Polynucleotide compositions and methods for treating neurodegenerative diseases

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 having a recruitment spliceosome and a targeting sequence complementary to the target sequence at the exon-intron splice point, and may include nucleotides having a 2'modification and a thiophosphate linkage The engineered polynucleotides may be administered to a subject to treat a neurodegenerative disease.

Description

[0001] Cross-referencing

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 480,464, filed January 18, 2023, the entire contents of which are incorporated herein by reference. Background Technology

[0003] Aberrant splicing and premature polyadenylation are associated with many disease states. Proper splicing of precursor messenger RNA (mRNA) and inhibition of premature polyadenylation are crucial for accurate protein translation. Splicing efficiency and inhibition of premature polyadenylation must be modulated to ensure proper splicing of mRNA precursors, thereby avoiding misfolded and potentially pathogenic proteins. Gene regulation at the ribonucleic acid (RNA) level remains limited in its efficiency. 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 neurodegenerative diseases in a subject of need, the method comprising administering to the subject a pharmaceutical composition comprising an engineered polynucleotide comprising: (1) 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 (2) a recruitment portion configured to recruit a spliceosome portion wherein, when bound to 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 a neurodegenerative disease in a subject of need, the method comprising administering to the subject a pharmaceutical composition comprising an engineered polynucleotide, the 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 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 neurodegenerative disease is selected from: Alzheimer's disease, Pick's disease, chronic traumatic encephalopathy, progressive supranuclear palsy, aerobatic granuloma, amyotrophic lateral sclerosis (ALS), and frontotemporal dementia. In some embodiments, the neurodegenerative disease is a neurodegenerative disease associated with the presence of Tau in the subject's brain. In some embodiments, Tau expression is altered. In some embodiments, Tau expression is reduced. In some embodiments, the total amount of Tau in the subject's brain is reduced. In some embodiments, the neurodegenerative disease is a neurodegenerative disease associated with the presence of amyloid β in the subject's brain. In some embodiments, the method alters the expression of amyloid β. In some embodiments, the method reduces the expression of amyloid β. In some embodiments, the method reduces the total amount of amyloid β in the subject's brain. In some embodiments, the method reduces the total amount of amyloid plaques in the subject's brain. In some embodiments, the engineered polynucleotide is administered at a concentration of at least 5 nM. In some embodiments, the engineered polynucleotide is administered at a concentration of at least 10 nM. In some embodiments, the engineered polynucleotide is administered at a concentration of about 5 nM to about 20 nM.

[0006] 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: 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 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. In some embodiments, the neuron is derived from an individual suffering from a neurodegenerative disease. In some embodiments, the neurodegenerative disease is Alzheimer's disease. In some embodiments, the neuron is a healthy neuron. In some embodiments, the neuron is a healthy neuron. In some embodiments, the neuron is derived from a healthy subject. In some embodiments, the engineered polynucleotide is administered at a concentration of at least 5 nM. In some embodiments, the engineered polynucleotide is administered at a concentration of at least 10 nM. In some implementations, the engineered polynucleotide is applied at a concentration of about 5 nM to about 20 nM.

[0007] In some aspects, this document describes a method for increasing fiber width in neurons, the method comprising administering an engineered polynucleotide to the neuron, the engineered polynucleotide comprising: 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 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. In some embodiments, the neuron is derived from an individual suffering from a neurodegenerative disease. In some embodiments, the neurodegenerative disease is Alzheimer's disease. In some embodiments, the neuron is a healthy neuron. In some embodiments, the neuron is a healthy neuron. In some embodiments, the neuron is derived from a healthy subject. In some embodiments, the engineered polynucleotide is administered at a concentration of at least 5 nM. In some embodiments, the engineered polynucleotide is administered at a concentration of at least 10 nM. In some implementations, the engineered polynucleotide is applied at a concentration of about 5 nM to about 20 nM.

[0008] In some aspects, this document describes a method for increasing the number of branches in a neuron, the method comprising administering an engineered polynucleotide to the neuron, the 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 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. In some embodiments, the neuron is derived from an individual suffering from a neurodegenerative disease. In some embodiments, the neurodegenerative disease is Alzheimer's disease. In some embodiments, the neuron is a healthy neuron. In some embodiments, the neuron is a healthy neuron. In some embodiments, the neuron is derived from a healthy subject.

[0009] In some respects, this document describes a method for increasing neuronal electrical activity, the method comprising administering an engineered polynucleotide to the neuron, the 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 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.

[0010] In some embodiments, the neurons are derived from an individual suffering from a neurodegenerative disease. In some embodiments, the neurodegenerative disease is selected from: Alzheimer's disease, Pick's disease, chronic traumatic encephalopathy, progressive supranuclear palsy, auricularylidosis, amyotrophic lateral sclerosis (ALS), and frontotemporal dementia. In some embodiments, the neurons are healthy neurons. In some embodiments, the neurons are derived from a healthy subject.

[0011] In some embodiments, the method alters gene expression in the neurons. In some embodiments, the method alters gene expression as part of the MAPK signaling pathway. In some embodiments, the method alters gene expression as part of the cholesterol metabolism pathway. In some embodiments, the method alters gene expression as part of the steroid biosynthesis pathway. In some embodiments, the method alters gene expression as part of the PI3K-Akt signaling pathway. In some embodiments, the method alters gene expression as part of cell cycle regulation or cellular senescence pathways. In some embodiments, the method alters gene expression as part of the pyruvate metabolism pathway. In some embodiments, the method alters gene expression as part of the Ras signaling pathway. In some embodiments, the method alters gene expression as part of the AMPK signaling pathway. In some embodiments, the method alters gene expression as part of the fatty acid metabolism pathway. In some embodiments, the method alters gene expression as part of the fatty acid synthesis pathway. In some embodiments, the method alters gene expression as part of the PPAR signaling pathway. In some embodiments, the method alters gene expression associated with mRNA transport. In some embodiments, the method increases mRNA transport in the neuron. In some embodiments, the method reduces excitotoxicity of the neuron. In some embodiments, the method increases synaptic integrity of the neuron. In some embodiments, the method reduces premature polyadenylation of one or more transcripts of the neuron.

[0012] In some aspects, this document describes a method for treating a subject with Alzheimer's disease, the method comprising administering to the subject a pharmaceutical composition comprising an engineered polynucleotide having the nucleotide sequence of SEQ ID NO:3, thereby modulating an mRNA precursor affected by U1 snRNP dysfunction in the subject's brain. In some embodiments, one or more (e.g., all) of the engineered polynucleotides comprise phosphate thioester bonds. In some embodiments, positions 1-5 and 20-24 of the engineered polynucleotide comprise a 2'-O-methyl moiety. In some embodiments, the mRNA precursor comprises tau, and the method reduces the level of tau protein in the brain.

[0013] In some respects, this document describes a method for reducing amyloid-β expression and / or aggregation 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 that may be a conserved splice site in a messenger ribonucleic acid precursor (mRNA precursor); and (ii) a recruitment portion configured to recruit a spliceosome portion, wherein, when bound to the mRNA precursor and the engineered polynucleotide, the spliceosome portion alters the mRNA precursor in or near the target sequence.

[0014] In some respects, this paper describes a method for reducing U1-70K aggregation and / or mislocalization 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, when bound to the mRNA precursor and the engineered polynucleotide, the spliceosome portion alters the mRNA precursor in or near the target sequence.

[0015] In some respects, this document describes a method for regulating astrocyte proliferation, 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.

[0016] In some embodiments, the engineered polynucleotide is administered intratumorally. In some embodiments, the engineered polynucleotide is administered intravenously. In some embodiments, the engineered polynucleotide is administered intrathecally. In some embodiments, the engineered polynucleotide is administered via subcutaneous injection, intramuscular injection, intradermal injection, percutaneous administration, intranasal administration, intralymphatic injection, intrathecal administration, pulmonary administration, rectal administration, gastric administration, or any other suitable parenteral administration.

[0017] 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.

[0018] In some respects, this document describes an engineered polynucleotide comprising: one or more targeting portions configured to specifically bind to a target sequence in a messenger ribonucleic acid precursor (mRNA precursor); and a recruitment portion configured to recruit a posttranscriptional regulatory portion (e.g., a spliceosome portion), wherein, upon binding to the mRNA precursor and the engineered polynucleotide, the posttranscriptional regulatory portion alters the mRNA precursor in or near the target sequence.

[0019] In some embodiments, the target portion of the one or more target portions is sufficiently identical or complementary to a common sequence in the target sequence of the target gene. In some embodiments, the target portion is complementary to and / or hybridizes with the target sequence. In some embodiments, the target portion is complementary to and / or hybridizes with a common sequence in the target sequence. In some embodiments, the target sequence contains a splicing site. In some embodiments, the splicing site is a conserved splicing site. In some embodiments, the splicing 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.

[0020] 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 first 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 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.

[0021] 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 moiety comprises AGGCC. In some embodiments, the recruitment portion comprises at least 80%, 90%, or identical nucleotide sequences of at least five consecutive nucleotides to the sequences shown in Tables 2-3. In some embodiments, the recruitment portion comprises at least 80%, 90%, or identical nucleotide sequences of about 5 to about 10 consecutive nucleotides to the sequences shown in Tables 2-3. In some embodiments, the recruitment portion comprises nucleotide sequences that are identical to or complementary to the sequences shown in Tables 2-3. In some embodiments, the engineered polynucleotide includes (e.g., secondary) structural features. In some embodiments, the engineered polynucleotide includes 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., a lower stem or an 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 portion of the stem-loop II (SL2) sequence of the U1 snRNA. In some embodiments, one side of the stem-loop structure of the engineered polynucleotide includes a nucleotide sequence complementary to a partial sequence of the stem-loop II (SL2) of the U1 snRNA. In some embodiments, the partial sequence includes a sequence corresponding to 5'-GGCCU-3' of the U1 snRNA's SL2. In some embodiments, the partial sequence does not include a sequence corresponding to 5'-CACGUUA-3' of the U1 snRNA's SL2. In some embodiments, the engineered polynucleotide substantially does not exhibit base pairing with the anchoring sequence of the U1 snRNA's SL2. In some embodiments, the inner loop of the engineered polynucleotide substantially does not exhibit base pairing with the anchoring sequence of the U1 snRNA's SL2.In some embodiments, the lower stem of the engineered polynucleotide does not substantially exhibit base pairing with the anchoring sequence of U1 snRNA at SL2. 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 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.

[0022] 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 internucleotide bonds of the engineered polynucleotide are chemically modified. In some embodiments, at least about 50%, 60%, 70%, 80%, or 90% of the internucleotide bonds are thiophosphates. In some embodiments, the 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 comprises about 10 to about 40 nucleotides, about 10 to about 35 nucleotides, about 10 to about 30 nucleotides, or about 10 to about 25 nucleotides. In some embodiments, the recruitment moiety comprises about 10 to about 30 nucleotides, or about 10 to about 20 nucleotides. In some embodiments, 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.

[0023] 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) comprises 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 that are 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 comprises 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 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.

[0024] 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 post-transcriptional regulatory moieties (e.g., spliceosome moieties) in 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 target moieties in the one or more target moieties are sufficiently identical or complementary to a common sequence in the target sequence of a target gene. In some embodiments, the mRNA precursor corresponds to a target gene. In some embodiments, the target gene comprises microtubule-associated protein tau (MAPT). In some embodiments, the method alters the expression or activity of the target gene. In some embodiments, prior to contact, the cell exhibits an aberrant messenger ribonucleic acid (mRNA) or protein corresponding to the target gene.

[0025] 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'.

[0026] In some respects, this document describes a set of engineered polynucleotides, each of which independently comprises: 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 comprising the target sequence.

[0027] Another aspect 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. Furthermore, when bound to the mRNA precursor and the engineered polynucleotide, the spliceosome portion 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 identical nucleotide sequences of at least five consecutive nucleotides as shown in Tables 2-3. In some embodiments, the recruitment moiety comprises at least 80%, 90%, or identical nucleotide sequences of about 5 to about 10 consecutive nucleotides as shown in Tables 2-3. In some aspects, the spliceosome moiety comprises U1 snRNA and U1-C protein.In some aspects, the upper or lower stem comprises two complementary sequences, each containing no more than 5 nucleotides; the inner loop comprises two nucleic acid sequences, each containing no more than 5 nucleotides; and the apical loop comprises a nucleic acid sequence of no more than 8 nucleotides. In other aspects, when bound to the engineered polynucleotide, the mRNA precursor substantially does not exhibit base pairing with the RNA-binding domain (RBD) of the U1 snRNA, and when bound to the engineered polynucleotide and the spliceosome, the spliceosome substantially does not exhibit base-specific interactions with the U1-C protein. 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 4 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

[0028] 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.

[0029] 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 within introns is clearly insufficient to distinguish numerous alternative splicing events, it is surprising how little other sequence information is known about what regulates alternative RNA splicing. 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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 U1-70K 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] Figure 11 A shows the mitochondrial activity of induced pluripotent stem cell (iPSC)-derived neurons from healthy patients (HDC) after incubation with ASMO1. Figure 11 B shows the mitochondrial activity of induced pluripotent stem cell (iPSC)-derived neurons from a patient diagnosed with Alzheimer's disease (AD) (ADC) after incubation with ASMO1.

[0040] Figure 12 Mitochondrial activity of neurons from healthy donors (HDCs) after incubation with ASMO1 is shown.

[0041] Figure 13 A shows the mitochondrial activity of iPSC-derived microglia from healthy patients (HDC) after incubation with ASMO1. Figure 13 B shows the mitochondrial activity of iPSC-derived microglia from a patient diagnosed with AD (ADC) after incubation with ASMO1.

[0042] Figure 14 The study showed mitochondrial activity in astrocytes after incubation with ASMO1.

[0043] Figure 15 A shows the extracellular glutamate levels of iPSC-derived neurons in healthy patients (HDC) after incubation with ASMO1. Figure 15 B shows the extracellular glutamate levels of iPSC-derived neurons in a patient diagnosed with AD (ADC) after incubation with ASMO1.

[0044] Figure 16 The image shows data from NeuroHTS. TM The technology's culture plate contains a schematic diagram of the pores of neurons derived from iPSCs, along with parameters that can be assessed using the system in three different regions of the neuron. In the upper region of the pore, the number of cells in the cell body and dendrites and nuclear aggregates are assessed; in the middle region of the channel (axon), the thickness of the axonal fiber is verified; and in the lower region where the axon and dendrites are located, the following four parameters are assessed: the number of branches, the number of branch junctions, the straightness of the neurite, and the axonal material.

[0045] Figure 17 A shows the number of iPSC-derived neurons in cultured healthy patients (HDC) after incubation with ASMO1. Figure 17 B shows the number of iPSC-derived neurons in cultured patients diagnosed with AD (ADC) after incubation with ASMO1.

[0046] Figure 18 A shows the formation of cell aggregates of iPSC-derived neurons in healthy patients (HDC) after incubation with ASMO1. Figure 18 B shows the formation of cell aggregates from neurons derived from patients diagnosed with AD (ADC) after incubation with ASMO1.

[0047] Figure 19A shows the fiber width of iPSC-derived neurons in healthy patients (HDC) after incubation with ASMO1. Figure 19 B shows the fiber width of neurons derived from patients diagnosed with AD (ADC) after incubation with ASMO1.

[0048] Figure 20 A shows the number of branches of iPSC-derived neurons in healthy patients (HDC) after incubation with ASMO1. Figure 20 B shows the number of branches of iPSC-derived neurons in a patient diagnosed with AD (ADC) after incubation with ASMO1. Figure 20 C shows the number of branches per cell in iPSC-derived neurons from healthy patients (HDC) after incubation with ASMO1. Figure 20 D shows the number of branches per cell in iPSC-derived neurons of a patient diagnosed with AD (ADC) after incubation with ASMO1.

[0049] Figure 21 A shows the branching connection points of iPSC-derived neurons in a healthy patient (HDC) after incubation with ASMO1. Figure 21 B shows the branching connection points of iPSC-derived neurons in a patient diagnosed with AD (ADC) after incubation with ASMO1. Figure 21 C shows the branching connection points of each cell in iPSC-derived neurons from healthy patients (HDC) after incubation with ASMO1. Figure 21 D shows the branching connection points of each cell in an iPSC-derived neuron diagnosed with AD (ADC) after incubation with ASMO1.

[0050] Figure 22 A shows the neurite straightness of iPSC-derived neurons in healthy patients (HDC) after incubation with ASMO1. Figure 22 B shows the neurite linearity of iPSC-derived neurons in a patient diagnosed with AD (ADC) after incubation with ASMO1.

[0051] Figure 23 A shows the total axonal material of iPSC-derived neurons in healthy patients (HDC) after incubation with ASMO1. Figure 23 B shows the total axonal material of iPSC-derived neurons in a patient diagnosed with AD (ADC) after incubation with ASMO1. Figure 23 C shows the axonal material of each cell of iPSC-derived neurons from a healthy patient (HDC) after incubation with ASMO1. Figure 23D shows the axonal material of each cell of an iPSC-derived neuron diagnosed with AD (ADC) after incubation with ASMO1.

[0052] Figure 24 Representative images of morphological changes in iPSC-derived neurons from healthy patients (HDC) after incubation in ASMO1 are shown.

[0053] Figure 25 Representative images of morphological changes in iPSC-derived neurons from patients diagnosed with AD (ADC) after incubation in ASMO1 are shown.

[0054] Figure 26 A shows the binding of ASMO1 to U1-70K protein in neuroblastoma cell lines as detected by Western blotting. Figure 26 B shows the binding of ASMO1 to U1-C protein in neuroblastoma cell lines as detected by Western blotting. Figure 26 C shows the binding of ASMO1 to U1-A protein in neuroblastoma cell lines as detected by Western blotting. Figure 26 D shows the binding of ASMO1 to GAPDH protein in neuroblastoma cell lines as detected by Western blotting.

[0055] Figure 27 A-27C shows the binding of ASMO1 to U1 snRNA and mRNA precursors. Figure 27 A U1 snRNA, Figure 27 BTau mRNA precursor, and Figure 27 C GAPDH.

[0056] Figure 28 A shows the difference in tau levels in iPSC-derived neurons of healthy patients (HDC) after incubation in ASMO1 compared with normalized tau levels in negative controls. Figure 28 B shows the difference in tau levels of iPSC-derived neurons in patients diagnosed with AD (ADC) after incubation in ASMO1 compared with normalized tau levels in negative controls.

[0057] Figure 29 The diagram shows a mapping curve (in percentage) of reads from the human reference genome obtained using the STAR RNA-seq alignment tool for RNA-seq differential gene expression analysis.

[0058] Figure 30The percentage of reads mapped to genomic features (such as genes, exons, promoters, gene bodies, genomic bins, and chromosome locations) is shown using featureCounts.

[0059] Figure 31 A shows the principal component analysis (PCA) plot of a single sample. Figure 31 B shows a PCA diagram where gray circles represent the top three clusters identified in the PCA results.

[0060] Figure 32 A shows a volcano plot of differentially expressed (DE) genes (DEG) in ADC cells treated with 18.52 nM ASMO1 compared to untreated ADC neurons. Figure 32 B shows a volcano plot of differentially expressed genes in ADC cells treated with 500 nM ASMO1 compared to untreated ADC neurons.

[0061] Figure 33 A shows a volcano plot of differentially expressed genes in HDC cells treated with 18.52 nM ASMO1 compared to untreated HDC neurons. Figure 33 B shows a volcano plot of differentially expressed genes in HDC cells treated with 500 nM ASMO1 compared to untreated HDC neurons.

[0062] Figure 34 A volcano plot of gene expression in untreated HDC neurons compared to untreated ADC neurons is shown.

[0063] Figure 35 The top-ranked gene ontology biological processes obtained from enrichment analysis of the DE gene (DEG) of ADC neurons treated with 18.52 nM ASMO1 are shown compared to untreated ADC (culture medium only).

[0064] Figure 36 The top-ranked gene ontology biological processes obtained from enrichment analysis of the DE gene (DEG) in HDC neurons treated with 18.52 nM ASMO1 are shown compared to untreated HDC (culture medium only).

[0065] Figure 37 The top-ranked gene ontology biological processes obtained from enrichment analysis of the DE gene (DEG) in HDC neurons treated with 500 nM ASMO1 compared to untreated HDC (culture medium only) are shown.

[0066] Figure 38All entries related to the Gene Ontology Biology Process (GO:BP) are shown, obtained from DEG of ADC neurons treated with 500 nM ASMO1 compared to untreated ADC (culture medium only).

[0067] Figure 39 A shows all entries from the Kyoto Encyclopedia of Genes and Genomes (KEGG), obtained from DEG enrichment analysis of ADC neurons treated with 18.52 nMASMO1 compared to untreated ADC (culture medium only). Figure 39 B shows all entries from the Kyoto Encyclopedia of Genes and Genomes (KEGG), obtained from DEG enrichment analysis of ADC neurons treated with 500 nM ASMO1 compared to untreated ADC (culture medium only).

[0068] Figure 40 A shows all KEGG entries obtained from the DEG enrichment analysis of HDC neurons treated with 18.52 nM ASMO1 compared to untreated HDC (culture medium only). Figure 40 B shows all KEGG entries obtained from the DEG enrichment analysis of HDC neurons treated with 500 nM ASMO1 compared to untreated HDC (culture medium only). Figure 40 C shows all KEGG entries obtained from the enrichment analysis of DEG in untreated neurons of HDC compared to untreated ADC (culture medium only).

[0069] Figure 41 The protein-protein interaction network and clusters of the DEG of the ADC treated with 18.52 nM ASMO1 are shown.

[0070] Figure 42 The 3'UTR / CDS expression ratios of aging-related genes are shown, indicating the potential profile of premature polyadenylation and the size of the resulting expressed transcripts. Aging.

[0071] Figure 43 The DE gene (DEG) in ADC neurons treated with 18.52 nM ASMO1 and its possible major role in the mechanism of action of ASMO1 are shown.

[0072] Figure 44 A-44B shows the spontaneous electrophysiological activity of iPSC-derived neurons of HDC after incubation with APT20TTMG (ASMO1).

[0073] Figure 45 A-45B illustrates the spontaneous electrophysiological activity of iPSC-derived neurons of the ADC after incubation with APT20TTMG (ASMO1).

[0074] Figure 46 A-46D shows the levels of soluble Aβ40 and Aβ42 in the cortex of an aging animal model (SAMP-8) measured by ELISA. The figure depicts the levels of (A) soluble Aβ40, (B) soluble Aβ42, (C) insoluble Aβ40, and (D) insoluble Aβ42 in cortical samples from n = 7 animals in each treatment group. Histologically identified biological anomalies were excluded from the analysis. Data are given as pg / mg protein (Aβ40 or Aβ42).

[0075] Figure 47 A-47D shows the levels of soluble Aβ40 and Aβ42 in the hippocampus of an aging animal model (SAMP-8) measured by ELISA. The figure depicts the levels of (A) soluble Aβ40, (B) soluble Aβ42, (C) insoluble Aβ40, and (D) insoluble Aβ42 in cortical samples from n = 7 animals in each treatment group. Histologically identified biological anomalies were excluded from the analysis. Data are given as pg / mg protein (Aβ40 or Aβ42).

[0076] Figure 48 A-48D shows the quantification of amyloid protein immunofluorescence in the cerebral cortex of an aging animal model (SAMP-8).

[0077] Figure 49 A-49D shows the quantification of amyloid protein immunofluorescence in the hippocampus of an aging animal model (SAMP-8).

[0078] Figure 50 A-50D shows the levels of soluble and insoluble tau and ptau (T231) in the cortex of an aging animal model (SAMP-8) as measured by ELISA.

[0079] Figure 51 A-51D shows the levels of soluble and insoluble tau and ptau (T231) in the hippocampus of an aging animal model (SAMP-8) measured by ELISA.

[0080] Figure 52 A-52D shows the quantification of pSer202 / pThr205-tau immunofluorescence in the cerebral cortex of an aging animal model (SAMP-8).

[0081] Figure 53A-53D shows the quantification of pSer202 / pThr205-tau immunofluorescence in the hippocampus of an aging animal model (SAMP-8).

[0082] Figure 54 A-54D shows the quantification of U1-70K immunofluorescence in the cerebral cortex of an aging animal model (SAMP-8).

[0083] Figure 55 A-55D shows the quantification of U1-70K immunofluorescence in the hippocampus of an aging animal model (SAMP-8).

[0084] Figure 56 A-56C shows the quantification of GFAP in the cerebral cortex of an aging animal model (SAMP-8) by ELISA (58A) and immunofluorescence (58B and 58C).

[0085] Figure 57 A-57C shows the quantification of GFAP in the hippocampus of an aging animal model (SAMP-8) by ELISA (58A) and immunofluorescence (58B and 58C).

[0086] Figure 58 A-58H demonstrates the quantification of pro-inflammatory cytokines in plasma.

[0087] Figure 59 A-59B shows immunofluorescence signals in an aging animal model (SAMP-8).

[0088] Figure 60 The ratio of the 3' untranslated region to the coding sequence (3'UTR / CDS) of TARDBP and STMN2 in HDC and ADC cells is shown.

[0089] 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

[0090] 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.

[0091] This document describes methods for treating neurodegenerative diseases and Alzheimer's disease using engineered polynucleotides described herein. The engineered polynucleotides described herein can effectively regulate the splicing of mRNA precursors by recruiting components of the spliceosome, or effectively inhibit premature polyadenylation by recruiting components of the spliceosome involved in telescripting processes. Improved recruitment of spliceosome components to mRNA precursors, without being constrained by a specific mechanism, can effectively modulate cellular mechanisms leading to neurodegenerative diseases. The engineered polynucleotides can be administered to subjects with neurodegenerative diseases and can treat these diseases, for example, by reducing the number of protein aggregates, fibromas, or plaques in the subject's brain, or by preventing the proliferation of fibromas, protein aggregates, plaques, or other structures associated with neurodegenerative diseases.

[0092] Neurodegenerative diseases are illnesses that affect the brain and nervous system, typically leading to the gradual degeneration of neurons and the cells that support them. This gradual degeneration of nervous system cells is often exacerbated by the aging process and can impair an individual's ability to interact with their environment and live independently. Alzheimer's disease (AD) is a neurodegenerative condition characterized by memory loss and a progressive decline in daily activities. Millions of people worldwide are affected by various manifestations of neurodegenerative diseases. Although numerous environmental and genetic causes of neurodegenerative diseases have been identified, there is currently no cure for these conditions.

[0093] Alzheimer's disease (AD) is one of many diseases classified as tau protein disorders. AD is characterized by the presence of amyloid-β plaques and hyperphosphorylated Tau accumulations in neurofibrillary tangles, neurofibrillary networks, and neuroinflammatory plaques within the brain. Progressive white matter loss has been observed in areas exhibiting tau pathology. Furthermore, AD is also characterized by U1 small nucleoribonucleoprotein (snRNP) nuclear depletion, accumulation, and aggregation in the cytoplasm, as well as splicing disorders. In addition, the basic-acid dipeptide domain of U1-70K has been shown to interact with Tau in the AD brain, and both U1-70K and Tau are co-localized in neurofibrillary tangles in late-onset sporadic and familial AD cases.

[0094] In a meta-analysis of data from human post-mortem brain tissue and the Drosophila melanogaster model, AD Tau neurofibrillary tangles pathology has been shown to disrupt spliceosome activity, leading to transcriptomic dysfunction and ultimately CNS dysfunction and neurodegeneration. Tau may be involved in spliceosome cytoplasmic dissociation and disrupt snRNP assembly and / or stability. For example, some spliceosome components (including U1-70K) can physically bind to Tau in the human brain with AD pathology; and in Drosophila, genetic manipulation of these factors can enhance the neurotoxicity of Tau. Furthermore, increased occult splicing load has been observed in the post-mortem brain of humans with Tau pathology.

[0095] Furthermore, the molecular events involved in the transition from the initial stages of Alzheimer's disease (associated with Aβ accumulation) to mild cognitive impairment (MCI) and symptomatic Alzheimer's disease (primarily associated with Tau aggregation) remain unclear (Hales et al., 2016a). U1 small nucleoribonucleoprotein particles (snRNPs), particularly U1-70K and U1-A proteins, are enriched in the insoluble proteome of brain tissue from asymptomatic Alzheimer's disease, MCI, and post-mortem Alzheimer's disease. Moreover, insoluble U1-A and U1-70K are strongly correlated with the quantification of insoluble Aβ and Tau (Hales et al., 2016b). This evidence not only suggests that U1 snRNP aggregation may be involved in the mechanism linking plaque development to non-fine muscle factor (NFT) formation and thus in the pathogenesis of Alzheimer's disease, but also proposes that dysfunction of snRNPs (marked by aggregation, hyperactivation, and mislocalization) may represent upstream events that could significantly contribute to the early development of Alzheimer's disease.

[0096] The U1 snRNP complex is one of the five complexes (named U1, U2, U4, U5, and U6) that make up the human spliceosome. It is responsible for processing precursor messenger RNA (pre-mRNA), thereby removing intron regions and generating mature mRNA within the nucleus. The U1 snRNP complex plays a role in the initial steps of spliceosome assembly by specifically recognizing precursor mRNA splicing sites. Furthermore, U1 snRNP plays a crucial overall function in the 3' end processing of precursor mRNA by actively inhibiting premature 3' cleavage and polyadenylation, particularly in GU-rich regions of the intron. Due to this important physiological role, U1 snRNP dysfunction in Alzheimer's disease (AD) can lead to RNA splicing defects, as well as inhibition of premature cleavage and polyadenylation of RNA transcripts and inhibition of the autophagy-lysosomal system. In addition, mislocalization and depletion of U1 from the nucleus and translocation to the cytoplasm can lead to neuronal cell cycle reentry (CCR), causing mitotic catastrophe and potentially neuronal death. Furthermore, as previously described, U1 snRNP aggregation may be involved in mechanisms linking plaque development with neurofibrillary tangles (NFT) formation, and thus in the pathogenesis of Alzheimer's disease (AD). The disclosed engineered polynucleotides (e.g., ASMO1) can generally target mRNA precursors, such as Tau mRNA precursors, recruit the U1 complex, and help stabilize initial spliceosome assembly at splice sites, thereby improving the accuracy of splice site selection. Additionally, they can interact with key domains of the U1 complex, such as the U1-C zinc finger, which has been proposed to be associated with (if not essential) the binding of U1-C snRNPs to the U1 complex. These features allow engineered polynucleotides (e.g., ASMO1) to promote proper 5' end recognition and initiation of the splicing process (even under pathological conditions), and also prevent U1 nuclear depletion and cytoplasmic aggregation associated with reduced 3' processing / premature polyadenylation (in other words, reduced U1 telescript function).

[0097] Therefore, this disclosure provides engineered polynucleotides for use as therapies for neurodegenerative diseases, and corresponding methods for using these engineered polynucleotides. Engineered polynucleotides can have broad applications in the treatment of neurodegenerative diseases through mechanisms that homeostatically regulate the function of U1 snRNPs, regulate tau expression, and a combination of both (e.g., synergistically). To allow for such U1 regulation, engineered polynucleotides can be designed with size chemical modifications, conformational chemical modifications, and strategic chemical modifications, thereby allowing 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 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 with abnormal gene expression 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 the treatment of neurodegenerative diseases. In conclusion, the crucial roles of U1 snRNPs and tau proteins in the progression of neurodegenerative diseases, 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 neurodegenerative diseases.

[0098] Engineered polynucleotides

[0099] 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). In some embodiments described herein, the engineered polynucleotide comprises: (i) one or more targeting portions configured to (e.g., specifically) bind said RNA (e.g., messenger RNA, such as precursor mRNA) 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 precursor mRNA) and the engineered polynucleotide, the post-transcriptional regulatory portion alters the RNA (e.g., mRNA, such as precursor mRNA) 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 to the mRNA precursor refers to a portion that is at least 80%, 90%, or 100% complementary to the mRNA precursor sequence to which it specifically binds. In some embodiments, the RNA (e.g., mRNA, such as a mRNA precursor) encodes a target gene. In some embodiments, the mRNA precursor that binds 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" means modifying a nucleic acid molecule by cleaving the nucleic acid, reacting the nucleotides of the nucleic acid, or splicing the nucleic acid. In some embodiments, "nearby" means a distance of no more than 5 nucleotides.

[0100] Engineered polynucleotides, or engineered polynucleotides as 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 may be combined to produce engineered polynucleotides that perform a specific function. For example, an engineered polynucleotide may include the targeting part disclosed herein and the recruitment part disclosed herein.

[0101] Targeted portion

[0102] 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 sequences of the ribonucleic acid (e.g., a precursor mRNA), such that the engineered polynucleotide and the ribonucleic acid can interact or hybridize. The targeting moieties may be complementary to or identical to a 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 conserved regions present in 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. To avoid being bound by theory, the stable binding of one or two targeting moieties to the constitutive donor 5' of the target mRNA precursor allows for interaction with conserved sites on the constitutive donor and can silence the RNA-binding domain (RBD) of the U1 snRNA. The targeting moieties may contain sequences substantially identical to the U1 snRNA RBD. For example, the RBD may contain the sequence 3'-GUCCAUUCAUA-5', and the targeting moieties may contain GTCCA (or GUCCA). Based on the sequence similarity between the U1 snRNA RBD and the targeting moieties, the targeting moieties can effectively replace the U1 snRNA RBD, preventing it from binding to the mRNA precursor, or preventing the U1 snRNA RBD from binding to the mRNA precursor.

[0103] 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 novel splice sites or activation of shadow-hidden splice sites, leading to aberrant transcripts or nonfunctional proteins associated with 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 moieties are sufficiently identical or complementary to at least two common sequences in the target sequence of the target gene. In some embodiments, one or more target moieties are each independently sufficiently identical or complementary to a common sequence in the target sequence of the target gene.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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'.

[0109] 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'.

[0110] 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.

[0111] 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.

[0112] 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.

[0113]

[0114] 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.

[0115] 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.

[0116] target sequence

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] 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).

[0123] 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.

[0124] 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.

[0125] 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 gene (e.g., a target gene). Non-limiting examples of genes may include microtubule-associated protein tau (MAPT).

[0126] 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.

[0127] 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 sequences corresponding to exon-intron boundaries and sequences corresponding to specific genes or common / conserved sequences present in many genes.

[0128] 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.

[0129] Fundraising portion

[0130] 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.

[0131] In some embodiments of the engineered polynucleotides described herein, the recruitment portion comprises at least 70%, 80%, 85%, or 90% of a nucleotide sequence that is identical or complementary to the sequences shown in Table 2. In some embodiments, the recruitment portion comprises a nucleotide sequence that is identical or complementary 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 selected from SEQ ID NO:1-2. In some embodiments, the engineered polynucleotide comprises at least 70%, 80%, 85%, or 90% of a nucleotide sequence that is identical or complementary to any sequence selected from SEQ ID NO:1-2.

[0132] Table 2. Example recruitment portions of the engineered polynucleotides described in this paper.

[0133]

[0134] 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.

[0135] 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.

[0136] 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.

[0137] Structural configuration

[0138] 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.

[0139] 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.

[0140] Example polynucleotides

[0141] 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.

[0142] 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.

[0143] Table 3. Example sequences of engineered polynucleotides containing recruitment and targeting moieties.

[0144]

[0145] 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.

[0146] In some embodiments, 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 a top 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. In some embodiments, the recruitment portion of the polynucleotide comprises at least one, two, three, four, or more secondary structures. In some embodiments, the targeting portion does not have a secondary structure. In some embodiments, the secondary structure is 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 portion. In some embodiments, the top loop is neither complementary to nor binds to the regulatory portion. In some embodiments, the secondary structure is at least one stem. In some embodiments, the engineered polynucleotide comprises two stems, one being an upper stem near the apical loop and the other a lower stem closer to the target moiety. In some embodiments, the upper stem comprises at least two, four, six, eight, ten, or more nucleotides, wherein 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 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 comprises an inner loop between the upper and lower stems. In some embodiments, the inner loop comprises 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 comprises a secondary structure containing a top 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] 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.

[0152] 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.

[0153] 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.

[0154] 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.

[0155] 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.

[0156] 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. Chemical modification increases the half-life of the engineered polynucleotide.

[0157] 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.

[0158] Chemical modification

[0159] 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).

[0160] 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.

[0161] 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.

[0162] 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.

[0163] 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.

[0164] 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.

[0165] 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.

[0166] 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.

[0167] Table 4. Examples of chemical modifications

[0168]

[0169]

[0170] Modification of the phosphate backbone

[0171] 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 is denoted by methyl and morpholino. "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.

[0172] 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.

[0173] 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.

[0174] 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.

[0175] In some implementations, main chain modifications (e.g., nucleotide linkages of methylphosphonates, thiophosphates, aminophosphates, and dithiophosphates) can confer immunomodulatory activity and / or enhance the in vivo stability of the modified nucleic acid.

[0176] 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.

[0177] 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.

[0178] 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.

[0179] 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 (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.

[0180] 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).

[0181] 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.

[0182] Replacement of phosphate ester portion

[0183] 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'.

[0184] Substitution of phosphate groups

[0185] 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 comprising the substitution or replacement of a phosphate ester group. 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 a phosphate ester group may include methylphosphonates, hydroxyamino groups, siloxanes, carbonates, carboxymethyl groups, carbamates, amides, thioethers, ethylene oxide linkers, sulfonates, sulfonamides, thiomethyl acetals, methyl acetals, oximes, methyleneimino, methylenemethylimino, methylenehydrazine, methylenedimethylhydrazine, and methyleneoxymethylimino.

[0186] Modification of the ribophosphate backbone

[0187] 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.

[0188] Sugar modification

[0189] 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. Example 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 (also having 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).

[0190] Modification of ribose components

[0191] 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).

[0192] 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.

[0193] 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.

[0194] 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.

[0195] 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.

[0196] Modification of nucleotide bases

[0197] 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.

[0198] 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.

[0199] 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, lysicidine, 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.

[0200] 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.

[0201] 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.

[0202] 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.

[0203] 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.

[0204] 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.

[0205] 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.

[0206] Adjustment section

[0207] 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.

[0208] 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).

[0209] 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.

[0210] 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.

[0211] Molecular interactions

[0212] 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.

[0213] 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.

[0214] 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.

[0215] 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.

[0216] 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.

[0217] In some of the embodiments described herein, the engineered polynucleotide does not contain any intramolecular disulfide bonds.

[0218] 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.

[0219] 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.

[0220] 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).

[0221] 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.

[0222] 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.

[0223] In some embodiments of the engineered polynucleotides described herein, the engineered polynucleotides substantially do not exhibit H-helical base pairing with U1 snRNA.

[0224] 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.

[0225] 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 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).

[0226] 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 ).

[0227] 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.

[0228] Table 5. Examples of interactions between U1-C and mRNA precursors mediated by the presence of engineered polynucleotides.

[0229]

[0230]

[0231]

[0232] 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 substantially does not exhibit base pairing with the H-helix of the U1 snRNA.

[0233] Engineered polynucleotide genome

[0234] 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).

[0235] carrier

[0236] Some of the implementation schemes described herein include vectors or plasmids containing nucleic acid sequences encoding the engineered polynucleotides described herein.

[0237] 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).

[0238] Pharmaceutical Composition

[0239] 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 methods of performing the treatment or use 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 as components of a mixture. The 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.

[0240] The pharmaceutical formulations described herein are administered to the target body via appropriate routes of administration, including but not limited to intravenous, intrathecal, intraventricular, intra-arterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intranasal, intravitreal, 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.

[0241] 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.

[0242] Reagent test kit

[0243] 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.

[0244] 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).

[0245] 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.

[0246] method

[0247] 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) 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 the RNA (e.g., mRNA, such as precursor mRNA), 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, methods for altering the expression or activity of a target gene. In some embodiments, prior to contact, the cell exhibits an abnormal messenger ribonucleic acid (mRNA) or protein corresponding to the target gene. In some implementations, the target portion of one or more target portions is sufficiently identical or complementary to a common sequence in the target sequence of a target gene (e.g., microtubule-associated protein tau (MAPT)).

[0248] 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)).

[0249] 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)).

[0250] 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.

[0251] 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.

[0252] 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.

[0253] 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.

[0254] 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.

[0255] 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.

[0256] 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, graft-like), such as two or more lactic acids, 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.

[0257] 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.

[0258] 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 messenger RNA precursor (mRNA precursor) in the cell. In some embodiments, the method includes contacting the cell with an engineered polynucleotide comprising one or more target moieties and a recruitment moieties. In some embodiments, one or more target moieties bind to the mRNA precursor at the target sequence within the mRNA precursor. In some embodiments, the recruitment moieties recruit post-transcriptional regulatory moieties (e.g., spliceosome moieties) 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 moieties and recruits them 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 moieties and recruits them 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 and are recruited to the target sequence.

[0259] 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.

[0260] 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.

[0261] In some implementations, methods for treating a disease or condition may include modulating the expression or activity of genes in cells. These genes may include non-target genes and / or genes that do not directly interact with engineered polynucleotides. Following the administration of engineered polynucleotides or pharmaceutical compositions containing engineered polynucleotides, gene expression in cells or objects may be altered, potentially resulting in different gene expression profiles. For example, genes in cells or objects may be downregulated or upregulated due to the administration of engineered polynucleotides. These alterations in genes within cells may provide benefits in alleviating symptoms or causes of a disease or condition.

[0262] 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.

[0263] In some embodiments, the engineered polynucleotide or pharmaceutical composition containing an engineered polynucleotide is administered at a concentration of at least 5 nM. In some embodiments, the engineered polynucleotide or pharmaceutical composition containing an engineered polynucleotide is administered at a concentration of at least 10 nM. In some embodiments, the engineered polynucleotide or pharmaceutical composition containing an engineered polynucleotide is administered at a concentration of at least 15 nM. In some embodiments, the engineered polynucleotide or pharmaceutical composition containing an engineered polynucleotide is administered at a concentration of at least 20 nM. In some embodiments, the engineered polynucleotide or pharmaceutical composition containing the engineered polynucleotide is administered at a concentration of at least 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 11 nM, 12 nM, 13 nM, 14 nM, 15 nM, 16 nM, 17 nM, 18 nM, 19 nM, 20 nM, 21 nM, 22 nM, 23 nM, 24 nM, 25 nM, 26 nM, 27 nM, 28 nM, 29 nM, 30 nM or higher. In some embodiments, the engineered polynucleotide or pharmaceutical composition containing an engineered polynucleotide is administered at a concentration not exceeding 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 11 nM, 12 nM, 13 nM, 14 nM, 15 nM, 16 nM, 17 nM, 18 nM, 19 nM, 20 nM, 21 nM, 22 nM, 23 nM, 24 nM, 25 nM, 26 nM, 27 nM, 28 nM, 29 nM, 30 nM, or lower. In some embodiments, the engineered polynucleotide or pharmaceutical composition containing an engineered polynucleotide is administered at a concentration of about 5 nM to 20 nM.

[0264] 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 involve administering engineered polynucleotides or pharmaceutical compositions containing engineered polynucleotides via intrathecal or intravenous (“iv”) administration. However, in some cases, the method includes administering engineered polynucleotides or pharmaceutical compositions containing engineered polynucleotides via intraperitoneal injection. In some cases, the method includes administering engineered polynucleotides or pharmaceutical compositions containing engineered polynucleotides via oral or intranasal 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, intraventricular injection, intravitreal injection, intradermal injection, transdermal administration, intranasal administration, intralymphatic administration, rectal administration, gastric administration, or any other suitable parenteral administration. In some embodiments, local delivery routes closer to the site of injury or inflammation are preferred compared to systemic routes. 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.

[0265] The appropriate dosage and frequency of administration to the subject are determined by factors including, but not limited to, the specific engineered polynucleotide, composition or pharmaceutical composition, disease condition and its severity, 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.

[0266] This article describes methods for treating neurodegenerative diseases using engineered polynucleotides or vectors encoding engineered polynucleotides. In some respects, neurodegenerative diseases are Alzheimer's disease. The engineered polynucleotide can be the engineered polynucleotide described in PCT / US2022 / 037391, the entire contents of which are incorporated herein by reference.

[0267] This article describes a method for treating neurodegenerative diseases in a subject of need, the method comprising: administering to the subject a pharmaceutical composition comprising: administering to the subject a carrier encoding an engineered polynucleotide. In some embodiments, the engineered polynucleotide comprises: (i) one or more targeting portions configured to specifically bind said 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.

[0268] In some embodiments, the method includes contacting the cells of the object with an engineered polynucleotide or a carrier encoding an engineered polynucleotide. In some embodiments, the cells are microglia. In some embodiments, the cells are astrocytes. In some embodiments, the cells are neurons. In some embodiments, the cells are excitatory neurons. In some embodiments, the cells are derived from a patient diagnosed with Alzheimer's disease. In some embodiments, the cells are neurons derived from a healthy patient. In some embodiments, the cells are neurons derived from a patient diagnosed with Alzheimer's disease. In some embodiments, the cells are excitatory neurons derived from a patient diagnosed with Alzheimer's disease. In some embodiments, the cells are derived from the hippocampus or a cortical region.

[0269] In some embodiments, the neurodegenerative disease is a neurodegenerative disease associated with the presence of Tau in the brain of the subject. For example, a neurodegenerative disease may be a tau proteinopathy or other disease in which the presence of tau indicates pathology. As described in other parts of this disclosure, when an engineered polynucleotide is applied to a subject or cells, it can reduce the amount of tau, alter tau expression, or decrease tau expression. Due to the altered presence of tau, the application of the engineered polypeptide can alleviate or improve the symptoms of the neurodegenerative disease, or otherwise treat the neurodegenerative disease. In some embodiments, the neurodegenerative disease is selected from: Alzheimer's disease, Pick's disease, chronic traumatic encephalopathy, progressive supranuclear palsy, aeruginosa granuloma, amyotrophic lateral sclerosis, and frontotemporal dementia.

[0270] In some embodiments, contacting cells with engineered polynucleotides or vectors encoding engineered polynucleotides increases cell fiber width, branching number, branch junctions, neurite straightness, and total axonal mass. In some embodiments, contacting cells with engineered polynucleotides or vectors encoding engineered polynucleotides improves axonal function associated with synaptic and cognitive functions. In some embodiments, contacting cells with engineered polynucleotides or vectors encoding engineered polynucleotides reduces the level of Tau protein in cells. In some embodiments, contacting cells with engineered polynucleotides or vectors encoding engineered polynucleotides leads to differential expression of genes associated with the regulation of neuronal generation, neurogenesis, neuronal differentiation, and synaptic assembly. In some embodiments, contacting cells with engineered polynucleotides or vectors encoding engineered polynucleotides leads to enrichment of genes associated with steroid biosynthesis pathways, MAPK signaling, cell cycle, PI3K-Akt signaling, cellular senescence pathways, fatty acid metabolism and biosynthesis, cholesterol metabolism, AMPK signaling pathways, and Ras signaling pathways. In some embodiments, contacting cells with engineered polynucleotides or vectors encoding engineered polynucleotides leads to enrichment of genes associated with increased mRNA transport. In some embodiments, contacting cells with engineered polynucleotides or vectors encoding engineered polynucleotides leads to prevention of Alzheimer's disease-related phenotypes in cells. In some embodiments, contacting cells with engineered polynucleotides or vectors encoding engineered polynucleotides leads to increased synapse formation. In some embodiments, contacting cells with engineered polynucleotides or vectors encoding engineered polynucleotides results in improved synaptic integrity and synaptic plasticity. In some embodiments, contacting cells with engineered polynucleotides reduces premature polyadenylation of one or more transcripts in cells. In some embodiments, contacting cells with engineered polynucleotides or vectors encoding engineered polynucleotides leads to reduced excitotoxicity. In some embodiments, contacting cells with engineered polynucleotides or vectors encoding engineered polynucleotides leads to improved synapse-related neural pathways and morphology in cells. In some embodiments, contacting cells with engineered polynucleotides or vectors encoding engineered polynucleotides leads to increased neuronal activity. In some embodiments, contacting cells with engineered polynucleotides or vectors encoding engineered polynucleotides leads to a reduction in the aggregation or mislocalization of U1-70K in the cells. In some embodiments, contacting cells with engineered polynucleotides or vectors encoding engineered polynucleotides leads to a reduction in the expression or aggregation of amyloid-β in the cells. In some embodiments, contacting cells with engineered polynucleotides or vectors encoding engineered polynucleotides leads to the regulation of astrogenesis.

[0271] In some embodiments, the cells are neurons derived from induced pluripotent stem cells. In some embodiments, the cells are excitatory neurons derived from induced pluripotent stem cells. In some embodiments, the cells are derived from healthy patients. In some embodiments, the cells are excitatory neurons derived from healthy patients. In some embodiments, the cells are derived from the hippocampus or cortical region.

[0272] 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.

[0273] The use of absolute or sequential terms, such as “will,” “will not,” “shall,” “shall not,” “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.

[0274] 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.”

[0275] 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, each of 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” refers 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.

[0276] 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.

[0277] 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.

[0278] 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.

[0279] 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.

[0280] 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.

[0281] 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.

[0282] List of Implementation Plans

[0283] 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.

[0284] 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.

[0285] 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.

[0286] Implementation Scheme 4. The engineered polynucleotide according to Implementation Scheme 3, wherein the first target sequence comprises a common sequence in the target sequence.

[0287] 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.

[0288] 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.

[0289] 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.

[0290] 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.

[0291] 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.

[0292] 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.

[0293] 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.

[0294] 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.

[0295] 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.

[0296] 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.

[0297] 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.

[0298] 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).

[0299] 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.

[0300] 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.

[0301] 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.

[0302] Implementation Scheme 20. The engineered polynucleotide according to Implementation Scheme 19, wherein the spliceosome portion comprises U1 snRNA and spliceosome protein.

[0303] 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.

[0304] 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.

[0305] 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.

[0306] 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.

[0307] 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.

[0308] 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.

[0309] 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.

[0310] 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.

[0311] 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).

[0312] 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.

[0313] 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.

[0314] 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.

[0315] 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.

[0316] 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.

[0317] 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.

[0318] 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.

[0319] 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.

[0320] 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.

[0321] 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).

[0322] 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).

[0323] 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.

[0324] 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.

[0325] 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.

[0326] 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.

[0327] 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.

[0328] 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.

[0329] 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.

[0330] 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'.

[0331] 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.

[0332] 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.

[0333] 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.

[0334] 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.

[0335] 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.

[0336] 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.

[0337] 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.

[0338] 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.

[0339] 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.

[0340] 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.

[0341] 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.

[0342] 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.

[0343] 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.

[0344] 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.

[0345] 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.

[0346] 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.

[0347] Implementation Scheme 65. The engineered polynucleotide according to Implementation Scheme 63 or 64, wherein the structural feature comprises one or more stem-loop structures.

[0348] 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.

[0349] 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.

[0350] 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.

[0351] 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).

[0352] 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.

[0353] 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.

[0354] 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.

[0355] 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.

[0356] 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.

[0357] 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.

[0358] Implementation Scheme 76. The engineered polynucleotide according to Implementation Scheme 74, wherein the target gene is microtubule-associated protein tau (MAPT).

[0359] 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.

[0360] Implementation Scheme 78. The engineered polynucleotide according to Implementation Scheme 77, wherein the engineered polynucleotide comprises at least one phosphate thioester nucleotide inter-bond.

[0361] 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.

[0362] 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.

[0363] 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.

[0364] 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.

[0365] 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.

[0366] 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.

[0367] 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.

[0368] 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.

[0369] Implementation Scheme 87. The method according to Implementation Scheme 85 or 86, wherein the mRNA precursor corresponds to the target gene.

[0370] Implementation Scheme 88. The method according to Implementation Scheme 87, wherein the target gene is microtubule-associated protein tau (MAPT).

[0371] 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.

[0372] 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.

[0373] 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.

[0374] Example

[0375] The following illustrative examples represent embodiments of the stimuli, systems, and methods described herein and are not intended to limit the scope of the invention in any way.

[0376] Example 1. Regulating target gene expression using engineered polynucleotides

[0377] Cells obtained from cells such as (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 examination of 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 associated with any of the target genes described herein (e.g., tau neurofibrillary tangles encoded by the MAPT target gene or amyloid plaques encoded by the APP target gene).

[0378] Example 2. Treatment of neurological diseases by editing RNA

[0379] The subject was diagnosed with Alzheimer's disease caused by aberrant splicing or polyadenylation activity of mRNA precursors in the subject's brain. A dosing regimen comprising a pharmaceutical composition of the chemically modified engineered polynucleotide disclosed herein was prescribed to the subject. This pharmaceutical composition recruits and stabilizes at least one regulatory moiety to reduce aberrant splicing or polyadenylation activity of multiple different mRNA precursors, thereby alleviating Alzheimer's disease symptoms. Some mRNA precursors (and others) may contain MAPT mRNA precursors. The chemically modified engineered polynucleotide recruits and stabilizes at least one regulatory moiety upon binding to the MAPT mRNA precursor to properly splice the MAPT mRNA precursor. In some embodiments, the chemically modified engineered polynucleotide improves the specificity or efficiency of recruiting and stabilizing at least one regulatory moiety RNA-editing entity. The regulation of the MAPT mRNA precursor by the engineered polynucleotide reduces the amount of tau plaques in the subject, thereby treating or alleviating the subject's Alzheimer's disease symptoms.

[0380] Example 3. Evaluation of mitochondrial activity of neurons derived from induced pluripotent stem cells (IPSCs) after co-incubation with ASMO1

[0381] Mitochondria are central to a variety of cellular processes, including ATP production, intracellular calcium signaling, and reactive oxygen species (ROS) generation. Neurons rely heavily on mitochondrial function to establish membrane excitability and perform complex neurotransmission and plasticity processes. In addition to assessing cellular metabolism, feasibility studies are used to determine whether specific molecules are toxic to the test system, which is crucial for determining working concentrations and safety profiles. Cell viability is defined as the number of healthy cells in a sample and can be used as a marker of cell death following exposure to toxic drugs.

[0382] The CyQUANT MTT cell viability assay uses MTT reagent to determine mammalian cell viability. Measuring changes in cell viability is a method for assessing cell health and determining genotoxicity. The redox potential in living mammalian cells causes the water-soluble MTT reagent (3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide) to convert into an insoluble formazan product. After dissolving the formazan in SDS (sodium dodecyl sulfate) reagent, the concentration of the colorimetric probe (CyQUANT) is determined by measuring the optical density at 540-570 nm. TM MTT cell viability assay.

[0383] Cells from healthy patients (HDC) and patients diagnosed with Alzheimer's disease (ADC) were cultured for 6 days, then cultured for 7 days (day 13) with ASMO1 (6.17, 18.52, 55.56, 166.67, 500 nM) or with culture medium alone. The culture medium was replaced with fresh medium containing MTT solution. Cells were incubated in a CO2 incubator (37°C, 5% CO2) for 4 hours, then SDS-HCl was added. The contents of the different wells were homogenized, and the absorbance was read at 540 nm on a microplate reader. The median of each biological replicate was obtained. Three independent experiments were performed in three technical replicates. Values ​​are expressed as mean ± standard error of mean (SEM). To assess statistical differences between groups, one-way ANOVA was performed, followed by Tukey's post-hoc test. A p-value less than 0.05 was considered significant. Statistical analysis was performed using GraphPad Prism 9 software (GraphPad Software Inc., San Diego, CA, USA).

[0384] like Figure 11 As shown in Figures A and B, excitatory neurons from (A) HDC or (B) ADC were cultured for 6 days and then incubated with APT20TTMG. After 7 days of treatment, mitochondrial activity of the neurons was assessed using a commercial kit.

[0385] No concentration of ASMO1 altered mitochondrial activity, indicating that it does not change the vitality of neurons derived from iPSCs in healthy patients and patients diagnosed with Alzheimer's disease.

[0386] Example 4. Assessment of mitochondrial activity in central nervous system cells after co-incubation with ASMO1

[0387] Mitochondria are increasingly recognized as a key hub in astrocyte- and microglia-mediated immune responses. A pathological feature of Alzheimer's disease (AD) is reduced mitophagy, the process by which damaged mitochondria are cleared from the cell through autophagy. Disruption of cellular capacity is a significant factor in AD pathogenesis, supporting the view that altered mitochondrial function may be a cause or consequence of the disease's pathological features.

[0388] Similar to Example 3, mammalian cell viability was determined using the CyQUANT MTT cell viability assay. Neurons and microglia from healthy patients (HDC) and microglia from patients diagnosed with Alzheimer's disease (ADC) were seeded and cultured for 6 days, then cultured for 7 days (day 13) with ASMO1 (18.52 and 500 nM) or with culture medium only. Astrocytes were seeded and cultured for 1 day, then cultured for 24 hours with ASMO1 (18.52 and 500 nM) or with culture medium only. The culture medium was then replaced with fresh medium containing MTT solution. Cells were incubated in a CO2 incubator (37°C, 5% CO2) for 4 hours, then SDS-HCl was added. The contents of the different wells were homogenized, and absorbance was read at 540 nm on a microplate reader. Two to three independent experiments were performed in three technical replicates, and statistical analysis was performed as described in Example 3.

[0389] like Figure 12 As shown, excitatory neurons from HDC were cultured for 6 days and then incubated with APT20TTMG. After 7 days of treatment, mitochondrial activity of the neurons was assessed using a commercial kit. Analysis of variance (ANOVA) was performed, followed by a Tukey post-hoc test. Data from three independent experiments conducted in three technical replicates were obtained. A p-value <0.05 was considered statistically significant compared to the control group (untreated cells).

[0390] like Figure 13As shown in Figures A and B, microglia from (A) HDC or (B) ADC were cultured for 6 days and then incubated with APT20TTMG. After 7 days of treatment, microglia were assessed using a commercial kit for measuring mitochondrial activity. Analysis of variance (ANOVA) was performed, followed by a Tukey post-hoc test. Data from two independent experiments performed in three technical replicates were obtained. A p-value <0.05 was considered statistically significant compared to the control group (untreated cells).

[0391] like Figure 14 As shown, astrocytes were cultured for 1 day and then incubated with APT20TTMG. Mitochondrial activity of the astrocytes was assessed 24 hours after treatment using a commercial kit. Analysis of variance (ANOVA) was performed, followed by a Tukey post-hoc test. Data from three independent experiments performed in three technical replicates were obtained. A p-value <0.05 was considered statistically significant compared to the control group (untreated cells).

[0392] No changes in mitochondrial activity were observed in neurons, microglia, and astrocytes after co-incubation with ASMO1. However, the parameter was slightly decreased at the highest concentration (500 nM) in neurons and microglia from healthy patients; and slightly decreased at both assessment concentrations in astrocytes, which may be related to reduced astrocyte responsiveness and the resulting decrease in cellular stress.

[0393] Example 5. Assessment of glutamate levels in neurons derived from induced pluripotent stem cells (iPSCs) after co-incubation with ASMO1.

[0394] Glutamate is the most abundant excitatory neurotransmitter in the mammalian central nervous system (CNS). It is widely distributed throughout the CNS, but almost entirely intracellular. The amount of extracellular glutamate available is tightly regulated to allow for appropriate signaling levels. Most excitatory neurotransmission in the mammalian CNS is mediated by glutamate and its receptors, primarily ligand-gated ionotropic glutamate receptors (iGluRs). These receptors also play a crucial role in synaptic plasticity (a fundamental molecular mechanism of learning and memory). A subgroup of iGluRs is selectively gated by the specific agonist N-methyl-D-aspartate (NMDA) and is therefore called NMDA receptors (NMDARs), which are essential for their critical role in synaptic function and plasticity. Insufficient synaptic NMDAR signaling impairs neuronal survival. However, excessive stimulation of glutamatergic signaling leads to excitotoxicity, in which neuronal damage or death occurs. In addition to acute effects, numerous studies have shown that glutamate excitotoxicity also plays a role in slowly progressive, delayed neurodegeneration.

[0395] Glutamate-Glo TM This assay is a bioluminescent assay for detecting glutamate in biological samples. The detection is based on the conversion of glutamine to glutamate via glutaminase. Subsequently, glutamate oxidation and NADH generation are combined with a bioluminescent NADH detection system. Glutamate dehydrogenase utilizes glutamate and NAD+ to generate α-ketoglutarate and NADH. In the presence of NADH, the proluciferin reductase substrate is converted to luciferin by the reductase, and then luciferin is detected by Ultra-Glo... TM Recombinant luciferase is used to produce light.

[0396] Cells from healthy patients (HDC) and patients diagnosed with Alzheimer's disease (ADC) were cultured for 6 days, then cultured for 7 days (day 13) with ASMO1 (6.17, 18.52, 55.56, 166.67 nM, and 500 nM) or with culture medium alone, and extracellular culture medium was collected for further analysis. Samples were prepared for use with Glutamate-Glo TM The relative quantification of glutamate was performed, and the glutamate level was reported in relative luminescent units (RLU). Three independent experiments were performed in six technical replicates, and statistical analysis was conducted as described in Example 3.

[0397] like Figure 15 As shown in Figures A and B, excitatory neurons from (A) HDC or (B) ADC were cultured for 6 days and then incubated with APT20TTMG. After 7 days of treatment, glutamate release from the neurons was assessed using a commercial kit.

[0398] No significant changes in extracellular glutamate levels were observed in neurons obtained from iPSCs of healthy patients and patients diagnosed with Alzheimer's disease after incubation with ASMO1, which confirms the previous data in Examples 3-4, suggesting that ASMO1 appears to be safe at these concentrations and under these conditions.

[0399] Example 6. Evaluation of morphological changes in neurons derived from induced pluripotent stem cells (iPSCs) after co-incubation with ASMO1.

[0400] Neuronal clusters infected with Alzheimer's disease (AD) are characterized by abnormal synaptic morphology and a significant reduction in the total number of synapses, which is considered the cause of the disease's characteristic memory and cognitive deficits. Normal function of axons and axon terminals depends on the transport of proteins, organelles, vesicles, and other components from their sites of synthesis within the cell body. Therefore, axonal transport is a key cellular process for axonal and synaptic function. Several studies have shown that malnourished axons can be observed before the detection of canonical tau and amyloid deposition. Understanding the temporal course of axonal pathology is crucial for comprehending disease progression over time and identifying potential therapeutic targets and the window of opportunity for treatment effectiveness.

[0401] Cells from healthy patients (HDC) and patients diagnosed with Alzheimer's disease (ADC) were processed in NeuroHTS. TM Neurons were cultured in plates for 6 days, then cultured for 7 days (day 13) with ASMO1 (6.17, 18.52, 55.56, 166.67 nM, and 500 nM) or with culture medium alone. Afterwards, all neurons were assessed using a 7-factor neuronal morphology profile via MetaXpress software. Figure 16 As shown, neurons were evaluated in three different regions and with different parameters. In the upper region of the foramen, the number of cells in the cell body and dendrites and nuclear aggregates were assessed; in the middle region of the channel (axon), the thickness of the axonal fibers was verified; and in the lower region where the axon and dendrites are located, the following four parameters were assessed: number of branches, number of branch junctions, neurite straightness, and axonal material. Three independent experiments were performed in up to three technical replicates.

[0402] Numerical values ​​are expressed as mean ± standard error of mean. One-way ANOVA was performed to assess statistical differences between groups, followed by Tukey's test (parametric test) for cell aggregates, fiber width, and neurite straightness, or Kruskal-Wallis post-hoc test (nonparametric test) for cell number, branch number, number of branches per cell, branch junctions, branch junctions per cell, total axonal material, and axonal material per cell. A p-value less than 0.05 was considered significant. Statistical analysis was performed using GraphPad Prism 9 software (GraphPad Software Inc., San Diego, CA, USA).

[0403] like Figure 17As shown in A and B, excitatory neurons were cultured for 6 days and then incubated with ASMO1. Neuron numbers were assessed using MetaXpress software after 7 days of treatment. (A) Cell number in healthy patient cells (HDC); (B) Cell number in AD patient cells (ADC). Cell number is an indirect indicator of cell death. This parameter is crucial for understanding the safety of ASMO1 in these cells and helps in selecting the concentration used in subsequent assays. Furthermore, some analytical parameters need to be normalized to the total cell count at the end of the experiment to avoid masking the results. While no concentration (in a statistically significant manner) reduced cell number, the two highest concentrations (166.67 and 500 nM) in both HDC and ADC appeared to cause a reduction.

[0404] like Figure 18 As shown in A and B, excitatory neurons were cultured for 6 days and then incubated with ASMO1. After 7 days of treatment, cell aggregates were assessed using MetaXpress software. (A) Cell aggregates from healthy patient cells (HDC); (B) Cell aggregates from AD patient cells (ADC).

[0405] This parameter may be related to the aggregation of neurons in culture, i.e., due to the presence of cell debris or even free DNA. Both of these may appear as indicators of toxicity after the addition of the molecule. Incubation with different concentrations of ASMO1 did not change cell aggregation, which is a novel safety indicator in the presence of ASMO1.

[0406] like Figure 19 As shown in A and B, excitatory neurons were cultured for 6 days and then incubated with ASMO1. After 7 days of treatment, fiber width was assessed using MetaXpress software. (A) Fiber width of healthy patient cells (HDC); (B) Fiber width of AD patient cells (ADC).

[0407] Fiber width / thickness is an indicator of axonal fasciculation, which consists of a group of intertwined axons. Any impairment of fiber thickness can lead to alterations in axonal transport, which plays a crucial role in the pathogenesis of Alzheimer's disease (AD). In both HDC and ADC, fiber thickness trended upward after treatment with all concentrations of ASMO1. Furthermore, ADC neurons treated with all evaluable concentrations of ASMO1 showed a profile similar to the negative HDC control. Even in HDC, ASMO1 incubation appeared to increase fiber thickness. Although the results did not show statistical differences, this parameter was considered in concentration selection in subsequent studies. These combined results support the hypothesis that ASMO1 can improve axonal transport.

[0408] like Figure 20As shown in AD, the number of branches is an indicator of neuronal growth, regeneration, and / or growth rate. Neurons output information through a wide variety of axonal morphologies. One of the processes that generates this diversity is axonal branching. From reaching multiple target sites to defining neural distribution areas within specific target sites, axonal branching allows neurons to establish unique connection patterns. Changes in axonal branching patterns are achieved by regulating various morphological processes, including branch initiation, branch elongation, arbor complexity formation, branch retraction / pruning, and terminal arborization. One class of proteins associated with regulating axonal branching are cytoskeleton-related proteins. Axonal degeneration, i.e., a decrease in the level of axonal branching, is directly related to neurodegeneration. Molecules that can prevent degeneration and even regenerate degeneratively vulnerable neurons are closely related to AD. Indirectly, this parameter may be associated with an increase in late synapses. For both analyses (number of branches and number of branches per cell), a concentration of 18.52 nM in HDC neurons showed an interesting profile, with an approximately 80% increase in the number of branches and a two-fold increase in the number of branches per cell. In ADC neurons, this same concentration increased the number of branches and the number of branches per cell by approximately 50% and 60%, respectively, suggesting that it can protect neurons from degeneration or promote axonal growth. (A) Number of branches in healthy patient cells (HDC); (B) Number of branches in AD patient cells (ADC); (C) Number of branches per cell in healthy patient cells (HDC); (D) Number of branches per cell in AD patient cells (ADC). Figure 21 As shown in AD, branching junctions are an indicator of the branching complexity and potential synaptic development of neurons. It is a complementary parameter to the number of branches; therefore, it refers to the likelihood that an axon of one neuron will communicate with the dendrites of another neuron. Thus, the more branching junctions, the greater the communication opportunities and the more synapses. The most interesting profiles are the treatments at 6.17 nM and 18.52 nM for two test systems (HDC and ADC). In terms of total branching junctions, HDC treated with a concentration of 6.17 nM doubled this parameter. For ADC, it increased to approximately 3.5 times. (A) Branching junctions of healthy patient cells (HDC); (B) Branching junctions of AD patient cells (ADC); (C) Branching junctions per cell of healthy patient cells (HDC); (D) Branching junctions per cell of AD patient cells (ADC).

[0409] A neurite is any protrusion from the neuron's cell body, a process by which neurons differentiate into dendrites and axons, and is potentially crucial for communication between different neurons. Therefore, the straightness of a neurite is a parameter for assessing the quality of molecular transport within a neuron and its internal action potentials. The straighter the neurite, the greater the chance of information being transmitted along the neuron. Figure 22 A and B show the neurite straightness of different cells. Compared to the negative control (untreated) in the same system, the straightness was statistically significantly increased in ADC treated with the highest concentration (500 nM) (**p = 0.0028). (A) Neuronal straightness of healthy patient cells (HDC); (B) Neuronal straightness of AD patient cells (ADC).

[0410] like Figure 23 As shown in the AD, both total axonal mass and axonal mass per cell showed an overall increase in neuronal content at concentrations of 6.17 and 18.52 nM. Regarding axonal mass per cell (cells normalized to total cell count), ADC treatment with the lowest concentration of ASMO1 increased this parameter by approximately 160%, while ADC treatment with 18.52 nM increased it by 130%. In HDC cells, treatment with the same concentrations increased axonal mass per cell by approximately 80% and up to 3.5 times, respectively. Besides the potential for some concentrations to increase axonal mass, it is worth noting that this parameter tended to improve in ADC treatments with the first three concentrations. (A) Total axonal mass of healthy patient cells (HDC); (B) Total axonal mass of AD patient cells (ADC); (C) Axonal mass per cell of healthy patient cells (HDC); (D) Axonal mass per cell of AD patient cells (ADC).

[0411] like Figure 24-25 As shown, excitatory neurons were cultured for 6 days and then incubated with ASMO1. After 7 days of treatment, all morphological changes were assessed. Parameters assessed included: cell body and dendrites (cell number and nuclear aggregates), axon (fiber width), and axon and dendrites (number of branches, number of branch junctions, neurite straightness, and axonal material).

[0412] As indicated by cell number and cell aggregate parameters, co-incubation with ASMO1 did not cause toxicity to HDC and ADC neurons. Following co-incubation with ASMO1, several analyzed parameters showed an increasing trend, which appears to be beneficial for preventing neurodegeneration and improving axonal function directly related to synaptic and cognitive functions, such as fiber width, number of branches, branch junctions, neurite straightness, and total axonal material. Therefore, it is certain that ASMO1 has the potential to be beneficial for both healthy neurons and neurons with AD-related phenotypes, helping to prevent the degeneration of vulnerable neurons in AD.

[0413] Example 7. Immunoprecipitation assay of complex U1 protein binding in neuroblastoma cell lines incubated with ASMO-1.

[0414] For protein immunoprecipitation assays, cell lysates from SK-H-SH (300 μg / IP) were added to 0.5 mg streptavidin beads (pre-conjugated with 90 pmol biotinylated APT20TTMG or conjugated beads only (negative control)) and incubated overnight at 4°C. The runoff was collected, and the beads were washed 10 times with ice-cold IP lysis buffer. Proteins were eluted from the beads for 5 min at 90°C in 30 μl of 4x Laemmli loading buffer / 2-mercaptoethanol. The sample (5 μg input sample, 2.5% runoff, and 50% eluent fraction) was separated on NuPAGE and electrophoresed. The isolated proteins were transferred to polyvinylidene fluoride (PVDF) membranes, blocked, and then incubated overnight at 4°C with the following primary antibodies: U1-70K (Abcam, catalog number ab83306), U1-A (Abcam, catalog number ab166890), U1-C (Abcam, catalog number ab192028), and GAPDH (Abcam, catalog number ab8245), followed by incubation with HRP-conjugated secondary antibodies. Protein visualization was achieved using enhanced chemiluminescence (ECL) reagents (ThermoFisher Scientific, USA), and images were acquired using the Jess Simple Western Imaging Kit. Evaluation was performed in triplicate (N=3).

[0415] For RNA immunoprecipitation using biotinylated APT20TTMG, cells were washed once with ice-cold PBS, collected, and precipitated by centrifugation at 500g for 5 minutes. Each sample was resuspended and lysed for 20 minutes at 4°C in 150 μl of ice-cold RSB-100 buffer (10 mM Tris-HCl pH 7.4, 100 mM NaCl, 2.5 mM MgCl2, 0.5% Triton X-100), containing EDTA-free Complete Ultra protease inhibitor mix mini-tablets (Roche) and 100 U / ml Super-ASE RNase inhibitor (Thermo Fisher Scientific). 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 20 minutes at 4°C. Collect the supernatant and determine the total protein concentration using the BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Adjust each sample to 1.00 mg / ml using RSB-100 lysis buffer and aliquot 100 μl from each sample (input) and store at -20°C until needed. For RNA manipulation, wash and prepare 0.5 mg Dynabeads M-270 streptavidin magnetic beads (Thermo Fisher Scientific) according to the manufacturer's instructions. Conjugate the beads with 90 pmol of biotinylated APT20TTDC or APT20TTMG (beads without oligonucleotides were used as a negative control) at room temperature for 30 minutes. After washing, add 100 μl of SK-N-SH cell lysate (100 μg) to the beads immobilized with biotinylated oligonucleotides (and controls), incubate overnight at 4°C, and gently rotate. Perform RNA analysis using Direct-zol RNA Microprep (Cambridge) according to the manufacturer's instructions. Bioscience isolates RNA from input samples, flow samples, and elution samples.

[0416] Following the manufacturer's instructions, RNA (50% input sample and flow buffer, and 100% eluent fraction) was converted to cDNA using the SuperScript IV first-strand synthesis system (Thermo Fisher). After RNA removal, 1 μl of cDNA was amplified by qPCR using the following TaqMan probes (Thermo Fisher): U1 (RNVU1-18, Hs04940459_gH), total tau (MAPT, assay ID: Hs00902193_m1), and GAPDH (GAPDH, assay ID: Hs99999905_m1). AppliedBiosystems QuantStudio was used. TM Gene expression was analyzed using the 12K Flex real-time PCR system.

[0417] Figure 26 AD demonstrates various Western blotting methods for detecting the presence of specific proteins in samples. Cell lysates from SK-H-SH (100 μg / IP) were added to 0.5 mg streptavidin beads (pre-conjugated with 90 pmol biotinylated APT20TTMG or beads only (negative control)) and incubated overnight at 4°C. Samples (5 μg input sample, 2.5% flow buffer, and 50% eluent fraction) were evaluated and electrophoresed. Immunoprecipitation of proteins was assessed using specific antibodies via Western blotting. GAPDH was used as a negative control (n = 3 technical replicates).

[0418] Figure 27 AC shows the relative expression of specific proteins in the samples. Human neuroblastoma SK-N-SH cells were lysed and prepared for qRT-PCR immunoprecipitation assays using RNA isolated from the following: input sample (50%), flow-through-FT sample (50%), and eluent sample (100%). (A) Relative expression of U1 snRNA, (B) Tau mRNA precursor, and (C) GAPDH. Data are expressed as mean ± standard error of mean. Figures show data from three independent experiments, analyzed using ANOVA followed by Tukey post-hoc test or t-test. **p<0.01, ****p<0.0001.

[0419] Immunoprecipitation assays were performed using SK-N-SH neuroblastoma cells to verify the assembly of the APT20TTMG / U1-mRNA precursor complex. These assays confirmed that APT20TTMG binds to key proteins of the U1 complex (e.g., U1-70K and U1-C proteins), but not to U1-A snRNP. The lack of binding to GAPDH protein was expected, as it was used as a negative control in the experiments. qRT-PCR confirmed the binding of APT20TTMG to U1 snRNA and to tau and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA precursors. This binding pattern indicates interaction with the functional U1 snRNP complex, which also contains a group of proteins called the Sm core that were not evaluated in this study. Furthermore, the binding of APT20TTMG to U1-70K provides evidence for the potential presence of Sm core assembly in the immunoprecipitation pool, as U1-70K is responsible for bridging the U1 precursor (U1 snRNA precursor) and U1 snRNA to the SMN complex, which mediates Sm core assembly. The assembled Sm core plays a crucial role in the stable binding of U1-70K to U1 snRNA. Therefore, the binding of APT20TTMG to these proteins, as well as to the mRNA precursor and U1 snRNA, strongly suggests the correct assembly of the spliceosome initiation mechanism, thus predicting subsequent spliceosome recruitment. This is noteworthy because the U1 complex requires proper assembly for proper splicing, and this process can be impaired by the typical U1 snRNP splicing dysfunction present in AD.

[0420] Evidence of direct or indirect interactions between ASMO1 and U1-C and U1-70K suggests that ASMO is correctly targeting the U1 complex and mRNA precursors (typically represented by MAPT and GAPDH mRNA precursors in these assays) to achieve proper assembly of the U1-mRNA precursor complex.

[0421] Example 8. Evaluation of tau protein regulation in neurons derived from induced pluripotent stem cells (iPSCs) after co-incubation with ASMO1.

[0422] In addition to the extracellular deposition of insoluble amyloid-β preceding early-onset Alzheimer's disease (AD), early AD is also characterized by the intracellular accumulation of neurofibrillary tangles (NFTs), which consist of aggregated hyperphosphorylated tau. Although tau plays a pathological role in AD, under physiological conditions, it is a phosphoprotein that promotes the assembly and stability of microtubules in neurons. Tau also functions in chromatin structure, signal transduction, synaptic plasticity, and nucleic acid protection. In AD, aberrant tau hyperphosphorylation impairs its ability to bind to microtubules, leading to abnormal tau self-assembly, accumulation, aggregation, and NFT formation in neurons. It has also been proposed that the pathogenic form of tau is involved in the disruption of axonal maintenance processes in AD, thereby triggering loss of neuronal connectivity, inhibiting axonal transport, and accumulating in the neurofibrillary network.

[0423] Cells from healthy patients (HDC) and patients diagnosed with Alzheimer's disease (ADC) were cultured for 6 days, then cultured for 7 days (day 13) with ASMO1 (6.17 nM, 18.52 nM, and 500 nM) or with culture medium alone. Afterwards, cells were lysed and cultured using commercially available ab273617 human Tau SimpleStep. The kit evaluated samples in each experimental group. A quantification curve was generated for each biological replicate. Three independent experiments were performed in three technical replicates. Values ​​are expressed as mean ± standard error of the mean. To assess statistical differences between groups, one-way ANOVA was performed, followed by Dunnett's multiple comparison test. A p-value less than 0.05 was considered significant. Statistical analysis was performed using GraphPad Prism 9 software (GraphPad Software Inc., San Diego, CA, USA).

[0424] like Figure 28 As shown in AB, excitatory neurons from HDC(A) or ADC(B) were cultured for 6 days and then incubated with culture medium (negative control). Tau levels were assessed using a commercial kit 7 days after treatment.

[0425] In neurons from healthy patients (HDC), tau protein levels remained unchanged after incubation with three concentrations of APT20TTMG. However, the same profile was not observed in neurons obtained from patients diagnosed with AD (ADC). Incubation with APT20TTMG at concentrations of 18.52 nM and 500 nM resulted in protein levels decreasing by 41.4 ± 2.9% (*p = 0.0196) and 60.4 ± 0.8% (**p = 0.0023), respectively, indicating a concentration-dependent effect. While a concentration of 6.17 nM also showed a decrease (33.7 ± 2.7%), this result was not statistically significant. Notably, the proposed mechanism of action of APT20TTMG, in addition to acting on mechanisms prior to amyloid β-plaque formation, also targets pathways prior to the formation of neurofibrillary tangles (composed of tau protein aggregates), two key features of AD. Therefore, through this study, we clearly understand that ASMO1 reduces tau protein levels in cultured neurons at stages and times prior to tau protein increase. Since tau protein levels were already low in cells mimicking some pathophysiological features of the disease, it can be concluded that APT20TTMG may play a role in the early stages of the disease, thereby preventing excessive tau protein formation.

[0426] ASMO1 reduced tau protein in neurons of patients diagnosed with AD in a concentration-dependent manner, but did not reduce tau protein in neurons of healthy patients, suggesting that it may play a role in the early stages of the disease and slow its progression.

[0427] Example 9. RNA-seq Differential Gene Expression Analysis of Neurons from Induced Pluripotent Stem Cells (iPSCs) after Co-incubation with ASMO1 This study involved RNA sequencing (RNA-seq) differential gene expression analysis of neurons from induced pluripotent stem cells (iPSCs) after co-incubation with APT20TTMG. Gene expression occurs when DNA is transcribed into RNA (primarily mRNA transcripts). The level of mRNA transcripts is commonly used to infer protein abundance. This set of genes expressed by the genome is called the transcriptome, and the measurement of RNA transcript levels can be accessed via RNA-Seq, an efficient method for sequencing the transcriptome using next-generation sequencing (NGS) technology. Once transcript levels are quantified and normalized, upregulated or downregulated genes in the transcriptome in response to ASMO1 treatment can be investigated (thus measuring differential expression). This differential expression analysis allows for deeper insights into the molecular mechanisms of action, theoretical safety assessments, and potential side effects of the therapeutic intervention.

[0428] Against this backdrop, identifying genes directly or indirectly affected by ASMO1 (APT20TTMG) may be crucial for understanding its potential role in target cells such as neurons. To this end, we selected iPSC-derived neurons from healthy patients and patients diagnosed with Alzheimer's disease to be RNA-seq-treated and untreated with APT20TTMG, and performed differential gene expression analysis to answer desired biological questions. The aim of this approach was to identify key differences in gene expression and related pathways in iPSC-derived neurons from healthy patients (HDC) and patients diagnosed with Alzheimer's disease (ADC) incubated with two concentrations of ASMO1 (APT20TTMG) compared to the untreated control. iPSC-derived neurons from healthy patients (HDC) and Alzheimer's disease patients (ADC) were cultured for 6 days, then cultured for 7 days (day 13) with treatments (APT20TTMG-18.52 nM, APT20TTMG-500 nM, or medium only). It is worth noting that this treatment time point was chosen to investigate upstream events that lead to disease development (before the increase of APP and MAPT in ADCs compared to HDCs), and thus to assess the effects of APT20TTMG on ADC cells prior to the formation of the disease phenotype.

[0429] Following RNA extraction, libraries were generated from 250 ng of total RNA as follows: mRNA enrichment was performed using a NEBNext Poly(A) magnetic separation module. cDNA synthesis was performed using the NEBNext RNA first-strand synthesis module and the NEBNext Ultra Directional RNA second-strand synthesis module. The remaining library preparation steps were performed using the NEBNext Ultra II DNA Library PrepKit for Illumina. Libraries were normalized and merged, then denatured in 0.02 N NaOH and neutralized with HT1 buffer. Libraries were loaded at 175 pM onto NovaSeq S4 lanes using the Xp protocol, and all sample libraries were sequenced using a NovaSeq 6000 (Illumina). Two x 100 cycles (paired-end mode) were run. The phiX library was used as a control and mixed with the libraries at a 1% level. Base calling was performed using RTA v3. The samples were then demultiplexed and FASTQ reads were generated using the bcl2fastq2 v2.20 program. The reads will be paired at both ends, with a read length of 101, aiming for 100 million reads per sample. The experiment will be conducted in 3 replicates and 1 replicate.

[0430] The quality of the raw reads (FASTQ files) was assessed using FASTQC v0.11.8 in conjunction with MultiQC. After reviewing the quality of the raw reads, no pruning was deemed necessary. The reads were aligned to the human reference genome using STAR v2.7.6a. The GTF annotation file for the human reference genome was downloaded from Ensembl (version 102). Raw counts were calculated using FeatureCounts v1.6.0. Differential expression analysis was performed using the DESeq2 R package to identify differentially expressed genes (DEGs). Five main comparisons were performed, each with its own DEG group: (i) ADC treated with 18.52 nM APT 20 TMG compared to untreated ADC (medium only); (ii) ADC treated with 500 nM APT 20 TMG compared to untreated ADC (medium only); (iii) HDC treate...

Claims

1. A method of treating a neurodegenerative disease in a subject of need, comprising administering to the subject a pharmaceutical composition comprising an engineered polynucleotide, said 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. The method according to claim 1, wherein the neurodegenerative disease is selected from: Alzheimer's disease, Pick's disease, chronic traumatic encephalopathy, progressive supranuclear palsy, auricula-judae disease, amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD).

3. The method of claim 1, wherein the neurodegenerative disease is a neurodegenerative disease associated with the presence or dysfunction of Tau accumulation or U1 snRNP accumulation or dysfunction in the brain of the subject.

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

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

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

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

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

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

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

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

12. A method for reducing Tau expression and / or aggregation in neurons, the method comprising: The engineered polynucleotide is administered to the neuron, 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 that may be a conserved splicing site 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.

13. A method for reducing amyloid-β expression and / or aggregation in neurons, the method comprising: The engineered polynucleotide is administered to the neuron, 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 that may be a conserved splicing site 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.

14. A method for reducing U1-70K aggregation and / or mislocalization in neurons, the method comprising: The engineered polynucleotide is administered to the neuron, 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.

15. A method for regulating astrocytosis, the method comprising: The engineered polynucleotide is administered to the neuron, 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.

16. A method for increasing fiber width in a neuron, 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 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.

17. A method for increasing the number of branches and branch connections of a neuron, 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 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.

18. A method for increasing total axonal material in a neuron, 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 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.

19. A method for increasing the electrical activity of neurons, the method comprising: The engineered polynucleotide is administered 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 the messenger ribonucleic acid precursor (mRNA precursor); and (ii) The fundraising portion, which is configured as a fundraising splice portion, wherein... When bound to the mRNA precursor and the engineered polynucleotide, the spliceosome portion alters the mRNA precursor in or near the target sequence.

20. The method according to any one of claims 12-19, wherein the neurons are derived from an individual suffering from neurodegenerative disease.

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

22. The method according to any one of claims 12-21, wherein the neuron is a healthy neuron.

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

24. The method according to any one of claims 12-23, wherein the method alters and / or regulates the expression of genes in the neurons.

25. The method of claim 24, wherein the method alters and / or regulates the expression of genes that are part of the MAPK signaling pathway.

26. The method of claim 24, wherein the method alters and / or regulates the expression of genes that are part of a cholesterol metabolism pathway.

27. The method of claim 24, wherein the method alters and / or regulates the expression of genes that are part of the steroid biosynthetic pathway.

28. The method of claim 24, wherein the method alters and / or regulates the expression of genes associated with mRNA transport.

29. The method of claim 24, wherein the method alters and / or regulates the expression of genes as part of cell cycle regulation or cell senescence pathways.

30. The method of claim 24, wherein the method alters and / or regulates the expression of genes that are part of the pyruvate metabolic pathway.

31. The method of claim 24, wherein the method alters and / or regulates the expression of genes that are part of the Ras signaling pathway.

32. The method of claim 24, wherein the method alters and / or regulates the expression of genes that are part of the AMPK signaling pathway.

33. The method of claim 24, wherein the method alters and / or regulates gene expression as part of a fatty acid metabolic pathway.

34. The method of claim 24, wherein the method alters and / or regulates the expression of genes that are part of the fatty acid synthesis pathway.

35. The method of claim 24, wherein the method alters and / or regulates the expression of genes that are part of the PPAR signaling pathway.

36. The method of claim 24, wherein the method alters and / or regulates the expression of genes that are part of the P13K-Akt signaling pathway.

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

38. The method according to any one of claims 12-37, wherein the method reduces the excitotoxicity of the neurons.

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

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

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

42. The method according to any one of claims 12-39, wherein the method reduces covert splicing of one or more transcripts of the neuron.

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

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

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

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

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

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

49. The method according to any one of claims 1 to 45, 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.

50. The method according to any one of claims 1 to 49, 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.

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

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

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

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

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

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

57. The method according to any one of claims 56, wherein the method alters the expression or activity of the target gene.

58. The method according to claims 1 to 57, 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.

59. The method of claim 53, wherein the first target sequence comprises a common sequence in the target sequences.

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

61. The method according to any one of claims 53 to 60, wherein the first and second target sequences are spacer sequences in the target sequence that are separated by no more than five nucleotides (e.g., one or two nucleotides).

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

63. The method of claim 62, wherein both the first and second target sequences are located at the 5' or 3' of the exon-intron boundary.

64. The method of claim 62, 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.

65. The method according to any one of claims 1 to 64, wherein the target sequence comprises a splicing site in the mRNA precursor.

66. The method of claim 65, wherein the first or second target sequence comprises a splicing site (e.g., 5'ss) in the mRNA precursor.

67. The method according to any one of claims 1 to 66, 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.

68. The method of claim 67, 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.

69. The method of claim 67, 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.

70. The method of claim 67, 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.

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

72. The method of claim 67, 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).

73. The method of claim 67, 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.

74. The method of claim 67, 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.

75. The method according to any one of claims 1 to 74, wherein the first targeting portion comprises at least 80%, 90%, or the same sequence as the ribosome binding site of the spliceosome snRNA, for example, U1 snRNA.

76. The method according to any one of claims 1 to 75, wherein the second targeting portion comprises at least 80%, 90%, or the same sequence as the ribosome binding site of the spliceosome snRNA, for example, U1 snRNA.

77. The method according to any one of claims 75 or 76, 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.

78. The method according to any one of claims 1 to 74, 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.

79. The method of claim 78, wherein the spliceosome portion comprises U1snRNA and spliceosome protein.

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

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

82. The method according to any one of claims 1 to 81, 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.

83. The method of claim 82, wherein the recruitment portion comprises a nucleotide sequence that is identical or complementary to any one of SEQ ID No: 1-2.

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

85. The method of claim 83, wherein the engineered polynucleotide comprises a top loop, an upper stem, an inner loop, a lower stem, or a combination thereof.

86. The method of claim 83, 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.

87. The method of claim 86, 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.

88. The method of claim 85 or 86, wherein 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).

89. The method of claim 88, wherein the inner loop comprises a nucleic acid sequence of no more than 10, 9, or 8 nucleotides.

90. The method of claim 88 or 89, wherein the stem sequence of the other stem (e.g., the upper stem) comprises no more than about five, four, or three nucleotides.

91. The method according to any one of claims 88 to 90, wherein the engineered polynucleotide further comprises a top loop.

92. The method of claim 91, wherein the top loop comprises a nucleic acid sequence of no more than 10, 9, 8, 7, 6 or 5 nucleotides.

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

94. The method according to any one of claims 1 to 93, 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.

95. The method according to any one of claims 1 to 94, 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.

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

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

98. The method of claim 96 or 97, 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).

99. The method according to any one of claims 96 to 98, 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).

100. The method according to any one of claims 96 to 99, wherein the recruitment portion comprises a nucleotide sequence containing an inter-nucleotide linker of a phosphate thioester that binds to a U1-C zinc finger.

101. The method according to any one of claims 1 to 100, wherein the engineered polynucleotide comprises a nucleotide sequence complementary to a partial sequence of the stem-loop II (SL2) of the U1snRNA.

102. The method of claim 101, 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.

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

104. The method according to any one of claims 101 to 103, wherein the partial sequence does not contain a sequence corresponding to the 5'-CACGUUA-3' of SL2 of U1snRNA.

105. The method according to any one of claims 1 to 104, wherein the recruitment portion is complementary to the stem-loop-II region of snRNA, such as U1 snRNA.

106. The method according to any one of claims 1 to 105, wherein the recruitment portion hybridizes with the stem-loop-II region of snRNA, such as U1 snRNA.

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

108. The method according to any one of claims 1 to 107, 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.

109. The method according to any one of claims 1 to 108, wherein 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.

110. The method according to any one of claims 1 to 104, 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.

111. The method according to any one of claims 1 to 104, wherein the engineered polynucleotide substantially does not exhibit base pairing with the anchoring sequence of SL2 of U1 snRNA.

112. The method of claim 111, 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.

113. The method according to claim 111 or 112, wherein the lower stem of said engineered polynucleotide does not substantially exhibit base pairing with the anchoring sequence of said SL2 of U1 snRNA.

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

115. The method according to any one of claims 1 to 114, wherein the engineered polynucleotide substantially does not exhibit base pairing with the H-helix of U1 snRNA.

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

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

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

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

120. The method according to any one of claims 118 or 119, wherein the 2'-modified nucleotide comprises 2'-methoxy, 2'-methoxymethyl, 2'-methoxyethyl, 2'-fluorine, or 2'-aminoethyl nucleotide.

121. The method according to any one of claims 116 to 120, wherein the engineered polynucleotide comprises nucleotides linked by internucleotide bonds, and at least one of the internucleotide bonds does not contain a phosphate ester.

122. The engineered polynucleotide according to claims 116 to 121, 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 (C); or NR2, wherein each R is independently selected from hydrogen, alkyl and aryl.

123. The method according to any one of claims 116 to 122, wherein the engineered polynucleotide comprises at least one phosphate thioester nucleotide bond.

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

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

126. The method according to any one of claims 116 to 125, 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.

127. The method according to any one of claims 1 to 126, 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.

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

129. The method according to any one of claims 1 to 128, 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.

130. The method according to any one of claims 1 to 129, 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.

131. The method according to any one of claims 1 to 130, wherein when bound to the engineered polynucleotide and the mRNA precursor, the spliceosome partially cleaves or splices the mRNA precursor in the target sequence.

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

133. The method according to any one of claims 1 to 132, 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.

134. The method according to any one of claims 1 to 133, wherein the engineered polynucleotide comprises a nucleotide sequence that is identical or complementary to any one of SEQ ID NO: 3 or 4.

135. The method according to any one of claims 1 to 134, 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'.

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

137. The method of claim 136, wherein all internucleotide bonds of the engineered polynucleotide comprise phosphate thioester bonds.

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

139. The method according to any one of claims 136 to 138, wherein the mRNA precursor comprises tau, and the method reduces the level of tau protein in the brain.

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