide bonds formed by the U1-C zinc finger amino acid side chains. Orange dashed lines: Disulfide bonds formed by atoms in the U1-C zinc finger backbone. 5'SS nucleotides are formed as follows... Figure 9B The kernel encoding in the text.

[0040] Figure 10A The fingerprint Z1 U1-C snRNP is shown, represented by 36 blue amino acid residues. Figure 10B The Z1 finger portion of the U1-C snRNP is shown, revealing the major residues that interact with the mRNA precursor / ASMO1 duplex in the 5' constitutive donor region. Figure 10C A representative sequence of U1-C snRNP containing 145-aa is shown, where 36-aa, highlighted in green, refers to the zinc finger portion.

[0041] Figure 11 AD shows the cell viability of the U-87 cell line at four time points after incubation in ASMO1 (also known as "APT20TTMG"). Figure 11 A shows the results after 24 hours of incubation. Figure 11 B shows the results after 48 hours of incubation. Figure 11 C shows the results after 72 hours of incubation. Figure 11D shows the results after 96 hours of incubation.

[0042] Figure 12 A-12D shows the cell viability of the MCF-7 cell line at four time points after incubation in ASMO1. Figure 12 A shows the results after 24 hours of incubation. Figure 12 B shows the results after 48 hours of incubation. Figure 12 C shows the results after 72 hours of incubation. Figure 12 D shows the results after 96 hours of incubation.

[0043] Figure 13 A-13D shows the cell viability of the SHSY5Y cell line at four time points after incubation in ASMO1. Figure 13 A shows the results after 24 hours of incubation. Figure 13 B shows the results after 48 hours of incubation. Figure 13 C shows the results after 72 hours of incubation. Figure 13 D shows the results after 96 hours of incubation.

[0044] Figure 14 AC shows the cell viability of the PC-3 cell line at three time points after incubation in ASMO1. Figure 14 A shows the results after 24 hours of incubation. Figure 14 B shows the results after 48 hours of incubation. Figure 14 C shows the results after 72 hours of incubation.

[0045] Figure 15 A-15C shows the cell viability of the 786-O cell line at three time points after incubation in ASMO1. Figure 15 A shows the results after 24 hours of incubation. Figure 15 B shows the results after 48 hours of incubation. Figure 15 C shows the results after 72 hours of incubation.

[0046] Figure 16 The kinetics of internalization of compound ASMO1 by the breast cancer cell line MCF-7 are shown.

[0047] Figure 17 An assessment of mitochondrial activity in the MCF-7 cell line after incubation with ASMO1 is shown.

[0048] Figure 18 The cell cycle phase distribution of the MCF-7 cell line after incubation with ASMO1 is shown.

[0049] Figure 19 The distribution of the MCF-7 stage after incubation with ASMO1 is shown.

[0050] Figure 20The mitochondrial membrane potential in the MCF-7 cell line after incubation with ASMO1 is shown.

[0051] Figure 21 The kinetics of internalization of compound ASMO1 by the neuroblastoma cell line SH-SY5Y are shown.

[0052] Figure 22 A-22B shows the assessment of mitochondrial activity in the SH-SY5Y cell line after incubation in ASMO1. Incubation for 24 hours ( Figure 22 A) and 48 hours ( Figure 22 B) Then perform MTT measurement.

[0053] Figure 23 The cell cycle phase distribution of the SH-SY5Y cell line after incubation with ASMO1 is shown.

[0054] Figure 24 The distribution of the SH-SY5Y stage after incubation with ASMO1 is shown.

[0055] Figure 25 The mitochondrial membrane potential in the SH-SY5Y cell line after incubation with ASMO1 is shown.

[0056] Figure 26 Cell viability results obtained in the neuroblastoma cell line (SK-N-SH) after incubation with ASMO1 (0.5 μM) for 24, 48, 96 and 144 hours are shown.

[0057] Figure 27 A-27B shows the MAPT expression and tau quantification obtained in neuroblastoma cell lines after incubation with ASMO1 (0.5 μM) for 24, 48, 96, and 144 hours. Figure 27 A shows MAPT expression at different time points after ASMO1 (0.5 μM). Figure 27 B shows the tau quantification at different time points after ASM01 (0.5 μM).

[0058] Figure 28 The kinetics of internalization of compound ASMO1 by the glioblastoma cell line U87-MG are shown.

[0059] Figure 29 The cell cycle phase distribution of the U87-MG cell line after incubation with ASMO1 is shown.

[0060] Figure 30 The distribution of the U87-MG stage after incubation with ASMO1 is shown. Figure 31 The quantification of mitochondrial membrane potential in the U87-MG cell line after incubation with ASMO1 is shown.

[0061] Figure 32 This shows an assessment of mitochondrial activity in the U87-MG cell line after incubation with APT20TTMG.

[0062] Figure 33 The proliferation of the U87-MG cell line was evaluated using APT20TTMG.

[0063] Figure 34 A-34C demonstrates the effect of intravenous administration of APT20TTMG in a glioblastoma xenograft model in athymic nude mice.

[0064] Figure 35 A-35B shows the effect of intravenous administration of APT20TTMG on the proliferation marker (Ki-67) in a glioblastoma xenograft model in athymic nude mice.

[0065] Figure 36 This study demonstrates the effects of intravenous administration of APT20TTMG on histopathological findings and overall tumor volume score.

[0066] Figure 37 A-37D shows the effect of APT20TTMG administration on signal transduction pathway proteins in a glioblastoma xenograft model in a thymic nude mouse.

[0067] The novel features of this disclosure are particularly set forth in the appended claims. A better understanding of the features and advantages of this disclosure will be obtained by referring to the following detailed description of illustrative embodiments. Detailed Implementation

[0068] This article describes (e.g., engineered) polynucleotides and (e.g., pharmaceutical) compositions and their methods of use, for example, to regulate gene expression or activity.

[0069] This document describes methods for treating cancer types using engineered polynucleotides described in this disclosure. These methods are applicable to a variety of cancer types, such as cancers of different organ or cell types, or cancers associated with or caused by different genetic aberrations. The engineered polynucleotides described herein can efficiently modulate the splicing of mRNA precursors by recruiting components of the spliceosome. Improved recruitment of spliceosome components to mRNA precursors, without being constrained by a specific mechanism, can effectively modulate cellular mechanisms leading to cancer. These engineered polynucleotides can be applied to subjects with cancer and can treat cancer, for example, by reducing the number of cancer cells or preventing cancer cell proliferation or metastasis.

[0070] Under physiological conditions, splicing is responsible for processing precursor messenger RNA (pre-mRNA), removing intron regions and joining exon regions to produce mature mRNA. Splicing is carried out by a complex of a series of snRNPs and small nuclear RNA (snRNA). The U1 snRNP complex is one of the five complexes that make up the human spliceosome (named U1, U2, U4, U5, and U6). The U1 snRNP complex consists of U1 snRNA, seven Sm proteins forming a heptameric loop, and three additional proteins: U1-70K, U1A, and U1C. In the spliceosome, the U1 snRNP complex plays a specific role in recognizing the pre-mRNA splice site (splicing donor site) in the early stages of spliceosome assembly. Then, other snRNPs, such as U2 snRNPs, are attracted to the splice site, and the interactions between their protein components complete the entire spliceosome assembly and splicing. Subsequently, the mature mRNA formed after capping and polyadenylation can leave the cell nucleus and be translated into protein. In addition to its role in splicing, U1snRNP also plays a role in actively inhibiting the polyadenylation mechanism by using early (primarily intron) polyadenylation signals (which lead to aberrant and truncated mRNA). Accurate splicing is a necessary step in gene expression and also enables alternative splicing, which allows for a balance between the accuracy and flexibility of splice site recognition and the creation of multiple isotypes from a single transcript with diverse, sometimes even antagonistic, biological functions.

[0071] In addition to its splicing function, the U1 snRNP complex also possesses telescripting capabilities, actively inhibiting the utilization of proximal polyadenylation signals (PAS, which are primarily located within introns) by the polyadenylation mechanism, thereby suppressing a process known as premature polyadenylation. Notably, polyadenylation is one of the major processes in mRNA maturation and involves terminating the mRNA precursor by removing the 3' end portion (typically located in the 3' untranslated region (UTR)) and adding a poly(A) tail. This process, along with splicing and 5' capping, produces mature mRNA, influencing transcript stability, export, and translation. The U1 snRNP complex's inhibition of premature polyadenylation prevents the production of aberrant and shortened mRNA.

[0072] In addition, U1 snRNP has been described as being associated with stress granules (cytoplasmic RNA granules containing mRNA, related translation initiation factors, and various RNA-binding proteins (such as snRNP) formed in response to various stresses) and with autophagy in lysosomal and autophagosome-lysosomal biogenesis.

[0073] Cell cycle reentry induced by U1 dysfunction has been hypothesized to be a contributing factor to excessive proliferation and cancer. In various cancer types, U1 snRNPs and snRNAs have been observed to play important roles in inhibiting premature RNA transcript cleavage and polyadenylation, and these snRNPs may also function in cell migration and proliferation.

[0074] In fact, numerous studies have demonstrated the importance of variable polyadenylation (APA) changes and dysregulation in cancer and its progression. Besides splicing processes, polyadenylation is also a source of transcriptobiological diversity, as mRNA precursors may possess multiple variable polyadenylation signals (PAS). Several studies have identified the potential impact of APA on gene expression regulation and function. For example, well-differentiated cells (such as neurons) typically use distal PAS located further downstream of the stop codon, resulting in transcripts with longer 3'UTRs and potentially lower protein expression levels. Conversely, faster-growing cells (primarily cancer cells) tend to use proximal PAS, producing shorter 3'UTRs and potentially higher protein expression levels. Increased use of proximal 3'UTR PAS in various cancer cell lines and tissues is associated with the promotion of cancer cell proliferation.

[0075] In addition to premature polyadenylation, there is evidence that spliceosome alterations also occur in cancers such as glioblastoma (GBM), thereby activating oncogenic splicing events associated with tumor progression and severity. In a well-defined group of adult diffuse gliomas (primarily GBM), impaired expression of spliceosome components (such as U1 small nuclear RNA (RNU1)) and splicing factors that co-recognize target introns has been observed. Notably, silencing specific splicing factors (such as RBM22, RBM3, PTBP1, and especially SRSF3) has resulted in reduced cell proliferation and migration, tumor spheroid formation, and apoptosis induction. These effects may be mediated by modulating key signaling pathways such as PDGFRB and PI3K-AKT / ERK, which are pro-oncogenic pathways in gliomas.

[0076] Multiple studies have demonstrated the importance of these mechanisms in cancers involving the assembly of snRNPs (i.e., the biogenesis of U1 snRNPs). For example, a potential association between these snRNPs and cell migration and proliferation has been identified in the human cervical epithelioid carcinoma (HeLa) cell line. This study shows that inhibition of U1 snRNA using antisense morpholino oligonucleotides (U1 AMO, designed to target U1 snRNA) leads to widespread premature transcriptional termination and shortened mRNA 3'-UTR, due to the use of more proximal PAS in introns and the last exon, resulting in shorter mRNA isoforms. Interestingly, even low concentrations of U1 AMO (affecting approximately 15-30% of U1 snRNA expression) enhance the migratory and invasive properties of cancer cells. Furthermore, this phenomenon is associated with the upregulation of oncogenes and the downregulation of tumor suppressor genes. When U1 snRNA is overexpressed, the opposite effect is observed, namely, reduced cancer cell migration and invasion. In a comparative study also using HeLa cells, overexpression of U1 snRNA reversed DNA damage, particularly damage induced by UV treatment. This DNA damage was characterized by decreased U1 snRNA levels, while significantly regulating intron alternative cleavage and 3'-related DNA damage. ′ Polyadenylation in the UTR leads to 3UTR shortening and gene elongation, as well as the expression of truncated transcripts. In summary, these studies highlight the crucial role of polyadenylation in regulating cancer cell fate.

[0077] Another study in hepatocellular carcinoma (HCC) showed that 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 understand the role of U1A in HCC, knockdown of U1A inhibited migration and cell cycle progression, while simultaneously promoting apoptosis in HCC cell lines. Furthermore, an association between U1A mRNA expression and tumor-infiltrating immune cells was observed. Another study involving HCC cell lines and HeLa cells showed that knockdown of U1A also led to a significant decrease in CCN2 expression and connective tissue growth factor (CTGF) secretion (CTGF protein is encoded by the CCN2 oncogene), thereby reducing cell migration and proliferation. Notably, CTGF is involved in cell proliferation, angiogenesis, and migration, a phenomenon crucial for epithelial-mesenchymal transition and eventual metastasis. Similarly, U1A was upregulated in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). High U1A expression was associated with first progression and poor survival after progression in LUAD cases. However, a 4% genetic alteration rate of U1A (such as missense mutations and deep deletions) was associated with overall survival in LUSC cases (but not LUAD cases). In both LUAD and LUSC, U1A expression was negatively correlated with M2 macrophage infiltration levels, but positively correlated with follicular B helper T cell infiltration levels. Furthermore, in the pathogenesis of LUAD, U1A expression was associated with genes involved in cell cycle and ubiquitin mechanisms, while in LUSC, it was primarily associated with RNA splicing-related cellular issues (e.g., spliceosomes, RNA splicing, mRNA precursor binding, variable mRNA splicing, etc.).

[0078] In summary, these studies on U1 snRNP in different cancer types may indicate that U1 homeostasis is crucial for maintaining the balance of normal gene isotype expression, while U1 snRNP imbalance may affect cancer cell proliferation, migration, and invasion. Therefore, regulating U1 may be a target for cancer therapy. In fact, regulating U1 snRNP activity in cancer may correct pathological effects caused by splicing and premature polyadenylation dysfunction, as U1 snRNP is involved in both mechanisms under physiological conditions. U1 snRNP is an essential component of the splicing mechanism responsible for selecting splice sites and promoting spliceosome assembly by binding to 5' splice sites (called donor splice sites) that typically contain GU sequences. It also plays a crucial role in inhibiting premature cleavage and polyadenylation of the 3' end of mRNA precursors, particularly in GU-rich regions located within introns. This inhibitory mechanism prevents the polyadenylation mechanism from recognizing these cryptic regions, which would otherwise lead to the production 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 splicing and polyadenylation. Therefore, the proper functioning of U1 snRNPs and their telescripting function (including inhibition of premature polyadenylation and occult splicing) protects mRNA precursors and helps regulate variable polyadenylation, thereby preventing oncogenic processes.

[0079] Beyond its complex regulatory role on U1 snRNP, further evidence revealing the potential of ASMO in cancer treatment lies in its ability to regulate tau expression: treatment of the neuroblastoma SK-N-SH cell line with different concentrations of APT20TTMG (ASMO1) showed decreased tau expression at both mRNA and protein levels, as well as reduced cell viability, suggesting a potential cytoseptic effect. Furthermore, in vivo studies using a human glioblastoma (U-87MG) xenograft mouse model also showed reduced tumor tau expression and tumor volume after APT20TTMG treatment compared to the control group. These results are significant given the growing evidence that tau protein plays a role in cancer. Moreover, as a microtubule-binding protein, tau may 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.

[0080] For example, in breast cancer cell lines, an association has been established between tau expression and response to taxanes: knockdown of tau via RNA interference enhances the sensitivity of ZR75-1 and MCF-7 cell lines to taxanes (paclitaxel and docetaxel), which may be related to tau protein isoforms smaller than 70 kDa. Furthermore, estrogen receptor (ER) signaling has been shown to affect the expression of tau protein isoforms smaller than 70 kDa, which may influence sensitivity to taxanes. Additionally, fulvestrant (a selective estrogen receptor degrader used to treat metastatic breast cancer) reduces ER and tau expression, and its combination with taxanes enhances the sensitivity of tau and ER-positive breast cancer cells to taxanes. Nevertheless, one study showed that patients with tau-positive cancer had better disease-free survival and overall survival than those with tau-negative tumors in ER-positive patients, but there was no significant interaction between tau expression and benefit from paclitaxel. In ER-negative patients in this study, tau expression had no prognostic value. One question raised by the authors is why a large randomized study did not confirm the previously described preclinical findings. One possible answer is that the role of tau in paclitaxel resistance (and other causes of resistance) may only be clinically significant for a small subset of patients. However, this difference could also be due to, for example, differences in chemotherapy regimens. Contrary to observations in patients with breast cancer, high tau expression was associated with poor prognosis in prostate cancer (PC). Furthermore, tau knockdown suppressed androgen receptor expression (AR plays a decisive role in the progression to castration-resistant PC) and increased sensitivity to picalutamide (an anti-androgen drug that blocks testosterone from reaching cancer cells). Studies using ALVA-31 derivatives (ALVA-NEO and ALVA-hCD40) of the prostate tumor cell line showed that all six alternatively spliced ​​adult brain tau isotypes were expressed and highly phosphorylated, among other isotypes, most of which do not bind to microtubules. Furthermore, ALVA-NEO tau interacts with PI3K / AKT, which is associated with cell survival and proliferation in many cancers. Consistent with this, in taxane-resistant prostate cell lines, downregulation of tau inhibits cell proliferation through the PI3K / Akt / mTOR signaling pathway, thereby increasing cytotoxicity to taxanes.

[0081] For example, in GBM, downregulation of tau in the U87-MG cell line via short hairpin interfering RNA (shRNA) significantly reduced 2D cell motility by repositioning the ROCK and inducing inefficient tail retraction. Rho-ROCK signaling is known to act on cell migration and invasion phenotypes by regulating the actin cytoskeleton. In this study, tau was observed to contribute to the remodeling of both microtubules and the actin cytoskeleton, both crucial for migration. This property may be attributed to limited activity of the Rho-ROCK pathway (which is involved in regulating posterior cell aggregation). All these effects are initiated upstream through tau-induced microtubule bundle formation and stabilization. Tau depletion disrupts this microtubule and actin assembly balance in the posterior U87MG cell line, as well as the crucial posterior cell retraction during migration. Notably, U87-MG cells expressed the highest levels of tau among the GBM cell lines (U118, U138, and U251). Another, more recent study (which used shRNA to knock down tau in a 3D model of multicellular spheroids (MCS) of U87-MG cells) showed that MCS growth and cell escape were inhibited by affecting cell migration and spheroid cohesion. Reduced MCS compactness due to N-cadherin mislocalization was also observed. Furthermore, in a glioblastoma xenograft model, mice injected with U87MG sh-tau cells (cells with reduced tau levels) had a significantly higher median survival than mice injected with U87MG control cells. Based on these results, the authors propose that tau plays a role in glioblastoma by controlling 3D cell organization and function via the PI3K / AKT signaling axis.

[0082] Furthermore, this MCS study proposed a model for the role of tau in controlling PI3K / AKT signaling and N-cadherin-β-catenin. N-cadherin junctions are responsible for intercellular adhesion essential for spherical compaction, and the cadherin-catenin complex on the membrane has been shown to recruit PI3K, thereby initiating a signaling cascade. Tau may enhance these interactions by stabilizing the microtubule network. Specifically, in GBM cells with PTEN null mutations (PTEN deletion is very common in GBM and may stimulate invasive behavior), it is proposed that tau enhances intercellular adhesion by promoting the stability and compaction of the N-cadherin / β-catenin complex through microtubule-dependent interactions. Tau is also proposed to participate in cell contraction by promoting actin assembly. Tau also stimulates the indirect activation cascade of the PI3K-AKT signaling pathway through MT-independent interactions or by increasing N-cadherin-β-catenin signaling, leading to cell survival and proliferation. In tau-depleted U87MG cells, a defective recruitment of the N-cadherin-β-catenin complex to the actin cytoskeleton was proposed. Under these conditions, N-cadherin is unstable on the membrane, leading to loss of MCS compaction. Furthermore, the loss of intercellular cohesion was proposed to be related to the isolation of β-catenin by mislocalized N-cadherin and caused by reduced PI3K-AKT signaling activity. A reduction in phosphorylated Akt kinase was also observed. Due to this proposed mechanism, cell proliferation and migration are expected to be inhibited in tau-depleted GBM cells. Moreover, it is noteworthy that tau proteins are described as having a broad interfacial group (including interactions with cancer-associated kinase proteins), which may also influence cellular pathways involved in other cascade mechanisms, such as cell signaling, cell movement, and cell metabolism.

[0083] Unlike studies on glioblastoma, research using renal cell carcinoma lines has shown that downregulation of tau enhances cell growth 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, knockout of tau via CRISPR-Cas9 and subsequent shRNA-mediated knockdown of tau led to dysregulation and altered activity of the pro-apoptotic tumor suppressor p53, resulting in reduced DNA damage-induced apoptosis and increased cellular senescence. Consistent with this beneficial role of tau in neuroblastoma, another study showed that patients with higher tau mRNA expression (measured by microarray and RNA sequencing data) were associated with significantly increased overall survival in children with neuroblastoma. The association of higher tau expression with better outcomes is consistent with the fact that samples with high tau expression also showed higher expression of certain apoptosis-effect genes (primarily CASP3 and CASP9), while samples with high tau expression showed lower expression of pro-proliferative histone genes. Patients with lower tau expression also had lower overall survival and a significantly higher incidence of MYCN amplification (often associated with worse outcomes in neuroblastoma). These cases where tau expression was associated with better outcomes may be related to microtubule stabilization, which leads to cell suppression through a mechanism similar to that of microtubule stabilizers such as paclitaxel, widely used in cancer treatment.

[0084] Therefore, this disclosure provides engineered polynucleotides for use in cancer therapy, and corresponding methods for using these engineered polynucleotides. Engineered polynucleotides can exert their effects through mechanisms that homeostatically regulate the function of U1 snRNPs, regulate tau expression, and a combination of both (e.g., synergistically), and produce cytotoxic or inhibitory effects against selected cancer types, thus having broad applicability in cancer therapy. To allow for such U1 regulation, engineered polynucleotides can be designed with size, conformation, and strategic chemical modifications, allowing for direct attraction and interaction with U1C and indirect attraction and interaction with U1-70K. Engineered polynucleotides (e.g., ASMO1) can have sequences complementary to highly conserved regions present in mRNA precursors, typically located at the exon-intron junction (called the donor splicing site) at the 5' end of introns. U1 regulation ensures the correct assembly (with the correct distance, position, and behavior) of the snRNPs that make up the U1 complex, which is highly correlated with the splicing process. Since splicing can control expression patterns at the posttranscriptional level, U1 regulation triggered by engineered polynucleotides (e.g., ASMO1) can help normalize protein expression in cells where gene expression is aberrant due to U1 dysfunction. Regulation of tau expression following treatment with engineered polynucleotides (e.g., ASMO1) may respond to this splicing regulation in dysregulated cells, potentially aiding in cancer therapy. In conclusion, the crucial roles of U1 snRNPs and tau proteins in cancer migration and progression, along with our in vitro and in vivo results, suggest that the engineered polynucleotides considered in this disclosure could be used as therapeutic agents for cancers such as GBM.

[0085] Engineered polynucleotides

[0086] This disclosure provides engineered polynucleotides and methods for using these engineered polynucleotides. Engineered polynucleotides generally refer to non-naturally occurring polynucleotides. 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 polynucleotide described herein, may include multiple parts. A “part” may refer to a region of the engineered polynucleotide. In some cases, a part may be described according to its function. For example, a “targeting part” may refer to a region of the engineered polynucleotide that is at least partially complementary to a target RNA; a “recruiting part” may refer to a part capable of recruiting any of the regulatory parts described herein; and a “spacer sequence” may refer to a part that provides a spacer between other parts. In some cases, the description of a part name does not limit the part to a specific function. For example, a “targeting part” that is at least partially complementary to a target RNA may, in some cases, recruit a regulatory part. The various parts described throughout this disclosure can be combined to produce engineered polynucleotides that perform specific functions. For example, an engineered polynucleotide may include a target part disclosed herein that has a recruitment part disclosed herein.

[0087] In some embodiments described herein, the engineered polynucleotide comprises: (i) one or more targeting portions configured to bind, for example, specifically, said RNA (e.g., messenger RNA, such as a precursor RNA) at a target sequence in ribonucleic acid (RNA). The engineered polynucleotide may also comprise (ii) a recruitment portion configured to recruit a post-transcriptional regulatory portion (e.g., a spliceosome portion) such that, upon binding with RNA (e.g., mRNA, such as a precursor RNA) and the engineered polynucleotide, the post-transcriptional regulatory portion alters the RNA (e.g., mRNA, such as a precursor RNA) at or near the target sequence. In some embodiments, “configured to specifically bind” refers to hybridization. In some embodiments, the portion configured to specifically bind a precursor RNA refers to a portion that is at least 80%, 90%, or 100% complementary to the precursor RNA sequence to which it specifically binds. In some embodiments, the RNA (e.g., mRNA, such as a precursor RNA) encodes a target gene. In some embodiments, the mRNA precursor bound to the engineered polynucleotide refers to an mRNA precursor that hybridizes to 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, causing a nucleotide reaction in the nucleic acid, or splicing the nucleic acid. In some embodiments, "nearby" refers to a distance of no more than 5 nucleotides.

[0088] Targeted portion

[0089] In various respects, the engineered polynucleotide includes one or more targeting moieties. The targeting moieties enable the engineered polynucleotide to interact with ribonucleic acid (e.g., a precursor mRNA). The targeting moieties may contain sequences complementary to or identical to the sequence of the ribonucleic acid (e.g., a precursor mRNA), allowing the engineered polynucleotide and ribonucleic acid to interact or hybridize. The targeting moieties may be complementary to or identical to the target sequence. In some embodiments of the engineered polynucleotide described herein, the targeting moieties in one or more targeting moieties are sufficiently identical to or complementary to a common sequence in the target sequence. The target sequence may be located in a target gene. The target sequence may be a conserved or common sequence present in multiple different precursor mRNAs. For example, the target sequence may enable the engineered polynucleotide to interact with a first precursor mRNA encoding one gene and a second precursor mRNA encoding another gene. The targeting moieties may be found at the 5' and 3' ends of the engineered polynucleotide and may also be referred to as the lower stem or leg. Not wishing to be bound by theory, stable binding of one or both targeting moieties to the constitutive donor 5' of the target mRNA precursor may allow interaction with conserved sites of the constitutive donor and may silence the RNA-binding domain (RBD) of the U1 snRNA. The targeting portion may contain a sequence substantially identical to that of the U1 snRNA RBD. For example, the RBD may contain the sequence 3'-GUCCAUUCAUA-5', and the targeting portion may contain GTCCA (or GUCCA). Based on the sequence similarity between the U1 snRNA RBD and the targeting portion, the targeting portion can effectively replace the U1 snRNA RBD, preventing it from binding to the mRNA precursor, or inhibiting the binding of the U1 snRNA RBD to the mRNA precursor.

[0090] Consensus sequences can be determined based on the identification of genetic variations (VUS) of unknown significance. Any exon or intron VUS can induce splicing by disrupting cis-DNA sequences that define the exons, introns, and regulatory sequences required for proper RNA splicing. Cis-DNA elements may include: exon-intron boundary core consensual nucleotides (e.g., GT at +1 and +2 of the 5' donor site and AG at -1 and -2 of the 3' acceptor site); or nucleotides of introns and exons adjacent to these invariant nucleotides that are also highly conserved and have been found to participate in splice site selection (e.g., CAG / GUAAGU in the donor site and NYAG / G in the acceptor site). Changes in any of these elements can lead to incorrect splice site recognition, resulting in new splice sites or activation of occult splice sites, leading to aberrant transcripts or nonfunctional proteins associated with a disease or condition. In some embodiments, at least one of the target motifs is sufficiently identical or complementary to a consensual sequence in the target ribonucleic acid or the target sequence of the target gene. In some embodiments, the common 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 of the one or more target portions are sufficiently identical or complementary to at least two common sequences in the target sequence of the target gene or target ribonucleic acid. In some embodiments, one or more target portions are each independently sufficiently identical or complementary to a common sequence in the target sequence of the target gene or target ribonucleic acid.

[0091] In some embodiments of the engineered polynucleotides described herein, 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 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.

[0092] In some embodiments of the engineered polynucleotides described herein, one or more targeting portions comprise (1) a first targeting portion configured to specifically bind a first target sequence in a target sequence of RNA (e.g., mRNA, such as a precursor mRNA), and (2) a second targeting portion configured to specifically bind a second target sequence in a target sequence of RNA (e.g., mRNA, such as a precursor mRNA). In some embodiments, the first target sequence comprises a common sequence in the target sequence. In some embodiments, the common sequence of the first target sequence comprises about 2 to about 15 nucleotides, about 2 nucleotides to about 10 nucleotides, or about 2 nucleotides to about 8 nucleotides. In some embodiments, the common sequence of the first target sequence comprises 2–3, 3–4, 4–5, 5–6, 6–7, 7–8, or 8–10 nucleotides. In some embodiments, the second target sequence comprises a common sequence in the target sequence. In some embodiments, the common sequence of the second target sequence comprises about 2 to about 15 nucleotides, about 2 nucleotides to about 10 nucleotides, or about 2 nucleotides to about 8 nucleotides. In some embodiments, the common sequence of the second target sequence comprises 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, or 8-10 nucleotides. In some embodiments, the common sequence of the first target sequence and the common sequence of the second target sequence have different nucleotide lengths. In some embodiments, one of the common sequences of the first and second target sequences comprises about 1 to about 5 nucleotides, and the other comprises about 4 to about 8 nucleotides. In some embodiments, one of the common sequences of the first and second target sequences comprises at least about 2 nucleotides, and the other comprises at least about 5 or 6 nucleotides. In some embodiments, one of the common sequences of the first and second target sequences comprises about 2 nucleotides, and the other comprises about 5 or 6 nucleotides.

[0093] In some embodiments of the engineered polynucleotide described herein, the first targeting portion and the second targeting portion have different nucleotide lengths. In some embodiments, one of the first targeting portion and the second targeting portion contains about 1 to about 5 nucleotides, and the other of the first targeting portion and the second targeting portion contains about 4 to about 8 nucleotides. In some embodiments, one of the first targeting portion and the second targeting portion contains at least about 2 nucleotides, and the other of the first targeting portion and the second targeting portion contains at least about 5 or 6 nucleotides. In some embodiments, one of the first targeting portion and the second targeting portion contains about 2 nucleotides, and the other of the first targeting portion and the second targeting portion contains about 5 or 6 nucleotides.

[0094] In some embodiments, (e.g., the first or second) targeting portion comprises a nucleic acid sequence having at least one, two, three, four, five, six, seven, eight, nine, ten, or more nucleotides. In some embodiments, (e.g., the first or second) targeting portion comprises a nucleic acid sequence having at most ten, nine, eight, seven, six, five, four, three, or two nucleotides. In some embodiments, (e.g., the first or second) targeting portion comprises a nucleic acid sequence having one, two, three, four, five, six, seven, eight, nine, or ten nucleotides, or any range between two of the aforementioned values.

[0095] In some embodiments, (e.g., a first or second) targeting portion comprises a nucleic acid sequence that targets nucleotides in an exon. In some embodiments, (e.g., a first or second) targeting portion comprises a nucleic acid sequence that targets nucleotides immediately adjacent to an intron in an exon. In some embodiments, (e.g., a first or second) targeting portion comprises a nucleic acid sequence that targets nucleotides adjacent to or immediately adjacent to the 3' end of an exon. In some embodiments, (e.g., a first or second) targeting portion comprises a nucleic acid sequence that targets nucleotides adjacent to or immediately adjacent to the 5' end of an exon. In some embodiments, (e.g., a first or second) targeting portion comprises a nucleic acid sequence that targets nucleotides in an exon, the 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'. In some embodiments, (e.g., a first or second) targeting portion comprises a nucleic acid sequence that targets nucleotides in an exon immediately adjacent to the 5' end of the exon. In some embodiments, (e.g., a first or second) targeting portion comprises a nucleic acid sequence that targets a nucleotide adjacent to an intron in an exon, the 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'. In some embodiments, (e.g., a first or second) targeting portion comprises a nucleic acid sequence that targets a nucleotide immediately adjacent to the 5' end of an exon. In some embodiments, (e.g., first or second) the targeting portion comprises a nucleic acid sequence that targets the nucleotides at the 5' end of the intron in the exon, the 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'.In some implementations, (e.g., first or second) the targeting portion comprises a nucleic acid sequence that targets nucleotides in the exon at the 3' of intron, the 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'. In some implementations, (e.g., first or second) the targeting portion comprises a nucleic acid sequence that targets nucleotides in the exon that are not immediately adjacent to introns, the 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'.

[0096] In some embodiments, (e.g., first or second) the targeting portion comprises a nucleic acid sequence that targets nucleotides within the intron. In some embodiments, (e.g., first or second) the targeting portion comprises a nucleic acid sequence that targets nucleotides adjacent to or immediately adjacent to exons within the intron. In some embodiments, (e.g., first or second) the targeting portion comprises a nucleic acid sequence that targets nucleotides adjacent to or immediately adjacent to the 5' end of the intron. In some embodiments, (e.g., first or second) the targeting portion comprises a nucleic acid sequence that targets nucleotides adjacent to or immediately adjacent to the 5' end of the intron. In some embodiments, (e.g., first or second) the targeting portion comprises a nucleic acid sequence that targets nucleotides entirely within the intron. In some implementations, (e.g., first or second) the targeting portion comprises a nucleic acid sequence of a nucleotide adjacent to an exon in a targeting intron, the 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'. In some embodiments, (e.g., first or second) the targeting portion comprises a nucleic acid sequence of nucleotides targeting an exon immediately adjacent to the 5' end of the intron, the 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'. In some embodiments, (e.g., first or second) the targeting portion comprises a nucleic acid sequence of nucleotides targeting an exon immediately adjacent to the 3' end of the intron, the 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'.In some implementations, (e.g., first or second) the targeting portion comprises a nucleic acid sequence of nucleotides in the targeting intron that are not adjacent to the exon, the 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'.

[0097] As described, the targeting portion (e.g., first or second) can be gene-agnostic, allowing it to target conserved regions shared (or substantially similar) in the mRNA precursors of multiple different genes. Furthermore, the targeting portion (e.g., first or second) can target a specific gene, or specific exons and introns of a gene. For example, Table 1 provides exon-intron linker sequences for the MAPT gene for multiple exon-intron linking. For example, using Table 1 (or other sequences from the gene or exon-intron linkers of the target gene), engineered polynucleotides can be designed to target specific exon-intron linkers, or they can be designed to target more than one exon-intron linker. In some embodiments of the engineered polynucleotides described herein, the (e.g., first or second) targeting portion 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 or complementary to the sequences shown in Table 1. In some embodiments, (e.g., a first or second) target portion 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 or complementary to the sequences selected from the "Exon Sequence" and "Intron Sequence" columns of Table 1.

[0098] In some implementations, the first targeting portion comprises 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 or complementary to the sequence selected from the "Exon Sequences" column of Table 1. The first targeting portion contains 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 or complementary to the sequence shown in the "Intron Sequences" column of Table 1. In some embodiments, the first targeting portion contains a sequence that is identical or complementary to the sequence selected from the "Exon Sequences" column of Table 1; and the second targeting portion contains a sequence that is identical or complementary to the sequence shown in the "Intron Sequences" column of Table 1. In some implementations, the first targeting portion comprises 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 the sequence shown in the "Intron Sequence" column of Table 1. The first targeting portion contains a sequence that is identical or complementary to the sequence shown in the "Exon Sequences" column of Table 1; and the second targeting portion contains a sequence that is identical or complementary to the sequence shown in the "Exon Sequences" column of Table 1. In some embodiments, the first targeting portion contains a sequence that is identical or complementary to the sequence shown in the "Intron Sequences" column of Table 1; and the second targeting portion contains a sequence that is identical or complementary to the sequence shown in the "Exon Sequences" column of Table 1. In some embodiments, (e.g., the first or second) targeting portion contains a sequence that is identical or complementary to the common sequence of the intron donor site (e.g., selected from GU, GT, GC, and CA). In some embodiments, (e.g., the first or second) targeting portion contains a sequence that is identical to or complementary to a common sequence (e.g., G) of the exon donor site. In some embodiments, (e.g., the first or second) targeting portion contains a sequence that is identical to or complementary to a common sequence selected from GU, GC, G, and CA.

[0099] Table 1. Examples of MAPT gene exon-intron linker sequences targeted by the 5' and 3'-targeting portions of engineered polynucleotides and the distance between the two target sequences.

[0100] Examples of common 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.

[0101] The targeting portion (e.g., first or second) can be gene-specific, or specific to the exons and introns of a gene. For example, the targeting portion can be specific to exon-intron junctions and portions of exons or introns. In some embodiments, the targeting portion is specific to a portion of a gene other than its splice site. In some embodiments, the targeting portion targets a specific gene. For example, the targeting portion can contain portions that are specific to splice sites and specific to the gene. As described elsewhere herein, the targeting portion can contain a sequence of a common splice site. By having a targeting portion specific to a common splice site, the targeting portion can be gene-agnostic and target multiple different mRNAs of different genes. Alternatively, the targeting portion can contain a portion specific to the gene. The engineered polynucleotide can then regulate the splicing of a specific target gene (rather than any gene / mRNA containing a common splice site). For example, the targeting portion can contain a sequence complementary to a MAPT-specific sequence.

[0102] target sequence

[0103] As described in this disclosure, engineered polynucleotides may include one or more targeting moieties. The targeting moieties may target (e.g., bind, hybridize, or otherwise configure to interact with) a target sequence, for example, wherein the targeting moieties target adjacent or nearby regions within the target sequence. The targeting moieties may be complementary to the target sequence. In some embodiments of the engineered polynucleotides described herein, the first target sequence and the second target sequence are spacer sequences separated by no more than five, four, or three nucleotides (e.g., one or two nucleotides) within the target sequence.

[0104] In some embodiments of the engineered polynucleotides described herein, the first target sequence and the second target sequence are consecutive or adjacent to each other.

[0105] In some embodiments, when the spacer sequence in the target sequence is adjacent to the 5'- or 3'-end of the target sequence of the target sequence, the spacer sequence may not be complementary to any target portion of the engineered polynucleotide.

[0106] In some embodiments, the spacer sequence separates the first target sequence and the second target sequence described herein. In some embodiments, the spacer sequence is neither complementary to nor binds to the target portion of the engineered polynucleotide. In some embodiments, the spacer sequence is neither complementary to nor binds to any target portion of the engineered polynucleotide.

[0107] In some embodiments of the engineered polynucleotides described herein, the target sequence may comprise an exon-intron boundary in RNA (e.g., mRNA, such as a precursor mRNA). In some embodiments, both the first and second target sequences are located at the 5' or 3' of the exon-intron boundary. In some embodiments, one of the first and second target sequences is located at the 5' of the exon-intron boundary, and the other is located at the 3' of the exon-intron boundary.

[0108] In some embodiments of the engineered polynucleotides described herein, the target sequence comprises a splicing site in RNA (e.g., mRNA, such as a precursor mRNA). In some embodiments, (e.g., a first or second) the target sequence comprises a splicing site (e.g., 5'ss) in RNA (e.g., mRNA, such as a precursor mRNA).

[0109] In some embodiments, the two target sequences (e.g., a first target sequence and a second target sequence) are part of a single nucleic acid molecule (i.e., RNA, e.g., mRNA, such as a precursor mRNA). In some embodiments, the first and second target sequences are separated by a single nucleic acid molecule. In some embodiments, the first and second target sequences cross the exon-intron boundary of the single nucleic acid molecule. In some embodiments, the first and second target sequences do not cross the exon-intron boundary of the single nucleic acid molecule. In some embodiments, the first and second target sequences are adjacent to the exon-intron boundary of the single nucleic acid molecule. In some embodiments, both the first and second target sequences target the introns of the single nucleic acid molecule. In some embodiments, the first and second target sequences cross splicing sites within the single nucleic acid molecule. In some embodiments, the first and second target nucleic acid sequences do not cross splicing sites within the single nucleic acid molecule.

[0110] In some embodiments of the engineered polynucleotides described herein, the common sequence in the target sequence comprises about 2 to about 15 nucleotides, about 2 to about 10 nucleotides, or about 2 to about 8 nucleotides.

[0111] In some embodiments, the engineered polynucleotide is complementary to and binds to the 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. Non-limiting examples of target genes may include the microtubule-associated protein tau (MAPT).

[0112] In some embodiments, the target sequence includes an RNA sequence. In some embodiments, the RNA is nuclear RNA, cytoplasmic RNA, or mitochondrial RNA. In some embodiments, the target RNA sequence includes messenger RNA (mRNA), precursor messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), ribozymes, recombinant polynucleotides, branched polynucleotides, isolated RNA, guide RNA, oligonucleotides, nucleic acid probes, primers, 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 an mRNA precursor. In some embodiments, the engineered polynucleotide is not an antisense oligonucleotide.

[0113] In some embodiments, the target RNA sequence contains at least one exon. In some embodiments, the target RNA sequence contains at least one intron. In some embodiments, the target RNA sequence contains at least one exon or at least one intron. In some embodiments, the target RNA sequence contains at least one exon-intron boundary. The target RNA sequence may contain a gene-specific sequence. For example, the target RNA sequence may contain a sequence encoding a polypeptide. For example, the target RNA sequence may contain a sequence encoding tau. The target sequence may contain a sequence corresponding to an exon-intron boundary and a sequence corresponding to a specific gene.

[0114] In some implementations, the target sequence is an endogenous nucleic acid molecule. In some implementations, engineered polynucleotides bind to the target sequence via base pairing, such as Watson-Crick base pairing.

[0115] Fundraising portion

[0116] As described in this disclosure, engineered polynucleotides may include a recruitment moiety. The recruitment moiety may recruit one or more components of the spliceosome. In some embodiments, a recruitment moiety configured to recruit a spliceosome portion refers to a recruitment moiety that hybridizes with the spliceosome portion. In some embodiments, a recruitment moiety configured to recruit a spliceosome portion refers to a recruitment moiety that is at least 80%, 90%, or 100% complementary to the spliceosome portion or a sequence thereof. In some embodiments, "recruitment" refers to the formation of at least one hydrogen bond between the recruitment moiety and the spliceosome portion. In some embodiments, "recruitment" refers to hybridization between at least one nucleotide of the recruitment moiety and at least one nucleotide of the spliceosome portion. The recruitment moiety includes a hairpin structure. The hairpin may be a complete hairpin or may consist of an inner loop intercalation. The hairpin structure may consist of 13 to 17 nucleotides. The recruitment moiety may interact with stem-loop II of the U1 snRNA. The recruitment moiety may include a sequence complementary to or partially complementary to stem-loop II. For example, the stem-loop II of U1 snRNA contains the sequence 5'-GUAGGCCUCACGUUACCUAU-3', and the recruitment moiety may contain 5'-CCGGA-3'. U1-A can also bind to the stem-loop II of U1 snRNA. Hydrogen bridges between the stem-loop II and the hairpin / inner loop region can indirectly regulate polyadenylation and acetylation signaling of U1-A. The recruitment moiety may not silence the anchoring domain of U1-A in the stem-loop II. This interaction can regulate gene expression and acetylation.

[0117] In some embodiments of the engineered polynucleotides described herein, the recruitment portion comprises at least 70%, 80%, 85%, or 90% identical or complementary nucleotide sequences to the sequences shown in Table 2. In some embodiments, the recruitment portion comprises identical or complementary nucleotide sequences to the sequences shown in Table 2. In some embodiments, the engineered polynucleotide comprises 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% of a nucleotide sequence that is identical or complementary to any sequence selected from SEQ ID NO:1-2.

[0118] Table 2. Example recruitment portions of the engineered polynucleotides described in this paper.

[0119]

[0120] In some embodiments described herein, the engineered polynucleotide (e.g., the recruitment moiety) includes (e.g., secondary) structural features (see...). Figure 2A-2D In some embodiments, the engineered polynucleotide (e.g., the recruitment portion) comprises a top loop, an upper stem, an inner loop, a lower stem, or a combination thereof. In some embodiments, the engineered polynucleotide (e.g., the recruitment portion) comprises a loop (e.g., an inner loop) adjacent to a stem (e.g., a lower or upper stem), the stem comprising two complementary stem sequences. In some embodiments, the stem sequence of the stem (e.g., a lower or upper stem) comprises no more than about five, four, or three nucleotides. In some embodiments, the loop is an inner loop adjacent to a stem (e.g., a lower stem) and another stem (e.g., an upper stem), the other stem comprising two complementary stem sequences. In some embodiments, the inner loop comprises a nucleic acid sequence having no more than 10, 9, or 8 nucleotides. In some embodiments, the stem sequence of the other stem (e.g., an upper stem) comprises no more than about five, four, or three nucleotides. In some embodiments, the engineered polynucleotide (e.g., the recruitment portion) also comprises a top loop. In some embodiments, the top loop comprises a nucleic acid sequence having no more than 10, 9, 8, 7, 6, or 5 nucleotides.

[0121] 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.

[0122] In some embodiments, the recruitment portion is partially complementary to a post-transcriptional regulatory portion (or regulatory portion) (e.g., a spliceosome portion) comprising a ribonucleoprotein complex. For example, the recruitment portion may be partially complementary to a regulatory portion comprising a spliceosome ribonucleoprotein complex comprising small nucleoribonucleonucleotide (snRNA). In some embodiments, the recruitment portion is neither complementary to nor binds to the target sequence described herein. For example, the recruitment portion is neither complementary to nor binds to the mRNA precursor described herein.

[0123] Structural configuration

[0124] In various respects, engineered polynucleotides comprise one or more functional portions. These one or more portions may be present in engineered polypeptides in various structural configurations to allow the engineered polynucleotide to perform a given function (e.g., recruiting components of regulatory moieties or spliceosomes, or binding mRNA precursors). In some embodiments of the engineered polynucleotides described herein, one of the first and second targeting portions is located at the 5' of the recruiting portion, and the other of the first and second targeting portions is located at the 3' of the recruiting portion.

[0125] In some embodiments, the engineered polynucleotide has a structural arrangement from the 5'-end to the 3'-end as follows: a first targeting moiety, a recruitment moiety, and a second targeting moiety. In some embodiments, the engineered polynucleotide has a structural arrangement from the 5'-end to the 3'-end as follows: a second targeting moiety, a recruitment moiety, and a first targeting moiety.

[0126] Example polynucleotides

[0127] In some embodiments of the engineered polynucleotides described herein, the engineered polynucleotides comprise 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 polynucleotides comprise a length of 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 some implementations, the engineered polynucleotide comprises a length of 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 some implementations, 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 nucleotides or a length between any two of the aforementioned values.

[0128] In some embodiments of the engineered polynucleotides described herein, the recruitment portion comprises at least 70%, 80%, 85%, or 90% identical or complementary nucleotide sequences to the sequences shown in Table 3. In some embodiments, the recruitment portion comprises identical or complementary nucleotide sequences to the sequences shown in Table 3.

[0129] Table 3. Example sequences of engineered polynucleotides containing recruitment and targeting moieties.

[0130]

[0131] In some implementations, engineered polynucleotides can be generated from precursors of engineered polynucleotides. In some cases, the precursors of engineered polynucleotides can be linear. For example, the precursor of an engineered polynucleotide can be a linear polynucleotide transcribed from a plasmid. In another example, the precursor of an engineered polynucleotide can be constructed as a linear polynucleotide having portions such as a ribozyme moiety and a linker moiety that allow the engineered polynucleotide to circularize in the cell. The linear engineered polynucleotide having the linker and ribozyme moiety can be transfected into cells, where it can be circularized. In some cases, the engineered polynucleotide can be circular. In some cases, the engineered polynucleotide comprises DNA, RNA, or both. In some cases, the precursor of an engineered polynucleotide comprises a precursor of an engineered polynucleotide. In some cases, the precursor of an engineered polynucleotide can be used to generate the engineered polynucleotide.

[0132] In some implementations, the engineered polynucleotide comprises at least one secondary structure (such as those described anywhere herein). For example, the engineered polynucleotide comprises at least one, two, three, four, or more secondary structures, wherein the secondary structure can be any or any combination of an apical loop, stem, stem-loop, or inner loop. One or more secondary structures can perform a variety of different functions. For example, the secondary structure can provide stability or otherwise help stabilize the engineered polynucleotide. The secondary structure can bind to or interact with polypeptides or other polynucleotides. For example, the secondary structure can interact with components of the spliceosome.

[0133] In some embodiments, the recruitment moiety of the polynucleotide comprises at least one, two, three, four, or more secondary structures. In some embodiments, the targeting moiety does not have a secondary structure. In some embodiments, the secondary structure is a top loop comprising at least two, three, four, five, six, seven, eight, nine, ten, or more nucleotides. In some embodiments, the top loop is complementary to and binds to the regulatory moiety. In some embodiments, the top loop is neither complementary to nor binds to the regulatory moiety. In some embodiments, the secondary structure is at least one stem. In some embodiments, the engineered polynucleotide comprises two stems, one being an upper stem closer to the top loop and the other a lower stem closer to the targeting moiety. In some embodiments, the upper stem comprises at least two, four, six, eight, ten, or more nucleotides, wherein the nucleotides pair to form the upper stem. In some embodiments, the upper stem is complementary to and binds to the regulatory moiety. In some embodiments, the upper stem is neither complementary to nor binds to the regulatory moiety. In some embodiments, the secondary structure is a lower stem, wherein the lower stem comprises at least two, four, six, eight, ten, or more nucleotides, wherein the nucleotides pair to form the lower stem. In some embodiments, the lower stem is complementary to and binds to the regulatory moiety. In some embodiments, the lower stem is neither complementary to nor binds to the regulatory moiety. In some embodiments, the engineered polynucleotide includes an inner loop between the upper and lower stems. In some embodiments, the inner loop includes at least two, three, four, five, six, seven, eight, nine, ten, or more nucleotides. In some embodiments, the inner loop is complementary to and binds to the regulatory moiety. In some embodiments, the inner loop is neither complementary to nor binds to the regulatory moiety. In some embodiments, the engineered polynucleotide includes a secondary structure containing a apical loop, an upper stem, an inner loop, and a lower stem, wherein the upper stem and inner loop are at least partially complementary to and bind to the regulatory moiety. In some embodiments, the upper stem and inner loop are complementary to and bind to the regulatory moiety comprising snRNA. In some embodiments, the snRNA is a U1 snRNA, such as US-A snRNA. In some embodiments, the snRNA is a U2 snRNA.

[0134] In some embodiments, the nucleic acid sequence of at least one secondary structure is partially complementary to the regulatory moiety comprising a ribonucleoprotein complex. In some embodiments, the nucleic acid sequence of at least one secondary structure is not complementary to the target nucleic acid sequence. In some embodiments, the engineered polynucleotide comprises a secondary structure of at least one nucleic acid. In some embodiments, the engineered polynucleotide comprises at least one, two, three, four, or more secondary structures of nucleic acids. In some embodiments, at least one secondary structure increases the binding between the recruitment moiety and the regulatory moiety. In some embodiments, at least one secondary structure stabilizes the assembly of the regulatory moiety. In some embodiments, at least one secondary structure stabilizes the assembly of the regulatory moiety with other additional moieties. In some embodiments, at least one secondary structure increases the regulatory efficiency of the expression or activity of a gene encoded by the target sequence. In some embodiments, at least one secondary structure increases the regulatory specificity of the expression or activity of a gene encoded by the target sequence. In some embodiments, 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 digestive degradation by nucleases. In some embodiments, at least one secondary structure increases the half-life of the engineered polynucleotide. In some implementations, at least one secondary structure reduces the immunogenicity induced by engineered polynucleotides.

[0135] 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 includes a apical loop, an upper stem, an inner 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 portion 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 that includes a stem-loop, a cross, a toe hold, a mismatched protrusion, or any combination thereof. In some embodiments, the engineered polynucleotide comprises a secondary structure that includes a apical loop, an upper stem, an inner loop, or a lower stem. In some embodiments, the engineered polynucleotide comprises a secondary structure that includes a apical loop, an upper stem, an inner loop, and a lower stem. In some cases, the secondary structure may include a stem, a hairpin loop, a pseudoknot, a protrusion, an inner loop, a polycyclic structure, a G-tetramer, or any combination thereof. In some embodiments, the engineered polynucleotide may be in the A-form, B-form, 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.

[0136] In some implementations, at least one secondary structure increases the binding between the fundraising and regulating portions. In some implementations, compared to the binding between the fundraising and regulating portions without a secondary structure, at least one secondary structure increases the binding between the fundraising and regulating portions by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, up to 2 times, 3 times, 4 times, 5 times, 10 times, or more.

[0137] In some embodiments, at least one chemical modification increases the binding between the recruiting portion and the regulating portion. In some embodiments, at least one chemical modification increases the binding between the recruiting portion and the regulating portion by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, up to 2, 3, 4, 5, 10, or more times compared to the binding between the recruiting portion without chemical modification of the regulating portion.

[0138] In some embodiments, when the regulatory portion binds to the target sequence, at least one chemical modification of the engineered polynucleotide stabilizes the assembly of the splice containing the regulatory portion. In some embodiments, the assembly of the splice containing the regulatory portion is stabilized by the engineered polynucleotide containing the chemical modification by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, up to 2, 3, 4, 5, 10, or more times compared to comparable polynucleotides without chemical modification. In some embodiments, the regulatory portion is U1 (U1SNP), U2 (U2SNP), U4, U5, U6, U11, U12, U14, or U16 of the splice. In some embodiments, the regulatory portion is U1SNP of the splice. In some embodiments, the regulatory portion is U1-A of the splice. In some embodiments, the regulatory portion is U2SNP of the splice. In some embodiments, at least one chemical modification of the engineered polynucleotide stabilizes the assembly of a splice comprising a regulatory portion and at least one additional portion. For example, at least one chemical modification of the engineered polynucleotide stabilizes the assembly of a splice comprising a regulatory portion containing U1-A and at least one additional portion containing 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 the splice.

[0139] In some embodiments, at least one chemical modification improves the efficiency of the engineered polynucleotide in regulating the expression or activity of a gene encoded by a target sequence compared to comparable polynucleotides without chemical modification. In some embodiments, the efficiency of the engineered polynucleotide in regulating the expression or activity of a gene encoded by a target sequence is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, up to 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more compared to the efficiency of comparable polynucleotides without chemical modification.

[0140] In some embodiments, at least one chemical modification increases the specificity of the engineered polynucleotide in regulating the expression or activity of the gene encoded by the target sequence compared to comparable polynucleotides without chemical modification. In some embodiments, the specificity of the engineered polynucleotide in regulating the expression or activity of the gene encoded by the target sequence is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, up to 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more compared to the efficiency of the engineered polynucleotide in regulating the expression or activity of the gene encoded by the target sequence.

[0141] In some embodiments, at least one chemical modification increases the resistance of the engineered polynucleotide to hydrolytic degradation (e.g., degradation by endonucleases). In some embodiments, at least one chemical modification increases the resistance of the engineered polynucleotide to hydrolytic degradation compared to the resistance of comparable engineered polynucleotides without chemical modification. In some embodiments, the resistance of engineered polynucleotides containing at least one chemical modification to hydrolytic degradation is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, up to 2, 3, 4, 5, 10, or more times compared to the resistance of comparable engineered polynucleotides without chemical modification.

[0142] In some embodiments, at least one chemical modification increases the resistance of the engineered polynucleotide to nuclease digestion and degradation. In some embodiments, at least one chemical modification increases the resistance of the engineered polynucleotide to nuclease digestion and degradation compared to comparable engineered polynucleotides without chemical modification. In some embodiments, the resistance of engineered polynucleotides containing at least one chemical modification to nuclease digestion and degradation is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, up to 2, 3, 4, 5, 10, or more times compared to comparable engineered polynucleotides without chemical modification.

[0143] In some embodiments, at least one chemical modification increases the half-life of the engineered polynucleotide compared to the half-life of a comparable engineered polynucleotide without chemical modification. In some embodiments, the half-life of an engineered polynucleotide containing at least one chemical modification is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, up to 2, 3, 4, 5, 10, or more times compared to the half-life of a comparable engineered polynucleotide without chemical modification. The chemical modification increases the half-life of the engineered polynucleotide.

[0144] 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 a comparable engineered polynucleotide without chemical modification. In some embodiments, the immunogenicity of an engineered polynucleotide containing at least one chemical modification is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, to 2, 3, 4, 5, 10, or more times compared to the immunogenicity of a comparable engineered polynucleotide without chemical modification.

[0145] Chemical modification

[0146] In some embodiments described herein, the engineered polynucleotide comprises at least one chemical modification. As described in this disclosure, the chemical modification may confer structural or functional advantages to the engineered polynucleotide (e.g., increased half-life, reduced immunogenicity, enhanced resistance to hydrolysis or enzymatic degradation, improved reactivity or binding with peptides (e.g., components of spliceosomes) or polynucleotides (e.g., components of mRNA precursors or spliceosomes).

[0147] In some embodiments, all nucleotides of the targeting moiety are linked by phosphate thioester bonds. In some embodiments, all nucleotides of the targeting moiety contain a 2'O-methyl modification. The 2' modification can prevent nuclease degradation and / or increase the affinity of the targeting moiety for the mRNA precursor target.

[0148] In some embodiments, all nucleotides of the recruitment moiety are linked by phosphate thioester bonds. In some embodiments, three nucleotides of the recruitment moiety contain a 2'O-methyl modification. The 2' modification can induce molecular dynamic changes in the recruitment moiety, thereby promoting a conformational change in stem-loop II of the U1-snRNA and the binding of the recruitment moiety to the U1-snRNA.

[0149] 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 in the engineered polynucleotide are chemically modified nucleotides. In some embodiments, at least about 50%, 60%, 70%, 80%, or 90% of the nucleotides in the engineered polynucleotide are 2'-modified (e.g., 2'-methoxy, 2'-methoxymethyl, or 2'-methoxyethyl) nucleotides. In some embodiments, the engineered polynucleotide comprises at least one phosphate thioester nucleotide linker. In some embodiments, at least about 50%, 60%, 70%, 80%, or 90% of the nucleotide links in the engineered polynucleotide are chemically modified. In some embodiments, at least about 50%, 60%, 70%, 80%, or 90% of the nucleotide links are phosphate thioesters.

[0150] In some embodiments, the engineered polynucleotide comprises at least one chemical modification of the nucleic acid. In some embodiments, the engineered polynucleotide comprises at least one, two, three, four, or more chemical modifications of the nucleic acid. In some embodiments, at least one chemical modification increases the binding between the recruitment moiety and the regulatory moiety. In some embodiments, at least one chemical modification stabilizes the assembly of the regulatory moiety. In some embodiments, at least one chemical modification stabilizes the assembly of the regulatory moiety with other additional moieties. In some embodiments, at least one chemical modification increases the efficiency of regulating the expression or activity of a gene encoded by a target sequence. In some embodiments, at least one chemical modification increases the specificity of regulating the expression or activity of a gene encoded by a target sequence. In some embodiments, at least one chemical modification increases the resistance of the engineered polynucleotide to hydrolytic degradation. In some embodiments, at least one chemical modification increases the resistance of the engineered polynucleotide to digestive degradation by nucleases. In some embodiments, at least one chemical modification increases the half-life of the engineered polynucleotide. In some embodiments, at least one chemical modification reduces the immunogenicity induced by the engineered polynucleotide.

[0151] In some embodiments, the chemical modification of the engineered polynucleotide includes substitution of at least one or both non-linked phosphate oxygen atoms in the phosphodiester backbone of the engineered polynucleotide. In some embodiments, at least one chemical modification of the engineered polynucleotide includes substitution of one or more linked phosphate oxygen atoms in the phosphodiester backbone of the engineered polynucleotide. A non-limiting example of chemical modification of the phosphate oxygen atom is a sulfur atom. Additional non-limiting examples are included in Table 3. In some embodiments, the chemical modification of the engineered polynucleotide includes at least one chemical modification of the sugar of the nucleotide of the engineered polynucleotide. In some embodiments, the chemical modification of the engineered polynucleotide includes at least one chemical modification of the sugar of the nucleotide, wherein the chemical modification includes at least one locked nucleic acid (LNA). In some embodiments, the chemical modification of the engineered polynucleotide includes at least one chemical modification of the sugar of the nucleotide of the engineered polynucleotide comprising at least one unlocking nucleic acid (UNA). In some embodiments, the chemical modification of the engineered polynucleotide includes at least one chemical modification of the sugar, including modification of the sugar component, wherein the sugar is a ribose sugar. In some embodiments, the chemical modification of the engineered polynucleotide includes at least one chemical modification of the ribose component of the nucleotide containing the engineered polynucleotide. In some embodiments, the chemical modification comprises 2'-F-RNA instead of 2'-O-methyl modification. In this case, the 2'-F-RNA and mRNA precursor duplex do not activate RNase H (which is degraded by nuclease digestion) and are determined to be more stable by a higher melting temperature (Tm) than the 2'-O-methyl-RNA and mRNA precursor duplex.

[0152] In some embodiments, the chemical modification of the engineered polynucleotide includes at least one chemical modification, which includes replacing the phosphate moiety of the engineered polynucleotide with a dephosphate linker. In some embodiments, the chemical modification of the engineered polynucleotide includes at least one chemical modification of the phosphate backbone of the engineered polynucleotide. In some embodiments, the engineered polynucleotide comprises a thiophosphate group. In some embodiments, the chemical modification of the engineered polynucleotide includes at least one chemical modification, which includes modification of the nucleotide bases of the engineered polynucleotide. In some embodiments, the chemical modification of the engineered polynucleotide includes at least one chemical modification comprising a non-natural base of the nucleotide. In some embodiments, the chemical modification of the engineered polynucleotide includes at least one chemical modification comprising a morpholino group, a cyclobutyl group, a pyrrolidine group, or a peptide nucleic acid (PNA) nucleoside substitute. In some embodiments, the chemical modification of the engineered polynucleotide includes at least one chemical modification comprising at least one stereopure nucleic acid. In some embodiments, at least one chemical modification may be located near the 5' end of the engineered polynucleotide. In some embodiments, at least one chemical modification may be located near the 3' end of the engineered polynucleotide. In some implementations, at least one chemical modification may be located near both the 5' and 3' ends of the engineered polynucleotide.

[0153] In some embodiments, at least one chemical modification of the engineered polynucleotide includes any one or any combination of modifications: modification of one or both non-linked phosphate oxygens in the phosphodiester backbone; modification of one or more linked phosphate oxygens in the phosphodiester backbone; modification of the ribose sugar component; replacement of the phosphate moiety with a "dephosphorylated" linker; modification or replacement of a naturally occurring nucleobase; modification of the ribose-phosphate backbone; modification of the 5' end of the polynucleotide; modification of the 3' end of the polynucleotide; modification of the deoxyribose-phosphate backbone; substitution of phosphate groups; modification of the ribose-phosphate backbone; modification of the nucleotide sugar; modification of the nucleotide base; or stereopurity of the nucleotide. Examples of chemical modifications to engineered polynucleotides are shown in Table 4.

[0154] Table 4. Examples of chemical modifications

[0155]

[0156]

[0157]

[0158] Modification of the phosphate backbone

[0159] In some embodiments, the chemical modification includes modification of one or both non-linked phosphate oxygen atoms in the phosphate diester backbone or modification of one or more linked phosphate oxygen atoms in the phosphate diester backbone. As used herein, "alkyl" means a straight-chain or branched saturated hydrocarbon group. Examples of alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl or isopropyl), butyl (e.g., n-butyl, isobutyl, or tert-butyl), or pentyl (e.g., n-pentyl, isopentyl, or neopentyl). The alkyl group may contain from 1 to about 20, from 2 to about 20, from 1 to about 12, from 1 to about 8, from 1 to about 6, from 1 to about 4, or from 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, anthracene, phenanthryl, indenyl, or indenyl. In some embodiments, the aryl group has from 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-chain or branched hydrocarbon chain containing 2 to 12 carbon atoms, characterized by having one or more triple bonds. Examples of alkynyl groups may include ethynyl, propynyl, 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, diphenylmethyl, and triphenylmethyl. “Cycloalkyl” refers to a cyclic, bicyclic, tricyclic, or polycyclic non-aromatic hydrocarbon group having 3 to 12 carbon atoms. Examples of cycloalkyl moieties include, but are not limited to, cyclopropyl, cyclopentyl, and cyclohexyl. “Heterocyclic” refers to a monovalent group in a heterocyclic system. Representative heterocyclic groups include, but are not limited to, tetrahydrofuranyl, tetrahydrothiophenyl, pyrrolylalkyl, pyrrolidone, piperidinyl, pyrrololinyl, piperazine, dioxane, dioxolane, and diazapyridine. Basic, oxygen and nitrogen Basic, sulfur-nitrogen The group consists of methyl and morpholinoyl groups. "Heteroaryl" refers to a monovalent group in a heteroaryl ring system. Examples of heteroaryl moieties include imidazolyl, oxazolyl, thiazolyl, triazolyl, pyrrolel, furanyl, indolyl, thienylpyrazolyl, pyridyl, pyrazinyl, pyridazinyl, pyrimidinyl, inazinyl, purinel, naphthidyl, quinolinyl, and pteridinyl.

[0160] In some embodiments, the phosphate group of a chemically modified nucleotide can be modified by replacing one or more oxygen atoms with different substituents. In some embodiments, the chemically modified nucleotide may include replacing an unmodified phosphate moiety with a modified phosphate ester as described herein. In some embodiments, modification of the phosphate backbone may include alterations resulting in uncharged joints or charged joints with an asymmetric charge distribution. Examples of modified phosphate groups may include thiophosphates, thioacetic acid phosphonates, selenophosphates, boran phosphates, boran phosphate esters, hydrophosphonates, aminophosphates, alkyl or aryl phosphonates, and phosphate triesters. In some embodiments, one of the non-bridging phosphate oxygen atoms in the phosphate backbone moiety may be replaced by any of the following groups: sulfur (S), selenium (Se), BR3 (where R may be, for example, hydrogen, alkyl, or aryl), C (e.g., alkyl, aryl, etc.), H, NR2 (where R may be, for example, hydrogen, alkyl, or aryl), or OR (where R may be, for example, alkyl or aryl). The phosphorus atom in the unmodified phosphate group may be achiral. However, replacing one of the non-bridging oxygen atoms with one of the aforementioned atoms or atomic groups can make the phosphorus atom chiral. The phosphorus atom in the phosphate ester group modified in this way is the stereocenter. The stereoisomeric phosphorus atom can have an "R" configuration (Rp in this document) or an "S" configuration (Sp in this document). In some cases, the engineered polynucleotide comprises a stereopure nucleotide comprising the S conformation of a thiophosphate or the R conformation of a thiophosphate. In some embodiments, the chiral phosphate ester product is present in a diastereomeric excess of 50%, 60%, 70%, 80%, 90%, or more. In some embodiments, the chiral phosphate ester product is present in a 95% diastereomeric excess. In some embodiments, the chiral phosphate ester product is present in a 96% diastereomeric excess. In some embodiments, the chiral phosphate ester product is present in a 97% diastereomeric excess. In some embodiments, the chiral phosphate ester product is present in a 98% diastereomeric excess. In some embodiments, the chiral phosphate ester product is present in a 99% diastereomeric excess. In some embodiments, both unbridged oxygen atoms of the dithiophosphate can be replaced by sulfur. The phosphorus center in the dithiophosphate can be achiral, which prevents the formation of diastereomers of oligonucleotides. In some embodiments, modification of one or both unbridged oxygen atoms may also include replacing the unbridged oxygen atoms with groups independently selected from S, Se, B, C, H, N, and OR (R may be, for example, alkyl or aryl). In some embodiments, the phosphate linker can also be modified by replacing the bridging oxygen (i.e., the oxygen atom linking the phosphate ester to the nucleoside) with nitrogen (bridged aminophosphate ester), sulfur (bridged thiophosphate ester), and carbon (bridged methylene phosphonate ester). The substitution can occur at one or both linked oxygen atoms.

[0161] In some embodiments, the nucleic acid comprises a linker nucleic acid. Nucleic acids can be linked together using any inter-nucleic acid linker. Two classes of inter-nucleic acid linker groups are primarily defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing inter-nucleic acid linkers include, but are not limited to, phosphodiesters, triphosphates, methylphosphonates, aminophosphates, and thiophosphates (P=S). Representative phosphorus-free inter-nucleic acid linker groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiesters (-OC(O)-S-), thiocarbamates (-OC(O)(NH)-S-); siloxanes (-O-Si(H)2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)). In some embodiments, inter-nucleic acid linkers with chiral atoms can be prepared as racemic mixtures, separate enantiomers, such as alkylphosphonates and thiophosphates. Non-natural nucleic acids can contain a single modification. Non-natural nucleic acids can contain multiple modifications within one part or between different parts.

[0162] Modification of the main chain phosphate esters of nucleic acids includes, but is not limited to, methylphosphonates, thiophosphates, aminophosphates (bridged or non-bridged), phosphate triesters, dithiophosphates, and boron phosphates, and can be used in any combination. Other non-phosphate linkages may also be used.

[0163] In some implementations, main chain modifications (e.g., nucleotide linkages of methylphosphonates, thiophosphates, aminophosphates, and dithiophosphates) can confer immunomodulation to the modified nucleic acids to regulate their activity and / or enhance their in vivo stability.

[0164] In some cases, phosphorus derivatives (or modified phosphate groups) are attached to sugar or sugar analog moieties, and can be monophosphates, diphosphates, triphosphates, alkylphosphonates, thiophosphates, dithiophosphates, aminophosphates, etc.

[0165] In some cases, backbone modifications involve replacing phosphodiester bonds with alternative moieties such as anionic, neutral, or cationic groups. Examples of such modifications include: anionic nucleotide interlinking; N3' to P5' aminophosphate modifications; boron phosphate DNA; oligonucleotide precursors; neutral nucleotide interlinking, such as methylphosphonates; amide-linked DNA; methylene (methylimino) bonds; methyl acetal and thiomethyl acetal bonds; backbones containing sulfonyl groups; morpholine oligomers; peptide nucleic acids (PNAs); and positively charged deoxyribonucleic acid guanidine (DNG) oligomers. Modified nucleic acids may contain chimeric or hybrid backbones containing one or more modifications, such as combinations of phosphodiester bonds, such as combinations of phosphodiester and thiophosphate bonds.

[0166] Phosphate ester substitutes include, for example, short-chain alkyl or cycloalkyl nucleotide linkages, mixed heteroatom and alkyl or cycloalkyl nucleotide linkages, or linkages between one or more short-chain heteroatom or heterocyclic nucleotides. These substitutes include those with morpholino linkages (partially formed from the sugar moiety of the nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; methylacetyl and thiomethylacetyl backbones; methylenemethylacetyl and thiomethylacetyl backbones; olefin-containing backbones; aminosulfonate backbones; methyleneimino and methylenehydrazine backbones; sulfonate and sulfonamide backbones; amide backbones; and other items with mixed N, O, S, and CH2 components. It should also be understood that in nucleotide substitutes, both the sugar and phosphate moiety of the nucleotide can be replaced by, for example, an amide-type linkage (aminoethylglycine) (PNA). Other types of molecules (conjugates) can also be linked to nucleotides or nucleotide analogs to enhance, for example, cellular uptake. Conjugates can be chemically linked to nucleotides or nucleotide analogs. Such conjugates include, but are not limited to, lipid moieties such as cholesterol moieties, thioethers such as hexyl-S-triphenylmethylthiol, thiocholesterol, aliphatic chains such as dodecyl glycol or undecyl residues, phospholipids such as di-hexadecyl-racemic-glycerol or triethylammonium 1-di-O-hexadecyl-racemic-glycerol-SH-phosphonate, polyamines or polyethylene glycol chains, or adamantaneacetic acid, palmityl moieties, or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties.

[0167] In some embodiments, the chemical modifications described herein include modifications to the phosphate backbone. In some embodiments, the engineered polynucleotides described herein comprise at least one chemically modified phosphate backbone. Example chemical modifications to the phosphate group or backbone may include replacing one or more oxygen atoms with different substituents. Furthermore, modified nucleotides present in the engineered polynucleotide may include replacing an unmodified phosphate moiety with a modified phosphate ester as described herein. In some embodiments, modifications to the phosphate backbone may include alterations resulting in uncharged joints or charged joints having an asymmetric charge distribution. Examples of modified phosphate groups may include thiophosphates, thioacetic acid phosphonates, selenophosphates, borane phosphates, borane phosphate esters, hydrophosphonates, aminophosphates, alkyl or aryl phosphonates, and phosphate triesters. In some embodiments, one of the non-bridged phosphate oxygen atoms in the phosphate backbone can be replaced by any of the following groups: sulfur (S), selenium (Se), BR3 (where R can be, for example, hydrogen, alkyl, or aryl), C (e.g., alkyl group, aryl group, etc.), H, NR2 (where R can be, for example, hydrogen, alkyl, or aryl), or OR (where R can be, for example, alkyl or aryl). The phosphorus atom in the unmodified phosphate group is achiral. However, replacing one of the non-bridged oxygen atoms with one of the aforementioned atoms or groups can make the phosphorus atom chiral; that is, the phosphorus atom in the phosphate group modified in this way is a stereocenter. The stereoisomeric phosphorus atom can have an "R" configuration (Rp in this document) or an "S" configuration (Sp in this document). In this case, the chemically modified engineered polynucleotide can be stereopure (e.g., confirmed by S or R). In some cases, the chemically modified engineered polynucleotide contains a stereopure phosphate modification. For example, the chemically modified engineered polynucleotide contains the S conformation of a thiophosphate or the R conformation of a thiophosphate.

[0168] Both unbridged oxygen atoms in the dithiophosphate are replaced with sulfur. The phosphorus center in the dithiophosphate is achiral, which prevents the formation of diastereomeric oligonucleotides. In some embodiments, modification of one or both unbridged oxygen atoms may further include replacing the unbridged oxygen atoms with groups independently selected from S, Se, B, C, H, N, and OR (R may be, for example, alkyl or aryl).

[0169] Phosphate linkers can also be modified by replacing the bridging oxygen (i.e., linking the phosphate ester to the oxygen of the nucleoside) with nitrogen (bridged aminophosphate), sulfur (bridged thiophosphate), and carbon (bridged methylene phosphonate). The substitution can occur at any one or both oxygen links.

[0170] Replacement of phosphate ester portion

[0171] In some embodiments, at least one phosphate group of the engineered polynucleotide may be chemically modified. In some embodiments, the phosphate group may be replaced by a phosphorus-free linker. In some embodiments, the phosphate moiety may be replaced by a dephosphated linker. In some embodiments, the charged phosphate group may be replaced by a neutral group. In some cases, the phosphate group may be replaced by methylphosphonate, hydroxyamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thiomethyl acetal, methyl acetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazine, methylenedimethylhydrazine, and methyleneoxymethylimino. In some embodiments, the nucleotide analogs described herein may also be modified at the phosphate group. Modified phosphate groups may include modifications at the link between two nucleotides using thiophosphates, chiral thiophosphates, dithiophosphates, triphosphates, aminoalkyl phosphates, methyl and other alkylphosphonates (including 3'-alkylene phosphonates) and chiral phosphonates, hypophosphonates, aminophosphates (e.g., 3'-aminoaminophosphates and aminoalkylaminophosphates), thioaminophosphates, thioalkylphosphonates, thioalkyl phosphates, and boron phosphates. The phosphate or modified phosphate link between the two nucleotides may be via a 3'-5' or 2'-5' link, and the link may contain reverse polarity, such as 3'-5' to 5'-3' or 2'-5' to 5'-2'.

[0172] Substitution of phosphate groups

[0173] In some embodiments, the chemical modifications described herein include modifications via the substitution of phosphate ester groups. In some embodiments, the engineered polynucleotides described herein comprise at least one chemical modification that includes the substitution or replacement of phosphate ester groups. Example phosphate ester group substitution may include a phosphorus-free linker. In some embodiments, phosphate ester group substitution or replacement may include replacing a charged phosphate ester group with a neutral moiety. Example moiety that may substitute the phosphate ester group may include methylphosphonate, hydroxylamine, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thiomethyl acetal, methyl acetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazine, methylenedimethylhydrazine, and methyleneoxymethylimino.

[0174] Modification of the ribophosphate backbone

[0175] In some embodiments, the chemical modifications described herein include 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 may include scaffolds that can mimic nucleic acids or be constructed, wherein the phosphate linker and ribose are replaced by nuclease-resistant nucleosides or nucleotide substitutes. In some embodiments, nucleobases may be bound by the substituted backbone. Examples may include morpholino, cyclobutyl, pyrrolidine, and peptide nucleic acid (PNA) nucleoside substitutes.

[0176] Sugar modification

[0177] In some embodiments, the chemical modifications described herein include modifications of sugars. In some embodiments, the engineered polynucleotides described herein comprise at least one chemically modified sugar. Example chemically modified sugars may include 2' hydroxyl groups (OH) modified or substituted with a number of different "oxygen" or "deoxy" substituents. In some embodiments, modification of the 2' hydroxyl group can enhance the stability of the nucleic acid because the hydroxyl group can no longer be deprotonated to form a 2'-alkoxide ion. The 2'-alkoxide can be catalytically degraded by intramolecular nucleophilic attack on the linker phosphorus atom. Examples of "oxygen"-2' hydroxyl group modifications may include alkoxy or aryloxy (OR, where "R" can be, for example, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar); polyethylene glycol (PEG); O(CH2CH2O). n CH2CH2OR, where R can be, for example, H or an optionally substituted alkyl group, and n can be from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20). In some embodiments, the "oxygen"-2' hydroxyl group modification can include (LNA, where the 2' hydroxyl group can be, for example, via Ci -6 An alkylene or Cj-6 heteroalkylene bridge is attached to the 4' carbon of the same ribose, wherein example bridges may include methylene, propylene, ether, or amino bridges; O-amino (wherein the amino group may be, for example, NH2; alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy, O(CH2). n-Amino (wherein the amino group can be, for example, NH2; alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino). In some embodiments, the "oxy"-2' hydroxyl group modification can include methoxyethyl (MOE) (OCH2CH2OCH3, for example, a PEG derivative). In some cases, deoxygenation modification can include hydrogen (i.e., deoxyribose, for example, in the protruding portion of a partial dsRNA); halogenation (e.g., bromine, chlorine, fluorine, or iodine); amino (wherein the amino group can be, for example, NH2; alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH (CH2CH2NH). n CH2CH2-amino (wherein the amino group can be, for example, as described herein), NHC(O)R (wherein the R group can be, for example, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl, and alkynyl groups, which may optionally be substituted with an amino group, for example, as described herein. In some cases, the glycosyl group may also contain one or more carbons having a stereochemical configuration opposite to that of the corresponding carbon in ribose. Thus, modified nucleic acids may include nucleotides containing, for example, arabinose as sugars. The nucleotide “monomer” may have an α-link at the Γ position on the sugar, for example, an α-nucleoside. Modified nucleic acids may also include “debased” sugars that lack a nucleobase at C-. Debased sugars may also be further modified at one or more constituent sugar atoms. Modified nucleic acids may also include one or more sugars in the form of L, such as L-nucleosides. In some aspects, the engineered polynucleotides described herein include glycosylribose, which is a 5-membered ring with oxygen. Examples of modified nucleosides and modified nucleotides may include the substitution of oxygen in the ribose (e.g., with sulfur (S), selenium (Se), or alkylene, such as methylene or ethylene); the addition of a double bond (e.g., replacing the ribose with a cyclopentenyl or cyclohexenyl group); ring contraction of the ribose (e.g., forming a 4-membered ring of cyclobutane or oxetane); ring expansion of the ribose (e.g., forming a 6- or 7-membered ring with additional carbon or heteroatoms, such as anhydride hexitol, araitol, mannitol, cyclohexyl, cyclohexenyl, and morpholino, which also has an aminophosphate backbone). In some embodiments, the modified nucleotides may include polycyclic forms (e.g., tricyclic) and “unlocked” forms, such as diol nucleic acids (GNAs) (e.g., R-GNA or S-GNA, wherein the ribose is replaced by a diol unit attached to a phosphodiester bond) and threonic acids. In some implementations, modification of the sugar in the engineered polynucleotide includes modifying the engineered polynucleotide to include locked nucleic acid (LNA), unlocked nucleic acid (UNA), or bridging nucleic acid (BNA).

[0178] Modification of ribose components

[0179] In some embodiments, the engineered polynucleotides described herein comprise at least one chemical modification of the ribose component. In some embodiments, the chemical modification of the ribose component may include 2'-O-methyl, 2'-O-methoxy-ethyl (2'-MOE), 2'-fluoro, 2'-aminoethyl, 2'-deoxy-2'-fluoroarabinonucleotide, 2'-deoxy, 2'-O-methyl, 3'-thiophosphate, 3'-phosphonoacetate (PACE), or 3'-phosphonothioacetate (thioPACE). In some embodiments, the chemical modification of the ribose component comprises a non-natural nucleic acid. In some cases, the non-natural nucleic acid includes modifications at the 5'- and 2'-positions of the sugar ring, such as 5'-CH2-substituted 2'-O-protected nucleosides. In some cases, non-natural nucleic acids include nucleoside dimers prepared for incorporation into oligonucleotides with amide linkages, wherein the 3' linking nucleoside (5' to 3') in the dimer comprises 2'-OCH3 and 5'-(S)-CH3. Non-natural nucleic acids may include 2'-substituted 5'-CH2 (or O)-modified nucleosides. Non-natural nucleic acids may include 5'-methylenephosphonate DNA and RNA monomers and dimers. Non-natural nucleic acids may include 2'-substituted 5'-phosphonate monomers and other modified 5'-phosphonate monomers. Non-natural nucleic acids may include 5'-modified methylenephosphonate monomers. Non-natural nucleic acids may include analogs of 5' or 6'-phosphonate ribonucleosides containing hydroxyl groups at the 5' and / or 6' positions. Non-natural nucleic acids may include 5'-phosphonate deoxyribonucleosides monomers and dimers having 5'-phosphate groups. Non-natural nucleic acids may include nucleosides having a 6'-phosphonate group, wherein the 5' or / and 6'-positions are unsubstituted or substituted with a thiotert-butyl group (SC(CH3)3) (and its analogues); a methylene amino group (CH2NH2) (and its analogues); or a cyano group (CN) (and its analogues).

[0180] In some embodiments, non-natural nucleic acids also include modifications to the sugar moiety. In some cases, the nucleic acid contains one or more nucleosides in which the sugar moiety has been modified. Such sugar-modified nucleosides can confer enhanced nuclease stability, increased binding affinity, or some other beneficial biological properties. In some embodiments, the nucleic acid comprises a chemically modified furanose ribosome moiety. Examples of chemically modified furanose ribosomes include, but are not limited to, the addition of substituents (including 5' and / or 2' substituents; bridging two ring atoms to form a bicyclic nucleic acid; using S, N(R), or C(R1)(R2)(R=H, C1-C)). 12 Alkyl or protecting group replacing ribosyl epoxy atoms; and combinations thereof.

[0181] In some cases, the engineered polynucleotides described herein contain modified sugars or sugar analogs. Therefore, in addition to ribose and deoxyribose, the sugar moiety can be a pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose, lythose, or a sugar “analyte” cyclopentyl group. The sugar can be in pyranosyl or furanosyl form. The sugar moiety can be a ribose, deoxyribose, arabinose, or a 2'-O-alkylribose furanoside, and the sugar can be attached to its respective heterocyclic base in an [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.

[0182] Modifications to the sugar moiety include both natural and non-natural modifications to ribose and deoxyribose. 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, wherein the alkyl, alkenyl, and alkynyl groups can be substituted or unsubstituted C1 to C2 groups. 10 Alkyl or C2 to C 10 Alkenyl and ynyl groups. 2' sugar modifications also include, but are not limited to, -O[(CH2)] n O] m CH3, -O(CH2) n OCH3, -O(CH2) n NH2, -O(CH2) n CH3, -O(CH2) n ONH2 and -O(CH2) n ON[(CH2) n CH3)]2, where n and m are from 1 to about 10. Other chemical modifications at the 2' position include, but are not limited to: C1 to C 10Lower alkyl groups, substituted lower alkyl groups, alkylaryl groups, arylyl groups, O-alkylaryl groups, O-arylyl groups, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocyclic alkyl groups, heterocyclic alkylaryl groups, aminoalkylamino groups, polyalkylamino groups, substituted silyl groups, RNA cleaving groups, reporter groups, intercalating agents, groups used to improve the pharmacokinetic properties of oligonucleotides, or groups used to improve the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. Similar modifications can also be made at other positions on the sugar, particularly at the 3' position of the sugar in 3' terminal nucleotides or 2'-5' linked oligonucleotides, and at the 5' position of the sugar in 5' terminal nucleotides. Chemically modified sugars also include sugars containing modifications such as CH2 and S at the bridging epoxy. Nucleotide sugar analogs can also have sugar mimics such as replacing the cyclobutyl moiety of furanylpentose 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'-OCH3, and 2'-O(CH2)2OCH3 substituents. The substituent at the 2' position may also be selected from allyl, amino, azide, thio, O-allyl, O-(C1-C)2OCH3, etc. 10 Alkyl), OCF3, O(CH2)2SCH3, O(CH2)2-ON(R) m (R) n ) and O-CH2-C(=O)-N(R m (R) n ), where each R m and R n Independently H or substituted or unsubstituted C1-C 10 alkyl.

[0183] In some embodiments, the nucleic acids described herein comprise one or more bicyclic nucleic acids. In some such embodiments, the bicyclic nucleic acid comprises a bridge between the 4' and 2' ribosyl ring atoms. In some embodiments, the nucleic acids provided herein comprise one or more bicyclic nucleic acids, wherein the bridge comprises a 4' to 2' bicyclic nucleic acid. Examples of such 4' to 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(CH2OCH3)-O-2' and their analogues; 4'-C(CH3)(CH3)-O-2' and their analogues.

[0184] Modification of nucleotide bases

[0185] In some embodiments, the chemical modifications described herein include modifications to nucleotide bases (e.g., nucleobases). Example nucleobases may include adenine (A), thymine (T), guanine (G), cytosine (C), and uracil (U). These nucleobases may be modified or substituted in the engineered polynucleotides described herein. The nucleobases of the nucleotide may be independently selected from purines, pyrimidines, purine or pyrimidine analogs. In some embodiments, the nucleobases may be naturally occurring or synthetic derivatives of the bases.

[0186] In some embodiments, the chemical modifications described herein include modifications to uracil. In some embodiments, the engineered polynucleotides described herein comprise at least one chemically modified uracil.Examples of chemically modified uracil may include pseudouridine, pyridine-4-ketoribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine, 4-thio-uridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 5-aminoallyluridine, 5-halouridine (e.g., 5-iodouridine or 5-bromouridine), 3-methyluridine, 5-methoxyuridine, uridine 5-oxyacetic acid, methyl uridine 5-oxyacetic acid, 5-carboxymethyluridine, 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyluridine, 5-carboxyhydroxymethyluridine methyl ester, 5-methoxycarbonylmethyluridine, 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-tauronic acid methyl-uridine, 1-tauronic acid methyl-pseudouridine, 5-tauronic acid methyl-2-thio-uridine, l-tauronic acid methyl-4-thio-pseudouridine, 5-methyl-uridine, 1-methyl-pseudouridine, 5-methyl-2-thio-uridine, l-methyl-4-thio-pseudouridine, 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine, 2-thio -1-Methyl-pseudouridine, 1-Methyl-1-denitro-pseudouridine, 2-Thio-1-methyl-1-denitro-pseudouridine, dihydrouridine, 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, 5-(isopentenylaminomethyl)uridine, 5-(isopentenylaminomethyl)-2-thio-uridine, α-thio-uridine, 2'-O-methyl-uridine 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-2'-O-methyluridine, 3,2'-O-dimethyluridine, 5-(isopentenylaminomethyl)-2'-O-methyluridine, l-thiouridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-methoxycarbonylvinyl)-uridine, 5-[3-(1E-propenylamino)]uridine, pyrazolo[3,4-d]pyrimidine, xanthine and hypoxanthine.

[0187] In some embodiments, the chemical modifications described herein include modifications to cytidine. In some embodiments, the engineered polynucleotides described herein comprise at least one chemically modified cytidine. Example chemically modified cytidines may include 5-aza-cytidine, 6-aza-cytidine, pseudocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-methylcytidine, 5-halo-cytidine, 5-hydroxymethylcytidine, 1-methyl-pseudocytidine, pyrrole-cytidine, pyrrole-pseudocytidine, 2-thiocytidine, 2-thio-5-methylcytidine, 4-thio-pseudocytidine, 4-thio-1-methyl-pseudocytidine, 4-thio-1-methyl-1-deazo-pseudocytidine, 1-methyl-1-deazo-pseudocytidine, zebularine, 5-aza-zebularine, 5- Methyl-zabraline, 5-aza-2-thio-zabraline, 2-thio-zabraline, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudo-cytidine, 4-methoxy-1-methyl-pseudo-cytidine, lysine, α-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.

[0188] In some embodiments, the chemical modifications described herein include modifications to adenine. In some embodiments, the engineered polynucleotides described herein comprise at least one chemically modified adenine. Example chemically modified adenines may include 2-amino-purine, 2,6-diamino-purine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deadenine, 7-deadenine-8-aza-adenine, 7-deadenine-2-amino-purine, 7-deadenine-8-aza-2-amino-purine, 7-deadenine-2, 6-Diaminopurine, 7-deazo-8-aza-2,6-diaminopurine, 1-methyl-adenosine, 2-methyl-adenosine, 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-glycylcarbamoyl-adenosine, N6-threonylcarbamoyl-adenosine N6-methyl-N6-threonylcarbamoyl-adenosine, 2-methylthio-N6-threonylcarbamoyl-adenosine, N6,N6-dimethyl-adenosine, N6-hydroxyn-valinecarbamoyl-adenosine, 2-methylthio-N6-hydroxyn-valinecarbamoyl-adenosine, N6-acetyl-adenosine, 7-methyl-adenosine, 2-methylthio-adenosine, 2-methoxy-adenosine, α-thio-adenosine, 2'-O-methyl-adenosine, N6,2'- O-dimethyl-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-pentaenodecyl)-adenosine.

[0189] In some embodiments, the chemical modifications described herein include modifications to guanine. In some embodiments, the engineered polynucleotides described herein comprise at least one chemically modified guanine. Examples of chemically modified guanines may include inosine, 1-methyl-inosine, wyoside, methyl-wyoside, 4-demethyl-wyoside, isowyoside, wyoside, peroxy-wyoside, hydroxy-wyoside, undeiodinated hydroxy-wyoside, 7-deazo-guanosine, piracene, epoxy-piracene, galactosyl-piracene, mannosyl-piracene, 7-cyano-7-deazo-guanosine, 7-aminomethyl-7-deazo-guanosine, archapurin, 7-deazo-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deazo-guanosine, 6-thio-7-deazo-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, etc. α-guanosine, N2,7-dimethylguanosine, N2,N2,7-dimethylguanosine, 8-oxoguanosine, 7-methyl-8-oxoguanosine, 1-methylthioguanosine, N2-methyl-6-thioguanosine, N2,N2-dimethyl-6-thioguanosine, α-thioguanosine, 2'-O-methylguanosine, N2-methyl-2'-O-methylguanosine, N2,N2-dimethyl-2'-O-methylguanosine, 1-methyl-2'-O-methylguanosine, N2,7-dimethyl-2'-O-methylguanosine, 2'-O-methylinosine, 1,2'-O-dimethylinosine, 6-O-phenyl-2'-deoxyinosine, 2'-O-ribosylguanosine, 1-thioguanosine, 6-O-methylguanosine, O 6 2'-methyl-2'-deoxyguanosine, 2'-F-ara-guanosine, and 2'-F-guanosine.

[0190] In some cases, chemical modification of engineered polynucleotides may include the introduction or substitution of nucleic acid analogs or non-natural nucleic acids into the engineered polynucleotide. In some embodiments, the nucleic acid analog may be any of the chemically modified nucleic acids described herein. Example nucleic acid analogs can be found in 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 explicitly incorporated herein by reference in their entirety. Chemically modified nucleotides described herein may include variants of guanosine, uridine, adenosine, thymidine, and cytosine, including any naturally occurring or non-natural guanosine, uridine, adenosine, thymidine, or cytosine that has been chemically altered (e.g., by acetylation, methylation, or hydroxylation).Examples of chemically modified nucleotides may 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'-ara adenosine, 2'-ara cytidine, 2'-ara uridine, 2'-azido-2'-deoxyadenosine, 2'-azido-2'-deoxycytidine, 2'-azido-2'-deoxyguanosine, 2'-azido-2'-deoxyguanosine, 2'-azido-2'-deoxyuridine Oxyuridine, 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-methylpseuuridine, 2-thiocytidine Glycosides, 2-thio-cytidine, 3-methyl-cytidine, 4-acetyl-cytidine, 4-thiouridine, 5-(carboxyhydroxymethyl)-uridine, 5,6-dihydrouridine, 5-aminoallylcytidine, 5-aminoallyldeoxyuridine, 5-bromouridine, 5-carboxymethylaminomethyl-2-thio-uridine, 5-carboxymethylaminomethyl-uridine, 5-chloro-ara-cytidine, 5-fluoro-uridine, 5-iodouridine, 5-methoxycarbonylmethyl-uridine, 5-methoxy-uridine, 5-methyl-2-thio-uridine, 6-azacytidine, 6-azauridine, 6-chloro-7-deazo-guanosine, 6-chloropurine riboside, 6-mercapto-guanosine, 6-methyl 1-Mercaptopurine-riboside, 7-denitro-2'-deoxyguanosine, 7-denitroadenosine, 7-methyl-guanosine, 8-azaadenosine, 8-bromo-adenosine, 8-bromo-guanosine, 8-mercapto-guanosine, 8-oxoguanosine, benzimidazole-riboside, β-D-mannosyl-piperidine, dihydro-uridine, inosine, N1-methyladenosine, N6-([6-aminohexyl]carbamoylmethyl)-adenosine, N6-isopentenyl-adenosine, N6-methyl-adenosine, N7-methyl-flavin, N-uracil-5-oxyacetic acid methyl ester, puromycin, piperidine, uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester, huaistin, flavin, and xylose-adenosine.In some embodiments, the chemically modified nucleic acids described herein comprise at least one chemically modified nucleotide selected from the following: 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 Acid, 5-iodouridine-5'-triphosphate, 5-iodo-2'-deoxyuridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5-methyluridine-5'-triphosphate, 5-propynyl-2'-deoxycytidine-5'-triphosphate, 5-propynyl-2'-deoxyuridine-5'-triphosphate, 6-azacytidine-5'-triphosphate, 6-azauridine-5'-triphosphate, 6-chloropurine riboside-5'-triphosphate, 7-deazoadenosine-5 '-Triphosphate, 7-dezoguanosine-5'-triphosphate, 8-azaadenosine-5'-triphosphate, 8-azidoadenosine-5'-triphosphate, benzimidazole-riboside-5'-triphosphate, N1-methyladenosine-5'-triphosphate, N1-methylguanosine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, 6-methylguanosine-5'-triphosphate, pseudouridine-5'-triphosphate, puromycin-5'-triphosphate, or flavin-5'-triphosphate. In some embodiments, the chemically modified nucleic acid described herein comprises at least one chemically modified nucleotide selected from the following: pyridine-4-ketoribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyluridine, 1-carboxymethyl-pseudouridine, 5-propynyluridine, 1-propynyl-pseudouridine, 5-taurate methyluridine, 1-taurate methyl-pseudouridine, 5-taurate methyl- 2-Thio-uridine, 1-Tauratemethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-denitro-pseudouridine, 2-thio-1-methyl-1-denitro-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine and 4-methoxy-2-thio-pseudouridine.In some embodiments, the artificial nucleic acid described herein comprises at least one chemically modified nucleotide selected from the following: 5-aza-cytidine, pseudocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudocytidine, pyrrolo-cytidine, pyrrolo-pseudocytidine, 2-thio-cytidine, 2-thio-5-methylcytidine, 4-thio-pseudocytidine, 4-Thio-1-methyl-pseudoisocytidine, 4-Thio-1-methyl-1-deazo-pseudoisocytidine, 1-methyl-1-deazo-pseudoisocytidine, zabrain, 5-aza-zabrain, 5-methyl-zabrain, 5-aza-2-thio-zabrain, 2-thio-zabrain, 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 comprise at least one chemically modified nucleotide selected from: 2-aminopurine, 2,6-diaminopurine, 7-deadenine, 7-deadenine-8-azaadenine, 7-deadenine-2-aminopurine, 7-deadenine-8-azaa-2-aminopurine, 7-deadenine-2,6-diaminopurine, 7-deadenine-8-aza-2,6-diaminopurine, 1-Methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenosine, 2-methylthio-adenosine, and 2-methoxy-adenosine. In other embodiments, the chemically modified nucleic acid described herein comprises at least one chemically modified nucleotide selected from the following: inosine, 1-methyl-inosine, wyoside, wyoside, 7-deazoguanosine, 7-deazo-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deazo-guanosine, 6-thio-7-deazo-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methyl-guanosine, N2-methyl-guanosine, N2,N2-dimethyl-guanosine, 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 some embodiments, the chemically modified nucleic acids described herein comprise at least one chemically modified nucleotide selected from the following: 6-aza-cytidine, 2-thio-cytidine, α-thio-cytidine, pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudo-uridine, 5,6-dihydrouridine, α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy- Thymidine, 5-methyl-uridine, pyrrolo-cytidine, inosine, α-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytidine, 8-oxo-guanosine, 7-deazo-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-deazo-adenosine.

[0191] Modified bases of non-natural nucleic acids include, but are not limited to, uracil-5-yl, hypoxanthine-9-yl (I), 2-aminoadenine-9-yl, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, and 5-propynyluracil and cytosine. 6-Azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halogenated, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halogen, especially 5-bromine, 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deadenine and 7-deadenine and 3-deadenine and 3-deadenine. Certain non-natural nucleic acids, such as 5-substituted pyrimidines, 6-azapyrimidines and N-2-substituted purines, N-6-substituted purines, O-6-substituted purines, 2-aminopropyladenine, 5-propynyluracil, 5-propynylcytosine, 5-methylcytosine, nucleic acids that increase the stability of duplex formation, universal nucleic acids, hydrophobic nucleic acids, hybrid nucleic acids, size-expanding nucleic acids, fluorinated nucleic acids, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6... Purines substituted with O-6, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl of adenine and guanine, other alkyl derivatives, 2-propyl of adenine and guanine and other alkyl derivatives, 2-thiouracil, 2-thiothymine and 2-thiocytosine, and 5-halouracil. 5-Halogenated cytosine, 5-propynyl (-C≡C-CH3)uracil, 5-propynylcytosine, other alkynyl derivatives of pyrimidine nucleic acids, 6-azouracil, 6-azocytosine, 6-azothymidine, 5-uracil (pseudouracil), 4-thiouracil, 8-halogenated, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halogens, especially 5-bromo, 5-trifluoromethyl, and others It contains 5-substituted uracil and cytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine, 8-azaadenine, 7-deadenine, 7-deadenine, 3-deadenine, 3-deadenine, tricyclic pyrimidines, phenoxazincytidine ([5,4-b][l,4]benzoxazin-2(3H)-one), phenothiazincytidine (1H-pyrimidino[5,4-b][l,4] benzothiazine-2(3H)-one), G-cup, phenoxazine cytosine (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-b][l,4]benzoxazine-2(3H)-one), carbazole cytosine (2H-pyrimido[4,5-b]indole-2-one), pyridoindole cytosine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimido-2-one), nucleic acids in which the purine or pyrimidine base is replaced by other heterocycles, 7-deadenine, 7-deadenine, 2-aminopyridine, 2-pyridone, azacytosine, 5-bromocytosine, bromouracil, 5-chlorocytosine Cytosine chloride, 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-propynyluracil, 4-thio-uracil, N4-ethylcytosine, 7-deazoguanine, 7-deazo-8-azoguanine, 5-hydroxycytosine, 2'-deoxyuridine or 2-amino-2'-deoxyadenosine.

[0192] In some embodiments, at least one chemical modification may include a chemically modified 5' or 3' end of the engineered polynucleotide, such as a 5' cap or a 3' tail. In some embodiments, the engineered polynucleotide contains a chemical modification containing a 3' nucleotide, which can be stabilized to prevent degradation, for example, by incorporation of one or more modified nucleotides described herein. In this embodiment, uridine may be replaced by modified uridines such as 5-(2-amino)propyluridine and 5-bromouridine or any modified uridine described herein; adenosine and guanosine may be replaced by modified adenosine and guanosine, such as having a modification at the 8-position, such as 8-bromoguanosine, or replaced with any modified adenosine or guanosine described herein. In some embodiments, a denitronucleotide, such as 7-denitro-adenosine, may be incorporated into the gRNA. In some embodiments, O- and N-alkylated nucleotides, such as N6-methyladenosine, may be incorporated into the gRNA. In some embodiments, a sugar-modified ribonucleotide may be incorporated, for example, wherein the 2'OH- group is selected from H, -OR, -R (where R can be, for example, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar), halogen, -SH, -SR (where R can be, for example, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar), amino (where the amino group can be, for example, NH2; alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); or a cyano (-CN) group. In some embodiments, the phosphate backbone may be modified as described herein, for example, with a thiophosphate group. In some implementations, the nucleotides in the gRNA overhang region can each be independently modified or unmodified nucleotides, including but not limited to 2'-sugar modifications such as 2-F 2'-O-methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine (Teo), 2'-O-methoxyethyl adenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo), or any combination thereof.

[0193] In some embodiments, all nucleotides in the targeting portion have a 2'O-methyl modification. The 2'O-methyl modification is believed to increase the affinity of the engineered polynucleotide for its mRNA precursor target and / or prevent the engineered polynucleotide from being degraded by nucleases. In some embodiments, all nucleotides in the targeting portion have a phosphate thioester modification.

[0194] Adjustment section

[0195] In some embodiments of the engineered polynucleotides described herein, the posttranscriptional regulatory portion (or regulatory portion) (e.g., the spliceosome portion) is selected from the spliceosome ribonucleoprotein complex, spliceosome small nucleoribonucleic acid (snRNA), spliceosome protein, functional variants thereof, or functional fragments thereof. In some embodiments, the spliceosome portion comprises U1 snRNA and spliceosome protein. In some embodiments, the spliceosome portion comprises U2 snRNA and spliceosome protein. In some embodiments, the spliceosome snRNA is selected from U1, U2, U4, U5, U6, U11, U12, U14atac, U6atac, and combinations thereof. In some embodiments, the spliceosome snRNA is U1 or U2. In some embodiments, the spliceosome protein is selected from Sm, U1-70k, U1A, U1C, and combinations thereof. Non-limiting examples of splice body portions include SmD1, SmD2, SmD3, SmE, SmF, SmG, U1, U2, U4, U5, U6, U11, U12, U14, or U16.

[0196] In some embodiments described herein, when bound to engineered polynucleotides and RNA (e.g., mRNA, such as mRNA precursors), the spliceosome portion cleaves or splices RNA (e.g., mRNA, such as mRNA precursors) in the target sequence. In some embodiments, the spliceosome portion also facilitates modification of the cleaved RNA (e.g., cleaved mRNA, such as cleaved mRNA precursors).

[0197] In some embodiments, engineered polynucleotides bind to target sequences via base pairing, such as Watson-Crick base pairing. The binding of the engineered polynucleotide to the recruitment motif provided herein can be used to regulate the expression or activity of the target gene. In some embodiments, the binding of the engineered polynucleotide to the recruitment motif enables the recruitment motif to splice the mRNA precursor encoding the target gene with increased specificity, thereby regulating the target gene. In some embodiments, the binding of the engineered polynucleotide to the recruitment motif enables the recruitment motif to splice the mRNA precursor encoding the target gene with increased efficiency, thereby regulating the target gene. Regulation can refer to increasing or decreasing the expression or activity of the target gene. Non-limiting examples of target genes may include microtubule-associated protein TAU (MAPT). In some embodiments, when the engineered polynucleotide binds to the recruitment moiety, the expression or activity of the target gene increases by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, to 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more, compared to the absence of the engineered polynucleotide binding to the recruitment moiety. In some embodiments, when the engineered polynucleotide binds to the recruitment moiety, the expression or activity of the target gene decreases by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, to 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 10, or less, compared to the absence of the engineered polynucleotide binding to the recruitment moiety.

[0198] In some embodiments, regulation of target expression or activity includes correcting aberrant expression of the target gene due to splicing variants. In some embodiments, when an engineered polynucleotide is bound to the recruitment motif, the expression or activity of a misfolded target gene or protein due to aberrant splicing variants is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, to 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 10, or lower. In some embodiments, the amount of misfolded protein aggregates due to anomalous splicing variants is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, to 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 10 or less when the engineered polynucleotide binds to the recruitment moiety, compared to the amount of misfolded protein plaques due to anomalous splicing variants in the absence of the engineered polynucleotide binding to the recruitment moiety. In some embodiments, the amount of plaques containing misfolded proteins due to anomalous splicing variants is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, to 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 10 or less when the engineered polynucleotide binds to the recruitment moiety, compared to the amount of plaques containing misfolded proteins due to anomalous splicing variants in the absence of the engineered polynucleotide binding to the recruitment moiety.

[0199] Molecular interactions

[0200] In various aspects of this disclosure, engineered polynucleotides may be able to engage in molecular interactions with peptides (e.g., U1-C) or other polynucleotides (e.g., mRNA precursors, U1 snRNA). Engineered polynucleotides may be configured or otherwise capable of interacting with other peptides or polynucleotides via one or more targeting or recruitment moieties. In some embodiments described herein, the targeting moieties comprise free 5' and 3' ends that interact with conserved sites of constitutive splicing donors. This interaction can silence the U1 snRNA RNA-binding domain. In some embodiments, the targeting moieties comprise 2'-modified nucleotides that increase affinity for mRNA precursor targets.

[0201] In some implementations, the binding of the targeting moiety to the mRNA precursor is stabilized through hydrogen bonding and electrostatic interactions between U1-C and the mRNA precursor backbone surrounding the mRNA precursor splice-joint region. In these implementations, U1-C may not make specific base contacts with the mRNA precursor. 2' nucleotide modifications can facilitate hydrogen bonding between the targeting moiety and U1-C. Therefore, the binding of the targeting moiety (free 5' and 3' ends) to the mRNA precursor duplex allows the targeting interaction of U1-C to be recognized and stabilized.

[0202] U1-C can stabilize the central core of the spliceosome. U1-C enhances the affinity of incompatible 5'-splicing and stabilizes the central core of the spliceosome mechanism through the interaction bridge between U1-70KD and the Sm ring.

[0203] In some implementations, the targeting moiety can interact with the zinc finger of U1-C. Phosphothionucleotide linkages within the targeting moiety can promote the interaction between the targeting moiety and the zinc finger. Engineered polynucleotides may contain phosphothionucleotide linkages at specific or particular sites that interact with the zinc finger.

[0204] In some implementations, the recruiting moiety forms hydrogen bonds with the stem-ring II of U1-A. This interaction can modulate the polyadenylation and acetylation signaling of U1-A because the stem-ring II of U1-A cannot be silenced by the recruiting moiety.

[0205] In some of the embodiments described herein, the engineered polynucleotide does not contain any intramolecular disulfide bonds.

[0206] In some embodiments of the engineered polynucleotides described herein, when bound to the engineered polynucleotide and spliceosome portion, the RNA (e.g., mRNA, such as mRNA precursors) substantially does not exhibit base pairing with the RNA-binding domain (RBD) of the U1 snRNA.

[0207] In some embodiments of the engineered polynucleotides described herein, when bound to the engineered polynucleotide and spliceosome portion, the RNA (e.g., mRNA, such as mRNA precursors) substantially does not exhibit base-specific interactions with the U1-C protein.

[0208] In some embodiments of the engineered polynucleotide described herein, the engineered polynucleotide is configured to specifically interact with the zinc finger of the U1-C protein, for example, 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 the zinc finger of the U1-C protein. In some embodiments, the engineered polynucleotide (e.g., its 5'-targeting portion) is configured to covalently interact with the zinc finger of the U1-C protein (e.g., via disulfide bonds). In some embodiments, the engineered polynucleotide (e.g., its 5'-targeting portion) is configured to non-covalently interact with the zinc finger of the U1-C protein (e.g., via hydrogen bonds).

[0209] In some embodiments, the engineered polynucleotide (e.g., ASMO1 as described herein) comprises a nucleotide sequence complementary to the U1 snRNA. In some embodiments of the engineered polynucleotide described herein, the engineered polynucleotide comprises a nucleotide sequence complementary to a partial sequence of the stem-loop II (SL2) of the U1 snRNA. In some embodiments, one side of the stem-loop structure of the engineered polynucleotide comprises a nucleotide sequence complementary to a partial sequence of the stem-loop II (SL2) of the U1 snRNA. In some embodiments, the partial sequence comprises a sequence corresponding to the 5'-GGCCU-3' of the SL2 of the U1 snRNA. In some embodiments, the partial sequence does not comprise a sequence corresponding to the 5'-CACGUUA-3' of the SL2 of the U1 snRNA.

[0210] In some embodiments of the engineered polynucleotide described herein, the engineered polynucleotide substantially does not exhibit base pairing with the anchoring sequence of the SL2 region of the U1 snRNA. In some embodiments, the anchoring sequence comprises a sequence corresponding to 5'-CACGUUA-3'. In some embodiments, the inner loop of the engineered polynucleotide substantially does not exhibit base pairing with the anchoring sequence of the SL2 region of the U1 snRNA. In some embodiments, the lower stem of the engineered polynucleotide substantially does not exhibit base pairing with the anchoring sequence of the SL2 region of the U1 snRNA.

[0211] In some embodiments of the engineered polynucleotides described herein, the engineered polynucleotides substantially do not exhibit H-helical base pairing with U1 snRNA.

[0212] In some embodiments, the engineered polynucleotide does not contain any intramolecular disulfide bonds. In some embodiments, when the described spliceosome moiety is recruited to a target sequence (such as a target mRNA precursor), the engineered polynucleotide does not exhibit base pairing with the RNA-binding domain (RBD) of the spliceosome moiety (such as U1snRNA). Figure 4 This lack of base pairing between the engineered polynucleotide and the spliceosome portion's RBD is illustrated, wherein the RBD site of the U1snRNA has the sequence 3'-GUCCAUCAUA-5' and forms a base pair with the target sequence. In some cases, when the engineered polynucleotide binds to the spliceosome portion, the engineered polynucleotide essentially does not exhibit base-specific interaction with the U1-C spliceosome portion. In some embodiments, the engineered polynucleotide is configured to specifically interact with the zinc finger or U1-1 spliceosome portion of the U1-C protein. Figure 10C A representative sequence of U1-C snRNP containing 145 amino acids is shown, of which the 36 amino acids highlighted (YYCDYCDTYLTHDSPSVRKTHCTGRKHRDNVKF(SEQ ID NO:5)) contain zinc finger domains.

[0213] In some embodiments, the engineered polynucleotide is configured to covalently interact (e.g., via disulfide bonds) with the zinc finger or U1-1 splice body portion of the U1-C protein. In some embodiments, the engineered polynucleotide is configured to non-covalently interact (e.g., via hydrogen bonds) with the zinc finger or U1-1 splice body portion of the U1-C protein. In some embodiments, the engineered polynucleotide (e.g., ASMO1 as described herein) comprises a nucleotide sequence complementary to the U1 snRNA. In some embodiments, the engineered polynucleotide comprises a nucleotide sequence complementary to a partial sequence of the stem-loop II (SL2) of the U1 snRNA. In some embodiments, one side of the stem-loop secondary structure of the engineered polynucleotide comprises a nucleotide sequence complementary to a partial sequence of the stem-loop II (SL2) of the U1 snRNA. In some embodiments, the partial sequence comprises a sequence corresponding to the 5'-GGCCU-3' of the SL2 of the U1 snRNA. In some embodiments, the partial sequence does not comprise a sequence corresponding to the 5'-CACGUUA-3' of the SL2 of the U1 snRNA. In some implementations, the engineered polynucleotide substantially does not exhibit base pairing with the anchoring sequence of the SL2 region of the U1 snRNA. In some cases, the inner loop of the engineered polynucleotide substantially does not exhibit base pairing with the anchoring sequence of the SL2 region of the U1 snRNA. In some aspects, the lower stem of the engineered polynucleotide substantially does not exhibit base pairing with the anchoring sequence of the SL2 region of the U1 snRNA. In some aspects, the anchoring sequence comprises a sequence corresponding to 5'-CACGUUA-3'. In some cases, the engineered polynucleotide substantially does not exhibit base pairing with the H-helix of the U1 snRNA, wherein the engineered polynucleotide does not contain any intramolecular disulfide bonds. For example, Figure 2A The presence of chemical modifications involving thiophosphate-type nucleotide internucleotides demonstrates the absence of intramolecular disulfide bonds. Stabilization of the U1 snRNP complex can be observed via the strong ionic attraction of the U1-C zinc finger, induced by disulfide bridging of the thiol at the 5'- or / and 3'-terminus of the engineered polynucleotide (ASMO) targeting moiety. The mRNA precursor / ASMO duplex bond is stabilized via hydrogen bonding and electrostatic interactions between U1-C and the mRNA precursor backbone around the splice site, but U1-C does not specifically contact the mRNA precursor bases. This structure suggests that the selection of 5'-splicing nucleotides by U1 snRNP is primarily achieved through interactions between the stem 5' / 3' and the mRNA precursor. Simultaneously, U1-C modulates the relative affinity of the 5'-splicing incompatible site and stabilizes the central core of the spliceosome mechanism via an interaction bridge between U1-70 kDa and the Sm ring (see [link to documentation]). Figure 7-9). Among U1 snRNP-specific proteins, U1-70K and U1-C play important roles in helping to recognize mRNA precursor transcripts. U1-70K has a highly conserved but expectedly unstructured N-terminus (residues approximately 2-60); an RNA-binding domain (or RBD) (residues 92-202) mediating its interaction with the stem-loop of U1 snRNA; and a C-terminus rich in arginine and serine residues (RS "domain") and R-(D / E) residue repeats. Although this C-terminal domain is not conserved, the RS "domain" is crucial for interaction with non-snRNP splicing factors such as ASF / SF2. Serine residues in this region undergo post-translational modification (phosphorylation) and are important for splicing activity. U1-C consists of an N-terminal zinc finger domain and a C-terminal region rich in RG residue repeats. Arginine residues in this region of U1-C undergo post-translational modification (methylation). Unlike U1-70K, U1-C does not bind to free U1 snRNA, but requires prior binding of the Sm protein to U1-70K. Mutations in the zinc finger region of U1-C significantly affect the recognition of the 5' splice site by U1 snRNP, indicating that this protein plays a direct role in this activity. The assembly and function of U1 snRNP have been greatly enhanced, initially through cryo-electron microscopy studies and more recently through X-ray crystallography elucidating its three-dimensional structure. Previously, the crystal structures of four of the seven Sm proteins led to modeling of the remaining three (Sm-F, Sm-E, and Sm-G), proposing that they would interact together to form a seven-membered ring. The crystal structure of fully recombinant human U1 snRNP reveals that the Sm protein does indeed form a heptameric ring, consisting of a single copy of each Sm protein, with the Sm site of the U1 snRNA passing through its center. In the crystal structure, the position of U1-C enables recognition of the double strand formed when the 5' end of the U1 snRNA pairs with the 5' splice site. The N-terminus of U1-70K extends from the RBD. And it wraps around one side of the Sm loop, passing through Sm-D2 and Sm-D3 / B. This discovery therefore ensures the correct structure and location of U1-C for interaction with the double strand of the U1 snRNA: 5' splice site (Figure 9).

[0214] In some implementations, the engineered polynucleotide, when bound to the spliceosome portion described herein, does not substantially exhibit base pairing with the RNA-binding domain (RBD) of the U1 snRNA. In some cases, the engineered polynucleotide, when bound to the spliceosome portion, does not substantially exhibit base-specific interactions with the U1-C protein. Figure 4This demonstrates that in the absence of engineered polynucleotides, the RBD of U1 snRNA binds to a conserved region of the constitutive donor. Conversely, in the presence of engineered polynucleotides, the stem 5' / 3' block the interaction between U1 snRNA and the RBD of the mRNA precursor (Figure 3 and...). Figure 6 ).

[0215] In some embodiments, the engineered polynucleotide is configured to interact specifically with the zinc finger of the U1-C protein. In some embodiments, the 5'-targeting portion of the engineered polynucleotide is configured to interact specifically with the zinc finger of the U1-C protein. In some embodiments, the engineered polynucleotide is configured to interact covalently with the zinc finger of the U1-C protein (e.g., via disulfide bonds). In some embodiments, the engineered polynucleotide is configured to interact non-covalently with the zinc finger of the U1-C protein (e.g., via hydrogen bonds). The formation of the mRNA precursor / engineered polynucleotide (ASMO) duplex interacting with the amino acid residues of the U1-C zinc finger stabilizes the 5' region (Fig. 9). Favorable molecular dynamics can then be observed for the formation of disulfide bonds formed by atoms in the main chain and side chains of the U1-C zinc finger with the stem 5' of the engineered polynucleotide. Strong ionic bonds can also be formed when the ASMO interacts with all the cysteine ​​residues present in the U1-C zinc finger (Figs. 9 and 10). Examples of additional interactions between U1-C and mRNA precursors, with or without the engineered polynucleotides described herein, are shown in Table 5.

[0216] Table 5. Examples of interactions between U1-C and mRNA precursors mediated by the presence of engineered polynucleotides.

[0217]

[0218]

[0219] In some embodiments, the engineered polynucleotide (e.g., ASMO1 as described herein) comprises a nucleotide sequence complementary to the U1 snRNA. In some aspects, the engineered polynucleotide comprises a nucleotide sequence complementary to a partial sequence of the stem-loop II (SL2) of the U1 snRNA. In other aspects, one side of the stem-loop structure of the engineered polynucleotide described herein comprises a nucleotide sequence complementary to a partial sequence of the stem-loop II (SL2) of the U1 snRNA. In some cases, the partial sequence comprises a sequence corresponding to 5'-GGCCU-3' of the SL2 of the U1 snRNA, wherein the partial sequence does not comprise a sequence corresponding to 5'-CACGUUA-3' of the SL2 of the U1 snRNA, and wherein the engineered polynucleotide substantially does not exhibit anchoring sequence base pairing with the SL2 of the U1 snRNA. In some aspects, the engineered polynucleotide comprises an inner loop of the engineered polynucleotide that substantially does not exhibit anchoring sequence base pairing with the SL2 of the U1 snRNA. In some embodiments, the engineered polynucleotide comprises a lower stem of an engineered polynucleotide that substantially does not exhibit base pairing with the anchoring sequence of the U1 snRNA at SL2. In some embodiments, the anchoring sequence comprises a sequence corresponding to 5'-CACGUUA-3', wherein the engineered polynucleotide then substantially does not exhibit base pairing with the H-helix of the U1 snRNA.

[0220] Engineered polynucleotide genome

[0221] In some implementations, the present invention includes engineered polynucleotide sets, each independently of the present invention. For example, the polynucleotide set may independently include: (i) one or more targeting portions (as described herein), the targeting portions being configured to bind ribonucleic acid (RNA) (as described herein) (e.g., messenger ribonucleic acid (mRNA), such as a precursor messenger ribonucleic acid (mRNA)) at a target sequence (as described herein), and (ii) a recruitment portion (as described herein), the recruitment portion being configured to recruit a post-transcriptional regulatory portion (e.g., a spliceosome portion) (as described herein), wherein the engineered polynucleotide set is configured to specifically bind RNA (e.g., mRNA, such as a precursor mRNA) at multiple target sequences containing a target sequence (as described herein).

[0222] carrier

[0223] Some of the implementation schemes described herein include vectors or plasmids containing nucleic acid sequences encoding the engineered polynucleotides described herein.

[0224] Some embodiments described herein include multiple vectors or plasmids containing multiple nucleic acid sequences, each encoding an engineered polynucleotide described herein. In some embodiments, the multiple vectors or plasmids contain multiple nucleic acid sequences encoding more than one engineered polynucleotide described herein. In some embodiments, the multiple vectors or plasmids contain multiple nucleic acid sequences encoding multiple engineered polynucleotides (each independently described herein).

[0225] Pharmaceutical Composition

[0226] In some embodiments, this document describes a pharmaceutical composition comprising the engineered polynucleotide, or plasmid, vector, or isolated DNA encoding its sequence as described herein. 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 other chemical components being, for example, carriers, excipients, binders, fillers, suspensions, flavoring agents, sweeteners, disintegrants, dispersants, surfactants, lubricants, colorants, diluents, solubilizers, wetting agents, plasticizers, stabilizers, penetration enhancers, humectants, defoamers, antioxidants, preservatives, or one or more combinations thereof. Optionally, the composition comprises two or more of the pharmaceutical compositions discussed herein. In carrying out the treatment methods or uses provided herein, a therapeutically effective amount of the pharmaceutical composition described herein is administered in pharmaceutical composition form to a mammal suffering from a disease, condition, or ailment 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 status of the subject, the potency of the pharmaceutical composition used, and other factors. Pharmaceutical compositions may be used alone or in combination with one or more pharmaceutical compositions that are components of a mixture. Pharmaceutical compositions described herein include engineered polynucleotides, compositions, cells in contact with engineered polynucleotides or cells in contact with compositions containing engineered polynucleotides, or combinations thereof.

[0227] The pharmaceutical formulations described herein are administered to the target body via appropriate routes of administration, including but not limited to intravenous, intrathecal, intra-arterial, intratumoral, oral, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, inhalation, or intraperitoneal administration. The pharmaceutical formulations described herein include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposome dispersions, aerosols, solid dosage forms, powders, immediate-release formulations, controlled-release formulations, fast-dissolving formulations, tablets, capsules, pills, delayed-release formulations, extended-release formulations, pulsatile-release formulations, multi-particle formulations, and a combination of immediate and controlled-release formulations.

[0228] The pharmaceutical composition, including the pharmaceutical composition, is manufactured in a conventional manner, such as, by way of example only, through conventional mixing, dissolving, granulation, sugar-coated pelleting, grinding, emulsification, encapsulation, embedding, or compression processes.

[0229] Reagent test kit

[0230] In some embodiments, this document describes a kit for use with the engineered polynucleotides, compositions, or pharmaceutical compositions described herein. In some embodiments, the kits disclosed herein are intended to treat a disease or condition in a subject. In some embodiments, the kit comprises a collection of materials or components other than engineered polynucleotides, compositions, or pharmaceutical compositions. In some embodiments, the kit comprises components for determining and selecting suitable oligonucleotides for treating a disease or condition. In some embodiments, the kit comprises 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 their intended use. For example, some embodiments are configured for the purpose of treating a disease or condition disclosed herein in a subject. In some embodiments, the kit is specifically configured for the purpose of treating mammalian subjects. In some embodiments, the kit is specifically configured for the purpose of treating human subjects.

[0231] The kit may include instructions for use. In some embodiments, the kit includes instructions for administering the composition to a subject in need. In some embodiments, the kit includes instructions for further engineering the engineered polynucleotide. In some embodiments, the kit includes instructions for thawing or otherwise restoring the bioactivity of the engineered polynucleotide, which may be cryopreserved or lyophilized during storage or transport. In some embodiments, the kit includes instructions for measuring its intended efficacy (e.g., therapeutic efficacy if used on a therapeutic subject).

[0232] Optionally, the kit may also contain other useful components such as diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipettes or measuring tools, bandage materials, or other useful equipment. The materials or components assembled in the kit may be provided to practitioners in any convenient and suitable manner to maintain their operability and usability. For example, engineered polynucleotides, compositions, or pharmaceutical compositions may be in dissolved, dehydrated, or lyophilized form. These components are typically contained in suitable packaging materials.

[0233] method

[0234] This document describes methods utilizing engineered polynucleotides (such as those described herein), such as methods for altering ribonucleic acid (RNA) (e.g., messenger ribonucleic acid (mRNA), such as precursor mRNA) in cells. The methods may include contacting cells with an engineered polynucleotide (such as those described herein) comprising one or more target moieties and recruitment moieties. One or more target moieties may bind to RNA at a target sequence (such as those described herein) in the RNA (e.g., mRNA, such as precursor mRNA) (such as those described herein), and the recruitment moieties recruit post-transcriptional regulatory moieties (e.g., spliceosome moieties) (such as those described herein) in the vicinity of the target sequence of the RNA (e.g., mRNA, such as precursor mRNA) to alter RNA (e.g., mRNA, such as precursor mRNA) in the cell, thereby producing one or more altered RNAs (e.g., one or more altered mRNAs, such as one or more altered precursor mRNAs). In some embodiments, the methods alter the expression or activity of a target gene. In some embodiments, prior to contact, the cells exhibit aberrant messenger ribonucleic acid (mRNA) or protein corresponding to the target gene. In some implementations, the target portion of one or more target moieties is sufficiently identical or complementary to a common sequence in the target sequence of a target gene (e.g., microtubule-associated protein TAU (MAPT)).

[0235] The methods described herein include methods for altering ribonucleic acid (RNA) (e.g., messenger RNA (mRNA), such as precursor RNA (mRNA)) at multiple locations within a cell. The methods may include contacting a cell with engineered polynucleotide sets (such as those described independently herein). The engineered polynucleotides may comprise one or more targeting and recruitment moieties. One or more targeting moieties may bind to RNA at multiple target sequences (such as those described herein) within the RNA (e.g., mRNA, such as precursor RNA) (such as those described herein). Each recruitment moieties may recruit post-transcriptional regulatory moieties (e.g., spliceosome moieties) (such as those described herein) within the vicinity of the target sequence of the RNA (e.g., mRNA, such as precursor RNA) to alter RNA (e.g., mRNA, such as precursor RNA) in the cell, thereby producing one or more altered RNAs (e.g., one or more altered mRNAs, such as one or more altered precursor RNAs). In some embodiments, the method alters the expression or activity of a target gene by altering (e.g., cleaving or / and chemically modifying) RNA (e.g., mRNA, such as precursor RNA) at multiple locations. In some embodiments, prior to contact, the cell exhibits an aberrant messenger RNA (mRNA) or protein corresponding to the target gene. In some embodiments, one or each of the one or more target motifs is sufficiently identical or complementary to a common sequence in the target sequence of the target gene (e.g., microtubule-associated protein TAU (MAPT)).

[0236] In some embodiments, the method includes delivering an engineered polynucleotide into a cell. In some embodiments, the method includes delivering a polynucleotide encoding the engineered polynucleotide into a cell and subsequently expressing the engineered polynucleotide to regulate the expression or activity of a gene encoded by a target sequence described herein. In some embodiments, the method includes using the engineered polynucleotide to treat a disease or condition of a subject in need. The disease or condition may be associated with the aberrant expression or activity of a target gene encoded by RNA (e.g., mRNA, such as a precursor mRNA). In some embodiments, the RNA (e.g., mRNA, such as a precursor mRNA) corresponds to a target gene (e.g., microtubule-associated protein TAU (MAPT)).

[0237] Figure 1A schematic diagram is shown of nucleotide sequences used to identify splice donors and acceptors for designing engineered polynucleotides, wherein the engineered polynucleotides or methods described herein represent an improvement over currently available methods for regulating gene expression or activity to treat diseases or conditions. In some embodiments, the methods described herein regulate gene expression or activity by targeting engineered polynucleotides to the transcripts of genes causing diseases or conditions. In some embodiments, the methods described herein include administering the engineered polynucleotides described herein to a subject in need. In some cases, the methods described herein include utilizing engineered polynucleotides to recruit regulatory portions to regulate the expression or activity of genes causing diseases or conditions, thereby treating the disease or condition. In some aspects, the methods described herein include utilizing engineered polynucleotides to stabilize the assembly of regulatory portions to regulate the expression or activity of genes causing diseases or conditions, thereby treating the disease or condition.

[0238] In some embodiments, methods for delivering the engineered polynucleotides described herein into cells are described herein. In some embodiments, the method includes delivering the engineered polynucleotide directly or indirectly into cells. In some embodiments, the method includes contacting cells with a composition containing the engineered polynucleotides described herein. In some embodiments, the method includes expressing the engineered polynucleotides described herein in cells. In some embodiments, the engineered polynucleotide or a vector encoding the engineered polynucleotide can be delivered into cells by any of the transfection methods described herein. In some embodiments, the engineered polynucleotide can be delivered into cells using an expression vector. In the case of an expression vector, the vector can be readily introduced into the cells described herein by any method in the art. For example, the expression vector can be transferred into cells by physical, chemical, or biological means.

[0239] Physical methods for introducing engineered polynucleotides or vectors encoding engineered polynucleotides into cells may include calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, gene gun, electroporation, etc. Methods for generating cells containing vectors and / or exogenous nucleic acids are applicable to the methods described herein. One method for introducing engineered polynucleotides or vectors encoding engineered polynucleotides into host cells is calcium phosphate transfection.

[0240] Chemical methods for introducing engineered polynucleotides or carriers encoding engineered polynucleotides into cells can include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, spherical nucleic acids (SNAs), liposomes, or lipid nanoparticles. An example colloidal system used as a delivery carrier in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). Other state-of-the-art nucleic acid targeted delivery methods are available, such as delivering engineered polynucleotides or carriers encoding engineered polynucleotides using targeted nanoparticles.

[0241] In the case of using non-viral delivery systems, an example of a delivery medium is liposomes. Consider using lipid formulations to introduce engineered polynucleotides or vectors encoding engineered polynucleotides into cells (in vitro, ex vivo, or in vivo). Alternatively, engineered polynucleotides or vectors encoding engineered polynucleotides can be bound to lipids. Lipid-bound engineered polynucleotides or vectors encoding lipid-bound engineered polynucleotides can be encapsulated within the aqueous interior of liposomes, dispersed within the lipid bilayer of liposomes, attached to liposomes via linker molecules binding both liposomes and engineered polynucleotides, embedded in liposomes, complexed with liposomes, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained in lipids as a suspension, containing micelles or complexed with micelles, or otherwise bound to lipids. Lipid, lipid / DNA, or lipid / expression vector binding compositions are not limited to any particular structure in solution. For example, in some embodiments, they exist in a bilayer structure, such as micelles, or have a “collapsed” structure. Alternatively, they may simply be dispersed in solution, forming aggregates of non-uniform size or shape. Lipids are fatty substances, which in some embodiments are naturally occurring or synthetic lipids. For example, lipids include fat droplets naturally occurring in the cytoplasm, as well as a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0242] Suitable lipids are obtained from commercial sources. Stock solutions of lipids in chloroform or chloroform / methanol are typically stored at approximately -20°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol. "Liposome" is a general term encompassing a variety of monolayer and multilayer lipid carriers formed by the formation of closed lipid bilayers or aggregates. Liposomes are typically characterized by a vesicle structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. They spontaneously form when phospholipids are suspended in excess aqueous solution. The lipid components undergo self-rearrangement before forming a closed structure, trapping water and dissolved solutes between the lipid bilayers. However, compositions having structures in solution that differ from normal vesicle structures are also included. For example, in some embodiments, lipids exhibit a micellar structure or exist solely as heterogeneous aggregates of lipid molecules. Lipid transfection reagent-nucleic acid complexes are also considered.

[0243] In some cases, non-viral delivery methods include lipid transfection, nuclear transfection, microinjection, biological projectiles, virions, liposomes, immunoliposomes, exosomes, polycationic or lipid:cargo conjugates (or aggregates), naked peptides (e.g., recombinant peptides), naked DNA, artificial viral particles, and drug-enhanced uptake of peptides or DNA. In some embodiments, delivery methods include 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 may include aliphatic polyesters, poly(amino acids), copoly(ether-esters), polyalkylene oxalates, polyamides, poly(imino carbonates), polyorthoesters, polyoxoesters, polyamide esters, polyoxoesters containing amine groups, and poly(anhydrides). Such synthetic polymers can be homopolymers or copolymers of various monomers (e.g., random, segmental, fragmented, grafted), such as two or more lactic acid, lactide, glycolic acid, glycolide, ε-caprolactone, trimethylene carbonate, p-dioxanone, etc. In examples, the scaffold can be composed of polymers containing glycolic acid and lactic acid, such as those with a glycolic acid to lactic acid ratio of 90 / 10 or 5 / 95. Non-limiting examples of naturally occurring biocompatible, biodegradable polymers can include glycoproteins, proteoglycans, polysaccharides, glycosaminoglycans (GAGs) and fragments derived from these components, elastin, laminin, decrorin, fibrinogen / fibrin, fibronectin, osteopontin, tendinin, hyaluronic acid, collagen, chondroitin sulfate, heparin, heparan sulfate, ORC, carboxymethyl cellulose, and chitin.

[0244] In some cases, the engineered polynucleotides described herein, or the vectors encoding engineered polynucleotides, can be packaged and delivered to cells via extracellular vesicles. Extracellular vesicles can be any membrane-bound particles. In some embodiments, extracellular vesicles can be any membrane-bound particles secreted by at least one cell. In some cases, extracellular vesicles can be any membrane-bound particles synthesized in vitro. In some cases, extracellular vesicles can be any membrane-bound particles synthesized without the need for cell synthesis. In some cases, extracellular vesicles can be exogenous bodies, microvesicles, retrovirus-like particles, apoptotic bodies, apoptotic vesicles, cancerous vesicles, exophers, enveloped viruses, exomeres, or other very large extracellular vesicles.

[0245] In some aspects, this document describes methods for regulating or altering the expression or activity of genes encoded by target sequences in cells. In some embodiments, the target sequence is a precursor messenger RNA (mRNA) in the cell. In some embodiments, the method includes contacting the cell with an engineered polynucleotide comprising one or more target portions and a recruitment portion. In some embodiments, one or more target portions bind to the mRNA precursor at the target sequence within the mRNA precursor. In some embodiments, the recruitment portion recruits a post-transcriptional regulatory portion (e.g., a spliceosome portion) within the vicinity of the target sequence of the mRNA precursor to alter the mRNA precursor in the cell, thereby producing one or more altered mRNA precursors. In some embodiments, the mRNA precursor 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 portion and recruits it to the target sequence. In some embodiments, the method decreases the expression or activity of the target gene when the engineered polynucleotide binds to the spliceosome portion and recruits it to the target sequence. In some implementations, the method corrects abnormal messenger RNA (mRNA) or protein corresponding to the target gene when engineered polynucleotides bind to spliceosome portions and recruit them to the target sequence.

[0246] In some embodiments, the method includes contacting or delivering two or more engineered polynucleotides into a single cell, wherein each engineered polynucleotide contains one or more target moieties configured to bind two or more target sequences. The two or more target sequences may be located on the same strand of an mRNA precursor encoding a target gene. The two or more target sequences may be located on different strands of an mRNA precursor encoding the same target gene. The two or more target sequences may be located on different strands of the mRNA precursor, wherein each strand of the mRNA precursor may encode a different target gene. In some embodiments, the method includes two or more engineered polynucleotides configured to specifically bind the mRNA precursor at multiple target sequences containing the target sequences.

[0247] In some embodiments, this document discloses methods for treating diseases or conditions by regulating the expression or activity of target genes in cells. In some embodiments, the methods include treating the disease or condition by correcting abnormal messenger RNA (mRNA) or proteins corresponding to the target genes. In some embodiments, the disease or condition is associated with increased expression or activity of any of the target genes described herein. In some embodiments, the disease or condition is associated with decreased expression or activity of any of the target genes described herein. In some embodiments, the disease or condition is associated with splicing of abnormal messenger RNA (mRNA) or proteins corresponding to any of the target genes described herein.

[0248] In some embodiments, engineered polynucleotides or pharmaceutical compositions containing engineered polynucleotides may be administered to the subject alone (e.g., as a standalone treatment). In some embodiments, engineered polynucleotides or pharmaceutical compositions containing engineered polynucleotides are administered in combination with an adjunct agent. In some cases, the adjunct agent, as used herein, is administered alone. Engineered polynucleotides or pharmaceutical compositions containing engineered polynucleotides and an adjunct agent may be administered together or sequentially. Combination therapy may be administered on the same day, or may be administered one or more days, weeks, months, or years apart.

[0249] In some embodiments, the engineered polynucleotide or pharmaceutical composition containing an engineered polynucleotide is administered at a concentration of at least 0.25 μM. In some embodiments, the engineered polynucleotide or pharmaceutical composition containing an engineered polynucleotide is administered at a concentration of at least 0.5 μM. In some embodiments, the engineered polynucleotide or pharmaceutical composition containing an engineered polynucleotide is administered at a concentration of at least 0.75 μM. In some embodiments, the engineered polynucleotide or pharmaceutical composition containing an engineered polynucleotide is administered at a concentration of at least 1 μM. In some embodiments, the engineered polynucleotide or pharmaceutical composition comprising the engineered polynucleotide is present in amounts 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 Apply at concentrations of 0.1 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 higher. In some embodiments, the engineered polynucleotide or pharmaceutical composition comprising the engineered polynucleotide is present in amounts not exceeding 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, ... The drug may be administered at concentrations 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 lower. In some embodiments, the engineered polynucleotide or pharmaceutical composition comprising an engineered polynucleotide may be administered at a concentration of about 0.25 μM to 1 μM.

[0250] In some embodiments, engineered polynucleotides or pharmaceutical compositions containing engineered polynucleotides are first-line treatments for diseases or conditions. In some embodiments, engineered polynucleotides or pharmaceutical compositions containing engineered polynucleotides are second-, third-, or fourth-line treatments. In some embodiments, engineered polynucleotides or pharmaceutical compositions containing engineered polynucleotides comprise at least one, two, three, four, five, six, seven, eight, nine, ten, twenty, thirty, or more oligonucleotides. Generally, the methods disclosed herein include administering engineered polynucleotides or pharmaceutical compositions containing engineered polynucleotides by oral administration. However, in some cases, the method includes administering engineered polynucleotides or pharmaceutical compositions containing engineered polynucleotides by intraperitoneal injection. In some cases, the method includes administering engineered polynucleotides or pharmaceutical compositions containing engineered polynucleotides by intravenous (“iv”) administration. In some cases, the method includes administering engineered polynucleotides or pharmaceutical compositions containing engineered polynucleotides by intratumoral administration. It is conceivable that the engineered polynucleotides disclosed herein, or pharmaceutical compositions comprising such engineered polynucleotides, can also be administered via other routes, such as subcutaneous injection, intramuscular injection, intradermal injection, transdermal administration, intranasal administration, intralymphatic injection, intrathecal administration, pulmonary administration, rectal administration, gastric administration, or any other suitable parenteral administration. In some embodiments, a local delivery route closer to the site of injury or inflammation is preferred compared to a systemic route. The route, dose, timing, and duration of administration of the therapeutic agent can be adjusted. In some embodiments, the therapeutic agent is administered before or after the onset of one or both of the acute and chronic symptoms of the disease or condition.

[0251] The appropriate dose (dose / dosage) administered to the subject is determined by factors including, but not limited to, the specific engineered polynucleotide, composition or pharmaceutical composition, disease condition and its severity, comorbidities, the identity of the subject requiring treatment (e.g., weight, sex, age), and may be determined based on the specific circumstances surrounding the case, including, for example, the specific agent administered, the route of administration, the condition being treated, and the subject being treated.

[0252] This article describes a method for treating cancer in patients of need using engineered polynucleotides or vectors encoding engineered polynucleotides. The engineered polynucleotide can be the one described in PCT / US2022 / 037391, the entire contents of which are incorporated herein by reference.

[0253] This article describes methods for treating cancer in subjects of need, including administering a pharmaceutical composition comprising an engineered polynucleotide to the subject. In some embodiments, the engineered polynucleotide comprises: (i) one or more targeting portions configured to specifically bind the mRNA precursor at a target sequence in a messenger ribonucleic acid precursor (mRNA precursor); and (ii) a recruitment portion configured to recruit a spliceosome portion, wherein, upon binding to the mRNA precursor and the engineered polynucleotide, the spliceosome portion alters the mRNA precursor in or near the target sequence.

[0254] In some embodiments, the cancer is selected from: 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 adenocarcinoma. In some embodiments, the cancer is kidney cancer. In some embodiments, the cancer is hepatocellular carcinoma. In some embodiments, the cancer is renal adenocarcinoma.

[0255] In some embodiments, this document provides a method comprising: contacting cells with the engineered polynucleotide described herein or a carrier encoding the engineered polynucleotide. In some embodiments, contacting cells with the engineered polynucleotide or a carrier encoding the engineered polynucleotide results in cytotoxicity or cell inhibition. In some embodiments, contacting cells with the engineered polynucleotide or a carrier encoding the engineered polynucleotide results in altered cell viability. In some embodiments, contacting cells with the engineered polynucleotide or a carrier encoding the engineered polynucleotide results in the internalization of the engineered polynucleotide. In some embodiments, contacting cells with the engineered polynucleotide or a carrier encoding the engineered polynucleotide results in reduced mitochondrial activity. In some embodiments, contacting cells with the engineered polynucleotide or a carrier encoding the engineered polynucleotide results in reduced cell proliferation. In some embodiments, contacting cells with the engineered polynucleotide or a carrier encoding the engineered polynucleotide results in a change in the distribution of cell cycle phases, wherein the cell cycle phases consist of: Sub / G1: non-proliferative state (quiescent); G1 / G0: cell growth; S: DNA replication; G2 / M: DNA separation and mitosis. In some implementations, contacting cells with engineered polynucleotides or vectors encoding engineered polynucleotides leads to an increased tendency for cells to be in the G2 / M phase.

[0256] In some embodiments, contacting cells with engineered polynucleotides or vectors encoding engineered polynucleotides leads to increased apoptosis or necrosis. In some embodiments, contacting cells with engineered polynucleotides or vectors encoding engineered polynucleotides leads to decreased MAPT expression. In some embodiments, contacting cells with engineered polynucleotides or vectors encoding engineered polynucleotides leads to a decrease in TAU levels. In some embodiments, contacting cells with engineered polynucleotides or vectors encoding engineered polynucleotides leads to changes in mitochondrial membrane potential.

[0257] In some embodiments, this document provides a method comprising contacting a plurality of cells with the 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 results in a change in the cell cycle phase distribution, wherein the cell cycle phases consist of: Sub / G1: non-proliferative state (quiescent); G1 / G0: cell growth; S: DNA replication; G2 / M: DNA separation and mitosis. In some embodiments, contacting a plurality of cells with the engineered polynucleotide or a vector encoding the engineered polynucleotide results in an increase in the number of cells in the G2 / M phase. In some embodiments, contacting cells with the engineered polynucleotide or a vector encoding the engineered polynucleotide results in an increase in the number of cells in the necrosis or apoptosis phase.

[0258] In some embodiments, this document provides a method for treating a subject who has or is suspected of having cancer. In some embodiments, this document provides a method for treating a tumor in a subject. In some embodiments, contacting the subject's tumor with an engineered polynucleotide or a carrier encoding an engineered polynucleotide results in a slowing of tumor progression. In some embodiments, the tumor is glioblastoma. In some embodiments, the engineered polynucleotide or the carrier encoding an engineered polynucleotide is administered intravenously. In some embodiments, the engineered polynucleotide or the carrier encoding an engineered polynucleotide is administered intratumorally. In some embodiments, the engineered polynucleotide or the carrier encoding an engineered polynucleotide is administered subcutaneously. In some embodiments, the engineered polynucleotide or the carrier encoding an engineered polynucleotide is administered once. In some embodiments, the engineered polynucleotide or the carrier encoding an engineered polynucleotide is administered once daily. In some embodiments, the engineered polynucleotide or the carrier encoding an engineered polynucleotide is administered once daily for 1 to 21 days. In some embodiments, application of the engineered polynucleotide or the carrier encoding an engineered polynucleotide to a subject in need results in a reduction in tumor volume. In some implementations, applying engineered polynucleotides or vectors encoding engineered polynucleotides to the recipient results in a reduction in tumor volume ratio.

[0259] In some embodiments, the cells are glioblastoma cells. In some embodiments, the cells are breast adenocarcinoma cells. In some embodiments, the cells are neuroblastoma cells. In some embodiments, the cells are prostate adenocarcinoma cells. In some embodiments, the cells are renal cell carcinoma cells. In some embodiments, the cells are hepatocellular carcinoma cells. In some embodiments, the cells are renal adenocarcinoma cells. In some embodiments, the cells are neurons. In some embodiments, the cells are derived from a subject diagnosed with cancer. In some embodiments, the cells are neurons derived from a subject diagnosed with cancer.

[0260] In some embodiments, the engineered polynucleotide is ASMO1. In some embodiments, ASMO1 interacts directly or indirectly with U1-C and U1-70K. In some embodiments, ASMO1 interacts directly or indirectly with the U1 complex and the MAPT mRNA precursor.

[0261] In various implementations, taxanes are administered or co-administered to the subject as if previously administered. Taxanes may include taxanes effective in treating cancer. Following administration of the engineered polynucleotide, the subject's prognosis may improve, for example, compared to before administration of the engineered polynucleotide, or compared to another subject who received a taxane but not the engineered peptide. The engineered peptide may alter the amount of TAU present in the subject. Taxanes may be able to bind to TAU, thereby reducing the amount of taxanes that can bind to other targets in the cell. By reducing the amount of TAU, off-target binding of taxanes may be reduced and their efficacy may be improved. In various implementations, taxanes are not administered or co-administered to the subject as if previously administered.

[0262] In various implementation schemes, engineered polynucleotides are administered to the subject without the administration of another anticancer drug or cancer treatment agent.

[0263] In various implementation schemes, another cancer therapy is administered to the subject, or co-administered, as if another cancer therapy (e.g., a drug, neoadjuvant therapy, adjuvant therapy) had been previously administered. Administering engineered polynucleotides can improve the subject's prognosis or enhance treatment efficacy compared to administering other therapies alone.

[0264] The use of absolute or sequential terms, such as “will,” “will not,” “shall,” “shall,” “must,” “must not,” “first,” “initially,” “next,” “subsequently,” “before,” “after,” “lastly,” and “finally,” is not intended to limit the scope of the embodiments disclosed herein, but is merely illustrative.

[0265] As used herein, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well. Furthermore, with regard to the terms “including,” “includes,” “having,” “has,” “with,” or variations thereof used in the detailed description and / or claims, these terms are intended to be inclusive, similar to the term “comprising.”

[0266] As used in this article, the phrases “at least one,” “one or more,” and “and / or” are open-ended expressions that are both conjunction and disjunctive in operation. For example, the expressions “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” refer to A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B, and C together.

[0267] As used herein, "or" can mean "and," "or," or "and / or," and all can be used exclusively and inclusively. For example, the term "A or B" can mean "A or B," "A but not B," "B but not A," and "A and B." In some cases, the context can determine the specific meaning.

[0268] Any systems, methods, software, and platforms described herein are modular. Therefore, terms such as “first” and “second” do not necessarily imply priority, order of importance, or order of action.

[0269] When referring to a number or range of values, the term "approximately" means that the number or range mentioned is an approximation within experimental variability (or statistical experimental error), and that the number or range may vary, for example, from 1% to 15% of the specified number or range. In the example, the term "approximately" refers to ±10% of the specified number or value.

[0270] As used herein, the term “increased / increasing / increase” generally refers to an increase in a statically significant amount. In some respects, the term “increase” means an increase of at least 10% compared to a reference level, such as an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or an increase of up to and including 100% or any increase between 10% and 100%. Other examples of “increase” include increases of at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times, at least 100 times, at least 1000 times, or more compared to a reference level.

[0271] As used herein, the term "decreased / decreasing / decrease" generally means a statistically significant reduction. In some respects, "decreased" means a reduction of at least 10% compared to a reference level, such as 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 and including 100% (e.g., no level or a level undetectable compared to the reference level), or any reduction between 10% and 100%. In the context of a biomarker or symptom, these terms imply a statistically significant reduction in this level. The reduction can be, for example, at least 10%, at least 20%, at least 30%, at least 40%, or more, and preferably a reduction to a level acceptable within the normal range for an individual without a given disease.

[0272] Although preferred embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. The invention is not intended to be limited to the specific examples provided in the specification. Although the invention has been described with reference to the foregoing description, the description and illustration of embodiments herein are not intended to be construed as limiting. Many variations, alterations, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it should be understood that all aspects of the invention are not limited to the specific descriptions, configurations, or relative proportions set forth herein, and depend on various conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in carrying out the invention. Therefore, it is contemplated that the invention should also cover any such alternatives, modifications, variations, or equivalents. The appended claims are intended to define the scope of the invention and thereby cover the methods and structures within the scope of these claims and their equivalents.

[0273] List of Implementation Plans

[0274] Implementation Scheme 1. An engineered polynucleotide comprising: one or more targeting portions configured to specifically bind the mRNA precursor at a target sequence in a messenger ribonucleic acid precursor (mRNA precursor); and a recruitment portion configured to recruit a post-transcriptional regulatory portion (e.g., a spliceosome portion), wherein, upon binding to the mRNA precursor and the engineered polynucleotide, the post-transcriptional regulatory portion alters the mRNA precursor in or near the target sequence.

[0275] Implementation Scheme 2. The engineered polynucleotide according to Implementation Scheme 1, wherein the target portion of one or more target portions is sufficiently identical or complementary to the common sequence in the target sequence of the target gene.

[0276] Implementation Scheme 3. The engineered polynucleotide according to Implementation Scheme 1 or 2, wherein the one or more targeting portions include (1) a first targeting portion configured to specifically bind a first target sequence in the target sequence of the mRNA precursor, and (2) a second targeting portion configured to specifically bind a second target sequence in the target sequence of the mRNA precursor.

[0277] Implementation Scheme 4. The engineered polynucleotide according to Implementation Scheme 3, wherein the first target sequence comprises a common sequence in the target sequence.

[0278] Implementation Scheme 5. The engineered polynucleotide according to Implementation Scheme 3 or 4, wherein the second target sequence comprises a common sequence in the target sequence.

[0279] Implementation Scheme 6. An engineered polynucleotide according to any one of Implementation Schemes 3 to 5, wherein the first and second target sequences are spacer sequences that are separated by no more than five nucleotides (e.g., one or two nucleotides) in the target sequence.

[0280] Implementation Scheme 7. An engineered polynucleotide according to any one of Implementation Schemes 1 to 6, wherein the target sequence comprises an exon-intron boundary in the mRNA precursor.

[0281] Implementation Scheme 8. The engineered polynucleotide according to Implementation Scheme 7, wherein both the first and second target sequences are located at the 5' or 3' of the exon-intron boundary.

[0282] Implementation Scheme 9. The engineered polynucleotide according to Implementation Scheme 7, wherein one of the first and second target sequences is located at the 5' of the exon-intron boundary; and wherein the other of the first and second target sequences is located at the 3' of the exon-intron boundary.

[0283] Implementation Scheme 10. An engineered polynucleotide according to any one of Implementation Schemes 1 to 9, wherein the target sequence comprises a splicing site in the mRNA precursor.

[0284] Implementation Scheme 11. The engineered polynucleotide according to Implementation Scheme 10, wherein the first or second target sequence comprises a splicing site (e.g., 5'ss) in the mRNA precursor.

[0285] Implementation Scheme 12. An engineered polynucleotide according to any one of Implementation Schemes 1 to 11, wherein one of the first and second targeting portions is located at the 5' of the recruitment portion, and the other of the first and second targeting portions is located at the 3' of the recruitment portion.

[0286] Implementation Scheme 13. The engineered polynucleotide according to Implementation Scheme 12, wherein the first targeting portion or the second targeting portion comprises a sequence that is identical to or complementary to the sequence shown in Table 1.

[0287] Implementation Scheme 14. The engineered polynucleotide according to Implementation Scheme 12, wherein the first targeting portion comprises a sequence that is identical or complementary to a sequence selected from the exon sequence column of Table 1; and wherein the second targeting portion comprises a sequence that is identical or complementary to a sequence shown in the intron sequence column of Table 1.

[0288] Implementation Scheme 15. The engineered polynucleotide according to Implementation Scheme 12, wherein the first targeting portion comprises a sequence that is identical or complementary to the sequence shown in the intron sequence column of Table 1; and wherein the second targeting portion comprises a sequence that is identical or complementary to the sequence shown in the exon sequence column of Table 1.

[0289] Implementation Scheme 16. The engineered polynucleotide according to Implementation Scheme 12, wherein the first targeting portion or the second targeting portion comprises a sequence that is identical or complementary to the common sequence of the intron donor site (e.g., selected from GU, GT, GC and CA).

[0290] Implementation Scheme 17. The engineered polynucleotide according to Implementation Scheme 12, wherein the first targeting portion or the second targeting portion comprises a sequence that is identical to or complementary to the common sequence (e.g., G) of the exon donor site.

[0291] Implementation Scheme 18. The engineered polynucleotide according to Implementation Scheme 12, wherein the first targeting portion or the second targeting portion comprises a sequence that is identical or complementary to a common sequence selected from GU, GC, G and CA.

[0292] Implementation Scheme 19. An engineered polynucleotide according to any one of Implementation Schemes 1 to 18, wherein the spliceosome portion is selected from spliceosome ribonucleoprotein complex, spliceosome small nucleoribonucleic acid (snRNA), spliceosome protein, functional variants thereof, or functional fragments thereof.

[0293] Implementation Scheme 20. The engineered polynucleotide according to Implementation Scheme 19, wherein the spliceosome portion comprises U1 snRNA and spliceosome protein.

[0294] Implementation Scheme 21. An engineered polynucleotide according to any one of Implementation Schemes 19 to 20, wherein the spliceosome snRNA is selected from U1, U2, U4, U5, U6, U11, U12, U14atac, U6atac and combinations thereof.

[0295] Implementation Scheme 22. An engineered polynucleotide according to any one of Implementation Schemes 19 to 21, wherein the spliceosome protein is selected from Sm, U1-70k, U1A, U1C and combinations thereof.

[0296] Implementation Scheme 23. An engineered polynucleotide according to any one of Implementation Schemes 1 to 22, wherein the recruitment portion comprises at least 70%, 80%, 85%, or 90% identical or complementary nucleotide sequences to any one of SEQ ID NO: 1-2.

[0297] Implementation Scheme 24. The engineered polynucleotide according to Implementation Scheme 23, wherein the recruitment portion comprises a nucleotide sequence that is identical or complementary to any one of SEQ ID NO:1-2.

[0298] Implementation Scheme 25. An engineered polynucleotide according to any one of Implementation Schemes 1 to 24, wherein the engineered polynucleotide comprises (e.g., secondary) structural features.

[0299] Implementation Scheme 26. The engineered polynucleotide according to Implementation Scheme 25, wherein the engineered polynucleotide comprises a top loop, an upper stem, an inner loop, a lower stem, or a combination thereof.

[0300] Implementation Scheme 27. The engineered polynucleotide according to Implementation Scheme 25, wherein the engineered polynucleotide comprises a loop (e.g., an inner loop) adjacent to a stem (e.g., a lower stem or an upper stem), the stem comprising two complementary stem sequences.

[0301] Implementation Scheme 28. The engineered polynucleotide according to Implementation Scheme 27, wherein the stem sequence of the stem (e.g., the lower stem or the upper stem) contains no more than about five, four or three nucleotides.

[0302] Implementation Scheme 29. An engineered polynucleotide according to Implementation Scheme 27 or 28, wherein the loop is an inner loop adjacent to the stem (e.g., the lower stem) containing two complementary stem sequences and another stem (e.g., the upper stem).

[0303] Implementation Scheme 30. The engineered polynucleotide according to Implementation Scheme 29, wherein the inner loop comprises a nucleic acid sequence of no more than 10, 9, or 8 nucleotides.

[0304] Implementation Scheme 31. The engineered polynucleotide according to Implementation Scheme 29 or 30, wherein the stem sequence of the other stem (e.g., the upper stem) contains no more than about five, four or three nucleotides.

[0305] Implementation Scheme 32. The engineered polynucleotide according to any one of Implementation Schemes 29 to 31, wherein the engineered polynucleotide further comprises a top loop.

[0306] Implementation Scheme 33. The engineered polynucleotide according to Implementation Scheme 32, wherein the top loop comprises a nucleic acid sequence of no more than 10, 9, 8, 7, 6 or 5 nucleotides.

[0307] Implementation Scheme 34. An engineered polynucleotide according to any one of Implementation Schemes 1 to 33, wherein the engineered polynucleotide does not contain any intramolecular disulfide bonds.

[0308] Implementation Scheme 35. An engineered polynucleotide according to any one of Implementation Schemes 1 to 34, wherein, when bound to the engineered polynucleotide and the spliceosome portion, the mRNA precursor substantially does not exhibit base pairing with the RNA-binding domain (RBD) of the U1 snRNA.

[0309] Implementation Scheme 36. An engineered polynucleotide according to any one of Implementation Schemes 1 to 35, wherein, when bound to the engineered polynucleotide and the spliceosome portion, the mRNA precursor substantially does not exhibit base-specific interactions with the U1-C protein.

[0310] Implementation Scheme 37. An engineered polynucleotide according to any one of Implementation Schemes 1 to 36, wherein the engineered polynucleotide is configured to specifically interact with the zinc finger of the U1-C protein.

[0311] Implementation Scheme 38. The engineered polynucleotide according to Implementation Scheme 37, wherein the 5'-targeting portion of the engineered polynucleotide is configured to specifically interact with the zinc finger of the U1-C protein.

[0312] Implementation Scheme 39. An engineered polynucleotide according to Implementation Scheme 37 or 38, wherein the engineered polynucleotide (e.g., its 5'-targeting portion) is configured to covalently interact with the zinc finger of the U1-C protein (e.g., via disulfide bonds).

[0313] Implementation Scheme 40. An engineered polynucleotide according to any one of Implementation Schemes 37 to 39, wherein the engineered polynucleotide (e.g., its 5'-targeting portion) is configured to interact non-covalently with the zinc finger of the U1-C protein (e.g., via hydrogen bonds).

[0314] Implementation Scheme 41. An engineered polynucleotide according to any one of Implementation Schemes 1 to 40, wherein the engineered polynucleotide comprises a nucleotide sequence complementary to a partial sequence of the stem-loop II (SL2) of the U1 snRNA.

[0315] Implementation Scheme 42. The engineered polynucleotide according to Implementation Scheme 41, wherein one side of the stem-loop structure of the engineered polynucleotide contains a nucleotide sequence complementary to a partial sequence of the stem-loop II (SL2) of the U1 snRNA.

[0316] Implementation Scheme 43. The engineered polynucleotide according to Implementation Scheme 41 or 42, wherein the partial sequence comprises a sequence corresponding to the 5'-GGCCU-3' of the SL2 of the U1 snRNA.

[0317] Implementation Scheme 44. An engineered polynucleotide according to any one of Implementation Schemes 41 to 43, wherein the partial sequence does not contain a sequence corresponding to the 5'-CACGUUA-3' of the SL2 of the U1 snRNA.

[0318] Implementation Scheme 45. An engineered polynucleotide according to any one of Implementation Schemes 1 to 44, wherein the engineered polynucleotide substantially does not exhibit base pairing with the anchoring sequence of U1 snRNA at SL2.

[0319] Implementation Scheme 46. The engineered polynucleotide according to Implementation Scheme 45, wherein the inner loop of the engineered polynucleotide substantially does not exhibit base pairing with the anchoring sequence of the SL2 of the U1 snRNA.

[0320] Implementation Scheme 47. An engineered polynucleotide according to Implementation Scheme 45 or 46, wherein the lower stem of the engineered polynucleotide substantially does not exhibit base pairing with the anchoring sequence of the SL2 of the U1 snRNA.

[0321] Implementation Scheme 48. An engineered polynucleotide according to any one of Implementation Schemes 45 to 47, wherein the anchoring sequence comprises a sequence corresponding to 5'-CACGUUA-3'.

[0322] Implementation Scheme 49. An engineered polynucleotide according to any one of Implementation Schemes 1 to 48, wherein the engineered polynucleotide substantially does not exhibit H-helical base pairing with U1 snRNA.

[0323] Implementation Scheme 50. An engineered polynucleotide according to any one of Implementation Schemes 1 to 49, wherein the engineered polynucleotide comprises at least one chemical modification.

[0324] Implementation Scheme 51. The engineered polynucleotide according to Implementation Scheme 50, wherein the engineered polynucleotide comprises at least one 2'-modified (e.g., 2'-methoxy, 2'-methoxymethyl, or 2'-methoxyethyl) nucleotide.

[0325] Implementation Scheme 52. The engineered polynucleotide according to Implementation Scheme 50 or 51, wherein at least about 50%, 60%, 70%, 80%, or 90% of the nucleotides of the engineered polynucleotide are chemically modified nucleotides.

[0326] Implementation Scheme 53. The engineered polynucleotide according to Implementation Scheme 50 or 51, 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.

[0327] Implementation Scheme 54. An engineered polynucleotide according to any one of Implementation Schemes 50 to 53, wherein the engineered polynucleotide comprises at least one phosphate thioester nucleotide linker.

[0328] Implementation Scheme 55. An engineered polynucleotide according to any one of Implementation Schemes 50 to 54, wherein at least about 50%, 60%, 70%, 80%, or 90% of the nucleotide linkages of the engineered polynucleotide are chemically modified.

[0329] Implementation Scheme 56. An engineered polynucleotide according to any one of Implementation Schemes 50 to 54, wherein at least about 50%, 60%, 70%, 80% or 90% of the nucleotides are linked by phosphate thioesters.

[0330] Implementation Scheme 57. An engineered polynucleotide according to any one of Implementation Schemes 1 to 56, wherein 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.

[0331] Implementation Scheme 58. An engineered polynucleotide according to any one of Implementation Schemes 1 to 57, wherein the recruitment portion comprises about 10 to about 30 nucleotides, or about 10 to about 20 nucleotides.

[0332] Implementation Scheme 59. An engineered polynucleotide according to any one of Implementation Schemes 1 to 58, wherein each of the one or more targeting portions independently comprises about 2 to about 15 nucleotides, about 2 nucleotides to about 10 nucleotides, or about 2 nucleotides to about 8 nucleotides.

[0333] Implementation Scheme 60. An engineered polynucleotide according to any one of Implementation Schemes 1 to 59, wherein one of the first and second targeting portions comprises about 2 nucleotides, and the other of the first and second targeting portions comprises about 5 or 6 nucleotides.

[0334] Implementation Scheme 61. An engineered polynucleotide according to any one of Implementation Schemes 1 to 60, wherein, when bound to the engineered polynucleotide and the mRNA precursor, the spliceosome partially cleaves or splices the mRNA precursor in the target sequence.

[0335] Implementation Scheme 62. An engineered polynucleotide according to any one of Implementation Schemes 1 to 61, wherein the spliceosome portion further promotes the modification of the cleaved mRNA precursor.

[0336] Implementation Scheme 63. An engineered polynucleotide comprising at least 70%, 80%, 85%, or 90% identical or complementary nucleotide sequences to any one of SEQ ID NO:1-4, wherein the engineered polynucleotide is characterized by (e.g., secondary) structural features.

[0337] Implementation Scheme 64. The engineered polynucleotide according to Implementation Scheme 63, wherein the nucleotide sequence is identical or complementary to any one of SEQ ID NO: 1-4.

[0338] Implementation Scheme 65. The engineered polynucleotide according to Implementation Scheme 63 or 64, wherein the structural feature comprises one or more stem-loop structures.

[0339] Implementation Scheme 66. The engineered polynucleotide according to Implementation Scheme 63 or 64, wherein the structural features include a top loop, an upper stem, an inner loop, a lower stem, or a combination thereof.

[0340] Implementation Scheme 67. An engineered polynucleotide according to any one of Implementation Schemes 63 to 66, wherein the engineered polynucleotide comprises a loop (e.g., an inner loop) adjacent to a stem (e.g., a lower stem or an upper stem), the stem comprising two complementary stem sequences.

[0341] Implementation Scheme 68. The engineered polynucleotide according to Implementation Scheme 67, wherein the stem sequence of the stem (e.g., the lower stem or the upper stem) contains no more than about five, four or three nucleotides.

[0342] Implementation Scheme 69. An engineered polynucleotide according to any one of Implementation Schemes 63 to 68, wherein the loop is an inner loop adjacent to the stem (e.g., the lower stem) containing two complementary stem sequences and another stem (e.g., the upper stem).

[0343] Implementation Scheme 70. The engineered polynucleotide according to Implementation Scheme 69, wherein the inner loop comprises a nucleic acid sequence of no more than 10, 9, or 8 nucleotides.

[0344] Implementation Scheme 71. The engineered polynucleotide according to Implementation Scheme 69 or 70, wherein the stem sequence of the other stem (e.g., the upper stem) contains no more than about five, four or three nucleotides.

[0345] Implementation Scheme 72. The engineered polynucleotide according to any one of Implementation Schemes 69 to 71, wherein the engineered polynucleotide further comprises a top loop.

[0346] Implementation Scheme 73. The engineered polynucleotide according to Implementation Scheme 72, wherein the top loop comprises a nucleic acid sequence of no more than 10, 9, 8, 7, 6 or 5 nucleotides.

[0347] Implementation Scheme 74. An engineered polynucleotide according to any one of Implementation Schemes 63 to 73, wherein the engineered polynucleotide further comprises one or more targeting portions that are sufficiently identical to or complementary to the target sequence of the target gene.

[0348] Implementation Scheme 75. The engineered polynucleotide according to Implementation Scheme 74, wherein the target portion of one or more target portions is sufficiently identical or complementary to a common sequence in the target sequence of the target gene.

[0349] Implementation Scheme 76. The engineered polynucleotide according to Implementation Scheme 74, wherein the target gene is microtubule-associated protein TAU (MAPT).

[0350] Implementation Scheme 77. An engineered polynucleotide according to any one of Implementation Schemes 63 to 75, wherein the engineered polynucleotide comprises at least one chemical modification.

[0351] Implementation Scheme 78. The engineered polynucleotide according to Implementation Scheme 77, wherein the engineered polynucleotide comprises at least one phosphate thioester nucleotide inter-bond.

[0352] Implementation Scheme 79. The engineered polynucleotide according to Implementation Scheme 77, wherein at least about 50%, 60%, 70%, 80%, or 90% of the nucleotide linkages of the engineered polynucleotide are chemically modified.

[0353] Implementation Scheme 80. The engineered polynucleotide according to Implementation Scheme 77, wherein at least about 50%, 60%, 70%, 80% or 90% of the nucleotides are linked by phosphate thioesters.

[0354] Implementation Scheme 81. An engineered polynucleotide according to any one of Implementation Schemes 77 to 80, wherein the engineered polynucleotide comprises at least one 2'-modified (e.g., 2'-methoxy, 2'-methoxymethyl, or 2'-methoxyethyl) nucleotide.

[0355] Implementation Scheme 82. An engineered polynucleotide according to any one of Implementation Schemes 77 to 80, wherein at least about 50%, 60%, 70%, 80%, or 90% of the nucleotides of the engineered polynucleotide are chemically modified nucleotides.

[0356] Implementation Scheme 83. An engineered polynucleotide according to any one of Implementation Schemes 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.

[0357] Implementation Scheme 84. An engineered polynucleotide according to any one of Implementation Schemes 64 to 84, wherein 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.

[0358] Implementation Scheme 85. A method for altering a messenger ribonucleic acid precursor (mRNA precursor) in a cell, the method comprising contacting the cell with an engineered polynucleotide comprising one or more target portions and a recruitment portion, wherein the one or more target portions bind the mRNA precursor at a target sequence in the mRNA precursor, and the recruitment portion recruits a post-transcriptional regulatory portion (e.g., a spliceosome portion) of the mRNA precursor in the vicinity of the target sequence to alter the mRNA precursor in the cell, thereby producing one or more altered mRNA precursors.

[0359] Implementation Scheme 86. The method according to Implementation Scheme 85, wherein the target portion of one or more target portions is sufficiently identical or complementary to the common sequence in the target sequence of the target gene.

[0360] Implementation Scheme 87. The method according to Implementation Scheme 85 or 86, wherein the mRNA precursor corresponds to the target gene.

[0361] Implementation Scheme 88. The method according to Implementation Scheme 87, wherein the target gene is microtubule-associated protein TAU (MAPT).

[0362] Implementation Scheme 89. The method according to any one of Implementation Schemes 85 to 88, wherein the method alters the expression or activity of the target gene.

[0363] Implementation Scheme 90. The method according to any one of Implementation Schemes 85 to 89, wherein, prior to the contact, the cell exhibits an abnormal messenger ribonucleic acid (mRNA) or protein corresponding to the target gene.

[0364] Implementation Scheme 91. A set of engineered polynucleotides, each engineered polynucleotide independently comprising: (i) one or more targeting portions configured to bind a messenger ribonucleic acid precursor (mRNA precursor) at a target sequence, and (ii) a recruitment portion configured to recruit a posttranscriptional regulatory portion (e.g., a spliceosome portion), wherein the set of engineered polynucleotides is configured to specifically bind the mRNA precursor at a plurality of target sequences including the target sequence.

[0365] Example

[0366] The following illustrative examples represent implementations of the stimuli, systems, and methods described herein and are not intended to limit in any way.

[0367] Example 1. Regulation of target gene expression using engineered polynucleotides

[0368] Cells obtained from cell samples (e.g., the HEK293 cell line) are cultured and maintained in cell culture medium. The cells can then be contacted with engineered polynucleotides or vectors encoding engineered polynucleotides to deliver the engineered polynucleotides or vectors encoding engineered polynucleotides into the cells using any of the delivery methods described herein. After delivery of the engineered polynucleotides into the cells, the cells can be cultured for a period of time to allow the engineered polynucleotides 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 for examining the abundance of mRNA precursors, mRNA, or proteins 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 abundance changes of inclusion bodies or amyloid plaques (e.g., TAU plaques encoded by the MAPT target gene) associated with any of the target genes described herein.

[0369] 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.

[0370] Cell lines were maintained in growth medium containing 10% fetal bovine serum (FBS) until the experimental time. Cells were passaged by trypsin digestion, aliquoting the cell suspension into fresh flasks, and replenishing with fresh medium. For assays, cells were trypsin-digested, neutralized with complete medium (10% FBS), centrifuged, counted using a hemocytometer, and seeded into appropriate medium containing 10% FBS. Except for the SH-SY5Y cell line, which was grown at 2.5 x 10⁻⁶ cells / mL... 4 In addition to seeding at a density of 1 x 10⁶ cells / well, other cell lines were seeded at a density of 1 x 10⁶ cells / well. 4 Cells were seeded at a density of [insert cell density here]. Different experimental groups were prepared using 0.1% FBS (U87MG, MCF-7, SH-SY5Y, and 786-O) and 1% FBS (PC-3). Untreated cells were used as negative controls (culture medium only). The effects of ASMO1 and controls on cell cytotoxicity were determined by MTT assay after 24, 48, 72, and 96 hours of incubation.

[0371] In short, remove the plate and add 20 μl of 5 mg / ml MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazol bromide solution to all wells. Incubate the cells at 37°C for 3 hours. After this period, aspirate the supernatant and add 100 μl of DMSO to each well to dissolve the formazan crystals. Then, read the absorbance of each well at 540 nm using a Synergy HT microplate reader.

[0372] The graphs were generated using GraphPad Prism 9 software (GraphPad Software Inc., San Diego, CA, USA).

[0373] Figure 11 AD shows the effect of ASMO1 APT20TTMG incubation in the U-87MG cell line. At 24 hours, it appeared to exert a cytotoxic effect, as cell viability tended to decrease compared to the negative control and the vector group. At 48 hours, absorbance values ​​decreased, which may indicate the presence of cytotoxic and / or cell-inhibiting effects. However, at 72 and 96 hours, these values ​​were similar to those of the vector group, meaning that the effect of reducing viability appeared to have ended. Cells were seeded and treated with seven different concentrations (0.03, 0.1, 0.3, 1, 3, 10, and 30 μM) of APT20TTMG with medium (0.1% FBS). MTT assays were performed at four different time points, and graphs are presented as absorbance (540 nm). (A) Incubation for 24 hours. (B) Incubation for 48 hours. (C) Incubation for 72 hours. (D) Incubation for 96 hours.

[0374] like Figure 12 As shown in AD, ASMO1 APT20TTMG did not appear to exert cytotoxic or inhibitory effects in the MCF-7 cell line. Cells were seeded and treated with seven different concentrations (0.03, 0.1, 0.3, 1, 3, 10, and 30 μM) of APT20TTMG in medium (0.1% FBS). MTT assays were performed at four different time points, and the results are presented in graphs as absorbance (540 nm). (A) 24 hours of incubation. (B) 48 hours of incubation. (C) 72 hours of incubation.

[0375] (D) Incubate for 96 hours.

[0376] like Figure 13As shown in AD, ASMO1 APT20TTMG appeared to be cytotoxic starting at a concentration of 1 μM for up to 72 hours. At 96 hours, the situation was slightly different, and a concentration of 0.3 μM appeared to begin to produce some effect. Cells were seeded and treated with seven different concentrations (0.03, 0.1, 0.3, 1, 3, 10, and 30 μM) of APT20TTMG in medium (0.1% FBS). MTT assays were performed at four different time points, and the results are presented in graphs as absorbance (540 nm). (A) 24 hours of incubation. (B) 48 hours of incubation. (C) 72 hours of incubation. (D) 96 hours of incubation.

[0377] like Figure 14 As shown in AC, ASMO1 APT20TTMG did not appear to exert cytotoxic or inhibitory effects in the PC-3 cell line. Cells were seeded and treated with seven different concentrations (0.03, 0.1, 0.3, 1, 3, 10, and 30 μM) of APT20TTMG in medium (1% FBS). MTT assays were performed at three different time points, and the results are presented in graphs as absorbance (540 nm). (A) 24 hours of incubation. (B) 48 hours of incubation. (C) 72 hours of incubation.

[0378] like Figure 15 As shown in AC, ASMO1 APT20TTMG appeared to alter cell viability in the 786-O cell line within 24 hours. However, no difference was observed at two other time points compared to the control group (mediator group), with differences only observed at the highest concentration (30 μM). Cells were seeded and treated with seven different concentrations (0.03, 0.1, 0.3, 1, 3, 10, and 30 μM) of APT20TTMG in medium (0.1% FBS). MTT assays were performed at three different time points, and graphs are presented as absorbance (540 nm). (A) 24 hours of incubation. (B) 48 hours of incubation. (C) 72 hours of incubation.

[0379] The results of this study indicate that the effect of ASMO1 depends on the type of cell line used, the time, and the concentration, and it has an enhancing effect on glioblastoma and neuroblastoma.

[0380] Example 3. Evaluation of the antitumor potential of ASMO1 in the breast cancer cell line (MCF-7)

[0381] MCF-7 cells were maintained in Roswell Park Memorial Institute 1640 medium (RPMI 1640) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin solution. Cells were cultured in 75 cm² cell culture flasks at 37°C, 5% CO₂, and controlled humidity. After reaching 80-90% confluence, cells were washed with 10 mL phosphate-buffered saline (PBS) and separated from the flasks with trypsin-EDTA solution (0.25% / 0.03%), then neutralized with complete medium. Cells were then transferred to conical tubes and centrifuged at 1000 rpm for 5 min. The pellet was resuspended in medium and stained with 0.4% trypan blue for subsequent counting and measurement at TC20. TM Cell viability was determined using an automated cell counter (BioRad, Hercules, CA, USA). The expected cell viability value is greater than 90%.

[0382] Because the molecular target of ASMO APT20TTMG is located in the cell nucleus, APT20TTMG is absorbed by the cell to initiate its intended action. Cellular uptake of oligonucleotides is not well understood, but it is expected to occur in two steps: adsorption and internalization. Adsorption of antisense oligonucleotides modified with phosphate thioesters to the cell surface is considered rapid and energy-free. After adsorption, various endocytic pathways (including pinocytosis) can internalize these oligonucleotides. Cell surface proteins can guide oligonucleotide internalization via unconventional endocytosis or via clathrin- or caveolin-dependent endocytosis. Furthermore, different cell types exhibit different oligonucleotide binding capacities, and rapidly growing cells (including malignant tumor cells) absorb oligonucleotides more efficiently than slowly growing cells.

[0383] To investigate the internalization of ASMO APT20TTMG in the MCF-7 cell line, intracellular fluorescence was quantitatively measured by flow cytometry. For this purpose, ASMO APT20TTMG was conjugated with the fluorescent dye carboxyfluorescein. It was hypothesized that the percentage of internalized ASMO molecules might be proportional to the quantitatively detected intracellular fluorescence signal. The cell line was cultured in 24-well plates for 24 hours, with 5 x 10⁶ cells per well. 4Cells were then incubated with 1 μM ASMO1 prepared in FBS-free medium for 0.5, 1, 2, 4, and 6 hours. Untreated cells were used as a negative control. After treatment incubation, cells were washed twice with 1 mL PBS / well and then incubated with 200 μL / well of trypsin / EDTA solution for approximately 3 minutes to depolymerize adherent cells. Trypsin was neutralized by adding complete medium, and cells were centrifuged at 1500 rpm for 5 minutes. Subsequently, cells were washed with 2 mL / sample of PBS and centrifuged again at 1500 rpm for 5 minutes. Finally, the supernatant was discarded, and cells were resuspended in 200 μL PBS. Cell fluorescence corresponding to cellular uptake of carboxyfluorescein-labeled APT20TTMG was analyzed using a BD FACSCANTO II flow cytometer (BD Biosciences, NJ, USA) by acquiring 10,000 gated events per sample.

[0384] MTT assays were performed to assess cell viability of the MCF-7 cell line after treatment with ASMO APT20TTMG and to infer the cytotoxicity of ASMO1. The tetrazolium salt reduction (MTT) assay assesses cell viability by evaluating mitochondrial integrity. Viable cells can reduce the yellow, water-soluble MTT reagent to an insoluble purple formazan product. Formazan quantification can be measured using a spectrophotometer at 560 nm. The amount of formazan is directly proportional to the number of viable cells in the culture.

[0385] To perform this assay, MCF-7 cells were prepared at a concentration of 1 x 10⁻⁶. 4 Cells were seeded at a density of 100 cells / well in 96-well plates and cultured for 24 hours for adhesion, followed by incubation for 48 hours with eight concentrations (0.0078–1 μM) of APT20TTMG (prepared in medium containing 2% FBS). Cells were then washed with 150 μL / well of PBS and incubated for 3 hours with 100 μL / well of MTT solution (prepared in medium at a concentration of 0.5 mg / mL). Afterward, the MTT solution was removed from the plates, and the resulting formazan crystals were dissolved in 100 μL / well of dimethyl sulfoxide (DMSO) and mixed at 300 rpm on a steady-state shaker for 20 minutes. Finally, the absorbance of the samples was measured at 560 nm using 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.

[0386] The cell cycle consists of DNA replication (S phase), mitosis (M phase), and cytokinesis, separated by two interphases (G1 and G2). A non-proliferative state, called G0 (or quiescent phase), may also occur during G1 phase. These interphases are crucial for cell cycle regulation and for decisions related to entering the cell cycle in G1 phase or initiating DNA separation in G2 phase. Cell cycle progression regulates cell proliferation in this way, and its dysfunction plays a key role in the development of cancer (primarily malignant tumors). Controlling cell cycle progression by triggering cell cycle arrest may be an important feature of cancer therapy.

[0387] To understand how ASMO APT20TTMG affects the cell cycle of the MCF-7 cell line and its potential mechanism of action, cell cycle assays were performed. First, cells were cultured at 5 x 10⁻⁶ cells / year. 4 Cells were seeded at a density of 10 cells / well in 12-well plates and cultured for 24 hours to allow adhesion. They were then treated with three different concentrations of APT20TTMG (0.25, 0.5, and 1.0 μM) for 48 hours. Cells were then collected 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. Afterward, cells were washed again with 2.0 mL of PBS and incubated for 1 hour at room temperature in the dark with propidium iodide solution (50 μg / mL) and RNase (200 μg / mL). Finally, cells were analyzed by flow cytometry (BD FACSCanto II, BD Biosciences) using 10,000 gated events per sample.

[0388] Although cell death takes many forms and occurs through various pathways, two distinct processes—apoptosis (also known as programmed cell death) or necrosis (uncontrolled cell death)—remain the dominant ones. Apoptosis is characterized by several characteristic morphological changes in cell structure, accompanied by several enzyme-dependent biochemical processes. The result is the clearance of cells 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 controlling the cell cycle and cell proliferation, which can lead to their uncontrolled growth and proliferation. Dysregulation of the apoptotic process may promote cancer development and progression; however, inducing apoptosis in cancer cells can also be an effective cancer treatment strategy.

[0389] The apoptosis-inducing potential of ASMOAPT20TTMG was evaluated using a dual labeling method employing Annexin V and propidium iodide. Annexin V is a peptide with a high affinity for phosphatidylserine, a phospholipid asymmetrically distributed in the inner lobe of the lipid bilayer of the plasma membrane under physiological conditions. During apoptosis-induced cell death, this asymmetrical phospholipid distribution disappears, and phosphatidylserine is translocated to the outer lobe of the plasma membrane via an exogenous process. Exposure to phosphatidylserine was measured using Annexin V conjugated with a fluorescent dye. On the other hand, propidium iodide is a DNA intercalator that can only penetrate the cell interior under conditions of impaired selective permeability of the plasma membrane (which apparently occurs in several stages of cell death).

[0390] The pro-apoptotic potential of APT20TTMG in the MCF-7 cell line was evaluated using the Annexin V / propidium iodide apoptosis assay kit (eBioScience). First, cells were loaded at 1 x 10⁻⁶ cells per cell line. 5 Cells were cultured at a density of 10 cells / well in 24-well plates for 24 hours to allow for adhesion. Cells were then treated with three concentrations of APT20TTMG (0.25, 0.5, and 1 μM) for 24 hours, followed by collection with 0.05% trypsin solution. After treatment, cells were washed once with 2 mL of ice-cold PBS and then again with the binding buffer provided in the kit. Samples were labeled with Annexin V conjugated with FITC for 15 minutes and washed again with binding buffer. Finally, samples were labeled with propidium iodide and analyzed by collecting 10,000 gated events / samples in a flow cytometer (BD FACSCanto II, BD Biosciences).

[0391] Mitochondria are essential for cell survival, and in cancer, they can contribute to the uncontrolled growth and proliferation of these cells. Mitochondria in cancer cells differ in function and structure from those in normal cells. One key difference is that the membrane potential in cancer cells is altered compared to healthy cells, and they generate energy for the cell in different ways. The mitochondrial membrane potential represents the difference in charge across the inner mitochondrial membrane and is crucial for ATP synthesis via oxidative phosphorylation. The elimination of the mitochondrial membrane potential is an early event during apoptosis, a common type of cell death in tumor cells in response to treatment.

[0392] The effect of ASMO1 APT20TTMG on mitochondrial membrane potential in the MCF-7 cell line was assessed using MitoTracker Red dye. MitoTracker Red is a cationic fluorophore that accumulates in the mitochondrial matrix, indicating the viability and integrity of this organelle.

[0393] Therefore, cells were divided into 1x10 5 Cells were cultured overnight in 24-well plates at a density of 10 cells / well to encourage cell adhesion. Cells were then treated with three concentrations of APT20TTMG (0.25, 0.5, and 1 μM) for 24 hours. After ASMO1 treatment, cells were collected using 0.05% trypsin solution and washed twice with 2.0 mL PBS buffer. Finally, the sample was incubated for 15 minutes with 500 μL of 50 nM MitoTracker Red fluorescent dye solution. Cells were then washed twice more with 2 mL PBS and analyzed by collecting 10,000 gated events / samples using a flow cytometer (BD FACSCantoII, BD Biosciences).

[0394] Numerical values ​​are expressed as mean ± standard error of mean. One-way ANOVA was performed to assess statistical differences between groups, followed by Dunnett's multiple comparison test. A p-value less than 0.05 was considered statistically significant. Statistical analysis was performed using GraphPad Prism 9 software (GraphPad Software Inc., San Diego, CA, USA).

[0395] like Figure 16 As shown, the internalization value of ASMO APT20TTMG was quite high, reaching 75.3 ± 1.4% within the first half hour after incubation with the MCF-7 cell line. After 1 hour of incubation, the degree of internalization remained unchanged during the subsequent evaluation period, with an approximate value of about 80%.

[0396] like Figure 17 As shown, after 48 hours of incubation, cell viability decreased at concentrations of 0.5 and 1 μM, at 86.4 ± 1.6% (p = 0.0235) and 85 ± 2.7% (p = 0.0120), respectively. New tests were performed at concentrations of 0.25, 0.5, and 1 μM after determining the concentration range potentially possessing antitumor activity. This cell viability assessment may represent mitochondrial activity, but also indirectly represents cell proliferation. At the two highest concentrations tested, cell numbers were likely reduced.

[0397] like Figure 18As shown, the only statistically significant change was observed in the S phase, the stage where DNA synthesis occurs, where cell concentrations were lower after incubation with 0.25 and 0.5 μM, indicating that they were not retained in this phase.

[0398] like Figure 19 As shown, no assessment parameters changed; that is, all treatments showed similar profiles to the negative control (untreated cells) in terms of the proportion of live cells, recent / late apoptosis, or necrosis.

[0399] like Figure 20 As shown, there was no change in mitochondrial membrane potential, meaning that all treatments showed a similar profile to the negative control (untreated cells).

[0400] After the study, it was found that ASMO1 was effectively internalized by the breast cancer cell line MCF-7. Furthermore, higher concentrations showed reduced cell viability, which may be related to decreased cell proliferation. Results showed no changes in mitochondrial membrane potential or apoptosis, but APT20TTMG appeared to reduce the number of cells in the S phase of the cell cycle.

[0401] Example 4. Evaluation of the antitumor potential of ASMO1 APT20TTMG in neuroblastoma cell lines (SH-SY5Y and SK-N-SH)

[0402] Under physiological conditions, TAU is a phosphoprotein that promotes the assembly and stabilization of intracellular microtubules. TAU also plays important roles in chromatin structure, signal transduction, and nucleic acid protection. In fact, this protein is described as having a broad interactome, including interactions with cancer-associated kinases, PI3K / AKT (associated with cell survival and proliferation in various cancers), and Rho-ROCK signaling (involved in cell migration and invasion phenotypes). Therefore, although TAU is well-known for its role in neurodegenerative diseases, recent studies suggest it may also be involved in the progression of various cancers as well as cell migration and invasion. Furthermore, as a microtubule-binding protein, TAU may interfere with the binding of taxanes (microtubule-stabilizing drugs) to tubulin, thus implicating resistance to taxanes used in cancer treatment. In this way, anti-TAU molecules could also be a strategy to improve the efficacy of taxane-based chemotherapy. Therefore, regulating TAU expression at both the mRNA and protein levels could be used in cancer chemotherapy.

[0403] Cells were washed once with ice-cold PBS, collected, and precipitated by centrifugation at 2500g for 5 minutes at 4°C. Each sample was resuspended and lysed for 20 minutes at 4°C in 50 μl of ice-cold radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific, USA), which contains an EDTA-free mixture of Halt protease and phosphatase inhibitors (Thermo Fisher Scientific, USA). To improve protein yield, each sample was homogenized using a 29G x 13mm needle and syringe. Cell lysates were clarified by centrifugation at 17,000g for 15 minutes at 4°C. The supernatant was collected, and total protein concentration was determined using the BCA Protein Assay Kit (Thermo Fisher Scientific, USA) according to the manufacturer's instructions. 15 μg of total protein from cell lysates was denatured in NuPAGELDS sample buffer (Thermo Fisher Scientific, USA) and NuPAGE sample reducing agent (DTT; Thermo Fisher Scientific, USA), and boiled at 90 °C for 5 min. The samples were separated using an XCell SureLock Mini-cell system (Thermo Fisher Scientific, USA) on a NuPAGE 4–12% Bis-Tris gel (Thermo Fisher Scientific, USA) and electrophoresed at 120 V in NuPAGE MESSDS running buffer (Thermo Fisher Scientific, USA). The dissociated proteins were transferred to a 0.45 μm polyvinylidene fluoride (PVDF) membrane (Thermo Fisher Scientific, USA) in NuPAGE transfer buffer containing 20% ​​methanol (Thermo Fisher Scientific, USA) at 120 V for 75 min at 4 °C. The PVDF membrane was blocked in TBS-T containing 5% (w / v) dry milk powder at room temperature for 1 hour, and then incubated overnight at 4°C with total TAU primary antibody (Cell Signaling Technology, catalog number 46687, or Abcam, catalog number ab80579), diluted in TBS-T containing 2% (w / v) BSA. The membrane was then washed in TBS-T and incubated for 1 hour at room temperature with HRP-conjugated secondary antibody at a 1:5000 dilution in TBS-T.The membrane was washed once more in TBS-T, and proteins were visualized using an enhanced chemiluminescence (ECL) reagent (Thermo Fisher Scientific, USA), with images captured using the Jess Simple Western Imaging Kit. Density determination was performed using Image J (v1.48k; NIH), and all quantifications were normalized to focal adhesion protein levels.

[0404] At all time points, cells were washed once with PBS, and RNA was extracted using the RNeasy Plus kit (Qiagen). Following the manufacturer's instructions, RNA (0.1–1 μg) was converted to cDNA using the SuperScript IV first-strand synthesis system (Thermo Fisher). After RNA removal, 5–10 ng of cDNA was amplified by qPCR. The following TaqMan probes (Thermo Fisher) were used: total TAU (MAPT, assay ID: Hs00902193_m1) and GAPDH (GAPDH, assay ID: Hs99999905_m1). Applied Biosystems QuantStudio was used. TM Gene expression was analyzed using a 12K Flex real-time PCR system. Gene expression was quantified using the comparable threshold cycle (2^-ΔΔCT) Livak method relative to cells treated with Lipofectamine 3000 alone. Gene expression was normalized relative to the reference gene GAPDH.

[0405] Numerical values ​​are expressed as mean ± standard error of mean. One-way ANOVA was performed to assess statistical differences between groups, followed by Dunnett's multiple comparison test. Western blotting and qPCR analyses of the final experiments were performed using two-tailed t-tests, and a p-value <0.05 was considered statistically significant compared to MOCK (cells treated only with control Lipofectamine 3000). Graphs were generated using GraphPad Prism 9 software (GraphPad Software Inc., San Diego, CA, USA).

[0406] like Figure 21 As shown, ASMO1 APT20TTMG was internalized, performing at approximately 50% across all evaluation periods. Figure 22 As shown in AB, cell viability remained unchanged at any assessed concentration and time. Figure 23 As shown, no changes were observed in cell cycle stages at any assessed concentration. Figure 24As shown, after incubation with ASMO1 APT20TTMG (0.25 μM) for 24 hours, the number of necrotic cells increased. Figure 25 As shown, there was no change in mitochondrial membrane potential, meaning that all treatments exhibited a similar profile to the negative control (untreated cells). Figure 26 As shown, after incorporating 0.5 μM APT20TTMG ASMO1, the number of viable cells remained almost unchanged over the four analytical time intervals, indicating a cellular inhibitory effect. Figure 27 As shown in AB, apart from the transient increase observed at 24 hours, the regulatory profile of MAPT expression at the two longest timeframes decreased by 82 ± 1.2% and 74 ± 4.3% at 96 and 144 hours, respectively. A similar profile was observed for the two highest concentrations in protein quantification, decreasing by 40 ± 13.3% and 46 ± 14.3%, respectively.

[0407] ASMO1 was internalized by the neuroblastoma cell line SH-SY5Y. However, cell viability or proliferation, cell cycle, and mitochondrial membrane potential remained unchanged. These subtle changes in necrotic cells may indicate cytotoxic or cytoseptic potential.

[0408] For the SK-N-SH neuroblastoma cell line, APT20TTMG has the potential to reduce TAU protein levels and MAPT gene expression in a time-dependent manner, with the number of viable cells remaining almost unchanged over four analytical periods (cell inhibition).

[0409] Example 5. Evaluation of the antitumor potential of ASMO APT20TTMG in the glioblastoma cell line (U87-MG)

[0410] The method in this embodiment is the same as that shown in Example 4. Since preliminary studies using five different human tumor lines indicated antitumor potential when incubated with ASMO1, further evaluation was conducted on three of the most promising tumor lines (including glioblastoma).

[0411] The in vitro effects of APT20TTMG on the viability and proliferation of the human cancer cell line U87MG (human glioblastoma) were also evaluated using MTT and BrdU assays. Cells were treated with different concentrations of APT20TTMG and a reference, with the technique repeated three times (the experiment was conducted in three different groups), for 48 hours, 96 hours, and 144 hours. APT20TTMG and the reference were read every 48 hours.

[0412] Numerical values ​​are expressed as mean ± standard error of mean. One-way ANOVA was performed to assess statistical differences between groups, followed by Dunnett's multiple comparison test. Graphs were generated using GraphPad Prism 9 software (GraphPad Software Inc., San Diego, CA, USA).

[0413] like Figure 28 As shown, ASMO1 internalization increased over the longest evaluation time. The obtained values ​​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 a 1 μM fluorescent dye conjugate compound FAM, and intracellular fluorescence was quantified at 1, 2, 4, and 6 hours using flow cytometry by collecting 10,000 gating events. Data from two independent experiments repeated using two techniques are presented.

[0414] like Figure 29 As shown, APT20TTMG at a concentration of 0.25 μM exhibited significant changes in both phases of the cell cycle (G1 / G0 and G2 / M). In the first phase, retention decreased (p = 0.0036), and in the G2 / M phase, retention increased (p = 0.0425), indicating that ASMO1 inhibited the proliferation of cells from this cell line. Cells were seeded and treated with three different concentrations (0.25, 0.5, and 1 μM) of APT20TTMG, and cell cycle phases were assessed after 48 hours of incubation. Cell proliferation was regulated by cell cycle progression. Phases: Sub / G1: non-proliferative state (quiescent); G1 / G0: cell growth; S: DNA replication; G2 / M: DNA separation and mitosis. Results are expressed as mean ± standard error of three independent experiments.

[0415] like Figure 30 As shown, apoptosis assays after incubation with APT20TTMG indicated that both peak concentrations reduced cell viability and clearly marked late apoptosis, while the 0.25 μM concentration just showed a tendency towards this state. Figure 31As shown, in addition to the results obtained in cell cycle and apoptosis, ASMO1 also altered mitochondrial membrane potential and reduced labeling at concentrations of 0.25 and 0.5 μM. Cells were seeded and treated with three different concentrations (0.25, 0.5, and 1 μM) of APT20TTMG, and cell cycle stages were assessed after 48 hours of incubation. Cell proliferation was regulated by cell cycle progression. Stages: Sub / G1: non-proliferative state (quiescent); G1 / G0: cell growth; S: DNA replication; G2 / M: DNA separation and mitosis. Results are presented as mean ± SEM from three independent experiments.

[0416] like Figures 32-33 As shown, MTT was determined using five other different concentrations of APT20TTMG (0.003, 0.03, 0.3, 3, and 30 μM) after incubation for 48, 96, and 144 hours. Figure 32 After 48 hours of incubation, only a 30 μM concentration of APT20TTMG was observed to reduce cell viability, while after 96 and 144 hours of incubation, the three highest concentrations of APT20TTMG (0.3, 3, and 30 μM) also exhibited this ability, suggesting its cytotoxic and / or cell-inhibiting potential. Finally, to confirm whether APT20TTMG's ability to reduce cell viability at specific concentrations is a cytotoxic or cell-inhibiting molecule, cell proliferation assays were performed using the U87-MG cell line under the same conditions as the MTT assay. Figure 33 At the lowest concentrations (0.003 and 0.03 μM) of APT20TTMG, although an increase in cell proliferation was observed during the first 48 hours of incubation at 0.03 μM, a statistically significant decrease in proliferation was observed during the last 144 hours of the assay. This decrease, along with no change in cell viability, suggests that APT20TTMG has a cytotoxic effect at its lowest concentrations (0.003 and 0.03 μM). At the highest concentrations of APT20TTMG (0.3, 3, and 30 μM), a significant decrease in cell proliferation was observed during the 144 hours of incubation, in addition to a transient increase in cell proliferation observed during the first 48 hours of the assay. At the highest concentrations of APT20TTMG (0.3, 3, and 30 μM), a significant decrease in cell proliferation was observed during the 144 hours of incubation, in addition to a transient increase in cell proliferation observed during the first 48 hours of the assay (which may be related to a favorable increase in the number of astrocytes in the astrogenesis activation pathway). This data ( Figure 33 ) and MTT results ( Figure 32The results showed that APT20TTMG was cytotoxic at the highest tested concentration. Cells were seeded and treated with five different concentrations (0.003, 0.03, 0.3, 3, and 30 μM). BrdU was measured after incubation for 48, 96, or 144 hours. Data were plotted as percentages of cell viability (%) compared to the negative control (mediator). Results are expressed as mean ± SEM of three independent experiments, which were repeated in triplicate. 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<0.0001.

[0417] Results obtained from incubation with ASMO1 in the glioblastoma cell line U87-MG strongly suggest its antitumor potential, as it exhibits decreased viable cell and mitochondrial membrane potential, and also increases cell retention in the G2 / M phase, thereby halting cell division. Furthermore, APT20TTMG enhances the labeling of late-apoptotic cells. Overall, the in vitro results from the GBM cell line (U87-MG) demonstrate that APT20TTMG is effectively internalized and exhibits cytotoxic and cell-inhibitory properties depending on the test conditions.

[0418] Example 6. Evaluation of the in vivo antitumor potential of APT20TTMG using a human glioblastoma carcinoma (U-87MG) xenograft model in at...

Claims

1. A method of treating cancer in a subject of need, the method comprising administering to the subject a pharmaceutical composition comprising an engineered polynucleotide, the engineered polynucleotide comprising: (i) one or more targeting portions configured to specifically bind the mRNA precursor at a target sequence in the messenger ribonucleic acid precursor (mRNA precursor); and (ii) The fundraising portion, which is configured as a fundraising splice portion. in, When bound to the mRNA precursor and the engineered polynucleotide, the spliceosome portion alters the mRNA precursor in or near the target sequence.

2. A method for treating cancer in a patient in need, the method comprising: The pharmaceutical composition comprising (a) a taxane drug and (b) an engineered polynucleotide is administered to the subject, the engineered polynucleotide comprising: (i) One or more targeting portions, said one or more targeting portions being configured to specifically bind said mRNA precursor at a target sequence in the messenger ribonucleic acid precursor (mRNA precursor); and (ii) The fundraising portion, which is configured as a fundraising splice portion. When the spliceosome binds to the mRNA precursor and the engineered polynucleotide, the spliceosome portion alters the mRNA precursor in or near the target sequence.

3. The method of claim 2, wherein the method improves the prognosis of the subject compared to a subject who has been given the taxane drug but not the engineered polynucleotide.

4. The method of claim 2, wherein the method reduces the total level of tau in the object, thereby reducing the amount of tau bound to the taxane drug.

5. A method for improving the prognosis of a subject with cancer who has been treated with taxane drugs, wherein the method comprises: The pharmaceutical composition comprising an engineered polynucleotide is administered to the object, the engineered polynucleotide comprising: (i) One or more targeting portions, said one or more targeting portions being configured to specifically bind said mRNA precursor at a target sequence in the messenger ribonucleic acid precursor (mRNA precursor); and (ii) The fundraising portion, which is configured as a fundraising splice portion. When the spliceosome binds to the mRNA precursor and the engineered polynucleotide, the spliceosome portion alters the mRNA precursor in or near the target sequence.

6. The method of claim 5, wherein the method reduces the total level of tau in the object, thereby reducing the amount of tau in the object that is bound to the taxane drug.

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

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

9. The method according to 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 method reduces the tumor volume ratio.

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

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

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

14. The method according to any one of claims 1 to 13, wherein the method reduces the total amount of TAU in the object.

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

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

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

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

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

20. The method according to any one of claims 1 to 16, wherein the engineered polynucleotide is administered via subcutaneous injection, intramuscular injection, intradermal injection, transdermal injection, intranasal administration, intralymphatic injection, intrathecal administration, pulmonary administration, rectal administration, gastric administration, or any other suitable parenteral administration.

21. The method according to any one of claims 1 to 20, wherein the method reduces premature polyadenylation of one or more transcripts of the object.

22. The method according to any one of claims 1 to 21, wherein the method reduces covert splicing of one or more transcripts of the object.

23. The method according to any one of claims 1 to 22, wherein the method improves the score associated with histopathological findings.

24. The method of any one of claims 23, wherein the histopathological findings include tumor grade, lipid content, necrosis, or nucleocytoplasmic ratio (N:C).

25. A method for reducing cell viability, the method comprising: The engineered polynucleotide is administered to cells, the engineered polynucleotide comprising: (i) One or more targeting portions, said one or more targeting portions being configured to specifically bind said mRNA precursor at a target sequence in the messenger ribonucleic acid precursor (mRNA precursor); and (ii) The fundraising portion, which is configured as a fundraising splice portion. When the spliceosome binds to the mRNA precursor and the engineered polynucleotide, the spliceosome portion alters the mRNA precursor in or near the target sequence.

26. A method for reducing cell proliferation rate, the method comprising: The engineered polynucleotide is administered to cells, the engineered polynucleotide comprising: (i) One or more targeting portions, said one or more targeting portions being configured to specifically bind said mRNA precursor at a target that may be a conserved splice site sequence in the messenger ribonucleic acid precursor (mRNA precursor); and (ii) The fundraising portion, which is configured as a fundraising splice portion. When the spliceosome binds to the mRNA precursor and the engineered polynucleotide, the spliceosome portion alters the mRNA precursor in or near the target sequence.

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

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

29. The method according to any one of claims 25 to 28, wherein the cell is a tumor cell.

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

31. A method for altering the distribution of cell cycle stages in multiple cells, the method comprising: The engineered polynucleotide is applied to the plurality of cells, the engineered polynucleotide comprising: (i) One or more targeting portions, said one or more targeting portions being configured to specifically bind said mRNA precursor at a target sequence in the messenger ribonucleic acid precursor (mRNA precursor); and (ii) The fundraising portion, which is configured as a fundraising splice portion. When the spliceosome binds to the mRNA precursor and the engineered polynucleotide, the spliceosome portion alters the mRNA precursor in or near the target sequence.

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

33. The method of claim 31, wherein the method increases the number of cells in the necrosis or apoptosis phase.

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

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

36. A method for reducing tau expression in neurons, the method comprising: The engineered polynucleotide is administered to the neuron, the engineered polynucleotide comprising: (ii) One or more targeting portions, said one or more targeting portions being configured to specifically bind said mRNA precursor at a target sequence in the messenger ribonucleic acid precursor (mRNA precursor); and (ii) The fundraising portion, which is configured as a fundraising splice portion. When the spliceosome binds to the mRNA precursor and the engineered polynucleotide, the spliceosome portion alters the mRNA precursor in or near the target sequence.

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

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

39. The method according to any one of claims 1 to 38, wherein the method regulates the formation of the U1 snRNP complex.

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

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

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

43. The method according to any one of claims 1 to 42, wherein the target portion of the one or more target portions is sufficiently identical or complementary to the common sequence in the target sequence of the target gene.

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

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

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

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

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

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

50. The method of any one of claims 49, wherein the method alters the expression or activity of the target gene.

51. The method according to any one of claims 1 to 52, wherein the one or more targeting portions comprise (1) a first targeting portion configured to specifically bind a first target sequence in the target sequence of the mRNA precursor, and (2) a second targeting portion configured to specifically bind a second target sequence in the target sequence of the mRNA precursor.

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

53. The method according to claim 51 or 52, wherein the second target sequence includes a common sequence in the target sequences.

54. The method according to any one of claims 51 to 53, wherein the first and second target sequences are spacer sequences that are separated by no more than five nucleotides (e.g., one or two nucleotides) in the target sequence.

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

56. The method of claim 55, wherein both the first and second target sequences are located at the 5' or 3' of the exon-intron boundary.

57. The method of claim 55, wherein one of the first and second target sequences is located at 5' of the exon-intron boundary; and wherein the other of the first and second target sequences is located at 3' of the exon-intron boundary.

58. The method according to any one of claims 1 to 57, wherein the target sequence comprises a splicing site in the mRNA precursor.

59. The method of claim 58, wherein the first or second target sequence comprises a splicing site (e.g., 5'ss) in the mRNA precursor.

60. The method according to any one of claims 1 to 59, wherein one of the first and second targeting portions is located at 5' of the recruitment portion, and the other of the first and second targeting portions is located at 3' of the recruitment portion.

61. The method of claim 60, wherein the first targeting portion or the second targeting portion comprises a sequence that is the same as or complementary to the sequence shown in Table 1.

62. The method of claim 60, wherein the first targeting portion comprises a sequence that is identical or complementary to a sequence selected from the exon sequence column of Table 1; and wherein the second targeting portion comprises a sequence that is identical or complementary to a sequence shown in the intron sequence column of Table 1.

63. The method of claim 60, wherein the first targeting portion comprises a sequence that is identical or complementary to the sequence shown in the intron sequence column of Table 1; and wherein the second targeting portion comprises a sequence that is identical or complementary to the sequence shown in the exon sequence column of Table 1.

64. The method of claim 60, wherein the first targeting portion or the second targeting portion comprises a sequence that is identical to or complementary to the common sequence of the intron donor site (e.g., selected from GU, GT, GC, and CA).

65. The method of claim 60, wherein the first targeting portion or the second targeting portion comprises a sequence that is identical to or complementary to a common sequence (e.g., CAG or AGG) of the exon donor site.

66. The method of claim 60, wherein the first targeting portion or the second targeting portion comprises a sequence that is identical or complementary to a common sequence selected from GU, GT, GC, G and CA.

67. The method according to any one of claims 1 to 66, wherein the first targeting portion comprises at least 80%, 90%, or the same sequence as the ribosome binding site of the spliceosome snRNA (e.g., U1 snRNA).

68. The method according to any one of claims 1 to 67, wherein the second targeting portion comprises at least 80%, 90%, or the same sequence as the ribosome binding site of the spliceosome snRNA (e.g., U1 snRNA).

69. The method according to any one of claims 67 or 68, wherein the sequence that is at least 80%, 90%, or identical to the ribosome binding site may be about 2 nucleotides to about 10 nucleotides.

70. The method according to any one of claims 1 to 69, wherein the spliceosome portion is selected from the spliceosome ribonucleoprotein complex, spliceosome small nucleoribonucleic acid (snRNA), spliceosome protein, functional variants thereof, or functional fragments thereof.

71. The method of claim 67, wherein the spliceosome portion comprises U1snRNA and spliceosome protein.

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

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

74. The method according to any one of claims 1 to 73, wherein the recruitment portion comprises at least 70%, 80%, 85%, or 90% identical or complementary nucleotide sequences to any one of SEQ ID NO: 1 or 2.

75. The method of claim 74, wherein the recruitment portion comprises a nucleotide sequence that is identical or complementary to any one of SEQ ID NO:1 or 2.

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

77. The method according to any one of claims 1 to 76, wherein the engineered polynucleotide comprises a top loop, an upper stem, an inner loop, a lower stem, or a combination thereof.

78. The method according to any one of claims 1 to 77, wherein the engineered polynucleotide comprises a loop (e.g., an inner loop) adjacent to a stem (e.g., a lower stem or an upper stem), the stem comprising two complementary stem sequences.

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 five, four or three nucleotides.

80. The method of claim 77 or 78, wherein the loop is an inner loop adjacent to the stem (e.g., the lower stem) and another stem (e.g., the upper stem) that comprises two complementary stem sequences.

81. The method of claim 80, wherein the inner loop comprises a nucleic acid sequence of no more than 10, 9, or 8 nucleotides.

82. The method of claim 80 or 81, wherein the stem sequence of the other stem (e.g., the upper stem) comprises no more than about five, four, or three nucleotides.

83. The method according to any one of claims 80 to 82, wherein the engineered polynucleotide further comprises a top loop.

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

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

86. The method according to any one of claims 1 to 85, wherein the mRNA precursor substantially does not exhibit base pairing with the RNA-binding domain (RBD) of the U1 snRNA when bound to the engineered polynucleotide and the spliceosome portion.

87. The method according to any one of claims 1 to 86, wherein the mRNA precursor substantially does not exhibit base-specific interactions with the U1-C protein when bound to the engineered polynucleotide and the spliceosome portion.

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

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

90. The method of claim 88 or 89, wherein the engineered polynucleotide (e.g., its 5'-targeting portion) is configured to covalently interact with the zinc finger of the U1-C protein (e.g., via disulfide bonds).

91. The method according to any one of claims 88 to 90, wherein the engineered polynucleotide (e.g., its 5'-targeting portion) is configured to interact non-covalently with the zinc finger of the U1-C protein (e.g., via hydrogen bonds).

92. The method according to any one of claims 88 to 91, wherein the aid recruitment portion comprises a nucleotide sequence containing an inter-nucleotide linker of a phosphate thioester that binds to the U1-C zinc finger.

93. The method according to any one of claims 1 to 92, wherein the engineered polynucleotide comprises a nucleotide sequence complementary to a partial sequence of the stem-loop II (SL2) of the U1snRNA.

94. The method of claim 93, wherein one side of the stem-loop structure of the engineered polynucleotide comprises a nucleotide sequence complementary to a partial sequence of the stem-loop II (SL2) of the U1 snRNA.

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

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

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

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

99. The method according to any one of claims 1 to 98, wherein the recruitment portion comprises AGGCC.

100. The method according to any one of claims 1 to 99, wherein the recruitment portion comprises at least 80%, 90%, or the same nucleotide sequence as at least five consecutive nucleotides shown in Tables 2-3.

101. The method according to any one of claims 1 to 100, wherein the recruitment portion comprises at least 80%, 90%, or the same nucleotide sequence as about 5 to about 10 consecutive nucleotides of the sequences shown in Tables 2-3.

102. The method according to any one of claims 1 to 101, wherein the recruiting nucleotide sequence comprises: (i) a nucleotide sequence complementary to at least four nucleotides of the stem-loop II (SL2) of the U1 snRNA.

103. The method according to any one of claims 1 to 102, wherein the engineered polynucleotide substantially does not exhibit base pairing with the anchoring sequence of SL2 of the U1 snRNA.

104. The method of claim 103, wherein the inner loop of the engineered polynucleotide does not substantially exhibit base pairing with the anchoring sequence of the SL2 of the U1 snRNA.

105. The method according to claim 103 or 104, wherein the lower stem of said engineered polynucleotide does not substantially exhibit base pairing with the anchoring sequence of said SL2 of U1 snRNA.

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

107. The method according to any one of claims 1 to 106, wherein the engineered polynucleotide substantially does not exhibit base pairing with the H-helix of U1 snRNA.

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

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

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

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

112. The method according to any one of claims 109 to 111, wherein the 2'-modified nucleotide comprises 2'-methoxy, 2'-methoxymethyl, 2'-methoxyethyl, 2'-fluorine, or 2'-aminoethyl nucleotide.

113. The method according to any one of claims 108 to 112, wherein the engineered polynucleotide comprises nucleotides linked by internucleotide bonds, and at least one of the internucleotide bonds does not contain a phosphate ester.

114. The engineered polynucleotide according to claims 108 to 113, wherein the engineered polynucleotide comprises nucleotides linked by internucleotide bonds, and at least one of the internucleotide bonds comprises sulfur (S); selenium (Se); BR3, wherein each R is independently selected from hydrogen, alkyl, and aryl; carbon Or NR2, where each R is independently selected from hydrogen, alkyl, and aryl.

115. The method according to any one of claims 108 to 114, wherein the engineered polynucleotide comprises at least one phosphate thioester nucleotide bond.

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

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

118. The method according to any one of claims 108 to 117, wherein the internucleotide linking comprises methylphosphonate, hydroxyamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thiomethyl acetal, methyl acetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazine, methylenedimethylhydrazine, or methyleneoxymethylimino.

119. The method according to any one of claims 1 to 118, wherein 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.

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

121. The method according to any one of claims 1 to 120, wherein each of the one or more targeting portions independently comprises about 2 to about 15 nucleotides, about 2 nucleotides to about 10 nucleotides, or about 2 nucleotides to about 8 nucleotides.

122. The method according to any one of claims 1 to 121, wherein one of the first and second targeting portions comprises about 2 nucleotides, and the other of the first and second targeting portions comprises about 5 or 6 nucleotides.

123. The method according to any one of claims 1 to 122, wherein when bound to the engineered polynucleotide and the mRNA precursor, the spliceosome partially cleaves or splices the mRNA precursor in the target sequence.

124. The method according to any one of claims 1 to 123, wherein the spliceosome portion further facilitates the modification of the cleaved mRNA precursor.

125. The method according to any one of claims 1 to 124, wherein the engineered polynucleotide comprises at least 70%, 80%, 85%, or 90% identical or complementary nucleotide sequences to any one of SEQ ID NO: 1-4.

126. The method according to any one of claims 1 to 125, wherein the engineered polynucleotide comprises a nucleotide sequence that is identical or complementary to any one of SEQ ID NO: 3 or 4.

127. The method according to any one of claims 1 to 126, wherein the engineered polynucleotide comprises: (i) A first targeting portion, configured to specifically bind to the mRNA precursor at a first target sequence in the messenger ribonucleic acid precursor (mRNA precursor), wherein the first targeting portion comprises a sequence identical to or complementary to 5'-GTCCA-3'. (ii) A recruitment portion comprising a sequence at least 90% similar to or complementary to SEQ ID NO:1, and configured to recruit a spliceosome portion comprising U1 snRNA and U1-C protein, wherein the recruitment portion comprises a top loop, an upper stem adjacent to the top loop, a lower stem, and an inner loop located between the upper stem and the lower stem, and (iii) A second targeting portion configured to specifically bind to the mRNA precursor at a second target sequence in the mRNA precursor, wherein the second targeting portion contains a sequence that is identical to or complementary to 5'-CG-3'.

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

3.

129. The method of claim 128, wherein all internucleotide bonds of the engineered polynucleotide comprise phosphate thioester bonds.

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

131. The method according to any one of claims 128 to 130, wherein the method further comprises administering a taxane drug.

132. A method of treating a subject suffering from cancer, wherein the subject has been administered a taxane drug, the method comprising administering a pharmaceutical composition to the subject, the pharmaceutical composition comprising (i) an engineered polynucleotide having a nucleotide sequence of SEQ ID NO:3, wherein the internucleotide bonds of the engineered polynucleotide comprise phosphate thioester bonds, and wherein the nucleotides at positions 1-5 and 20-24 of the engineered polynucleotide comprise a 2'-O-methyl moiety, thereby reducing TAU protein levels in the subject and TAU binding to the taxane drug.

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