Compositions for modulating CD33 expression

Abstract

Provided herein are compositions and methods for modulating CD33 expression. In some instances, compositions and methods are used to treat a neurodegenerative disorder. In some instances, the compositions comprise antisense oligonucleotides.

Description

COMPOSITIONS FOR MODULATING CD33 EXPRESSIONCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U. S. Provisional Application No. 63 / 613,551, filed December 21, 2023 which is incorporated herein by reference in its entirety.BACKGROUND

[0002] Neurodegenerative disorders are an umbrella term for diseases that, by conservative estimates, shorten the lifespan and significantly decrease the quality of life of >7.5 million individuals in the US and >80 million individuals globally. Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are among the most severe age-related neurodegenerative disorders with increasing prevalence in the aging global population that do not have adequate therapeutic options to date. The two diseases can occur separately, but their molecular and genetic causes are intertwined, and some patients can suffer from symptoms of both diseases simultaneously. ALS affects >35,000 individuals just in the US and the typical survival after the disease onset is 2-4 years due to complications from progressive degeneration of motor neurons in the spinal cord, brainstem, and motor cortex that result in paralysis. FTD is a result of progressive neuronal degeneration primarily in the frontal and temporal lobes of the brain. The variants of the disease can initially result in a range of symptoms including behavioral changes, emotional problems, speech and language pathology, apathy, tremor, muscle spasms, and difficulty walking. FTD affects >50,000 individuals in the US and the survival after onset ranges based on the disease variant from 2.5 years in patients with FTD-ALS to 8-12 years for patients with behavioral variant FTD or primary progressive aphasia. The common pathologic observations in patients with ALS, FTD, and related dementias including Parkinson’s (PD) and Alzheimer’s disease (AD) include elevated presence of protein aggregates associated with neuroinflammation and neuronal death. Importantly, hundreds of genetic mutations are associated with the both ALS and FTD and multiple types of protein aggregates are being observed in the tissue of the patients including TDP-43 (TARDBP), SOD1, FUS, and tau (MAPT) highlighting importance of either targeting multiple dysfunctions or modulating common pathologic pathways relevant in the symptomatic patients. Improved therapeutics are needed for treating these disorders.SUMMARY OF DISCLOSURE

[0003] Provided herein are compositions and methods for modulating CD33 expression. In some instances, compositions and methods are used to treat a neurodegenerative disorder. In some instances, a neurodegenerative disorder comprises neuroinflammation or neuronal degeneration.Provided herein, in some embodiments, is a compound comprising a modified oligonucleotide. In some embodiments, the modified oligonucleotide consists of 12 to 30 linked nucleotides and comprises a nucleic acid sequence complementary to at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, atleast 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1 , 2, or 3 mismatches from any one of SEQ ID NOs: 1 or 2. In some embodiments, the modified oligonucleotide comprises a nucleic acid sequence complementary to at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13 , at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1, 2, or 3 mismatches to a target sequence in positions 1-21, 29- 48, 94-116, 137-187, 211-241, 263-282, 392-417, 416-435, 456-523, 460-479, 504-671, 652-671, 683-721, 724-746, 730-753, 731-756, 770-819, 772-807, 857-876, 880-899, 1050-1138, 1073- 1108, 1165-1184, 1189-1266, 1194-1276, 1250-1334, 1270-1360, 1285-1332, 1336-1360, 1340- 1360, 1364-1383, 1388-1452, or 1370-1462 of SEQ ID NO: 1. In some embodiments, the modified oligonucleotide comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1, 2, or 3 mismatches from any one of nucleic acid sequences of SEQ ID NOs: 123-432. In some embodiments, the nucleic acid sequence of the modified oligonucleotide is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of the nucleic acid sequences of SEQ ID NOs: 123-432. In some embodiments, the modified oligonucleotide is a single -stranded modified oligonucleotide. In some embodiments, the modified oligonucleotide comprises a gapmer. In some embodiments, the modified oligonucleotide comprises: a gap segment consisting of 5-15 linked deoxynucleotides; a 5 ’ wing segment consisting of 4-10 linked nucleotides; and a 3 ’ wing segment consisting of 4-10 linked nucleotides. In some embodiments, the gap segment is positioned between the 5 ’ wing segment and the 3 ’ wing segment. In some embodiments, each nucleotide of 5’ and 3 ’ wing segment comprises at least one 2’-modified nucleotide. In some embodiments, the at least one 2 ’-modified nucleotide comprises a 2’-O-methoxyethyl (MOE) modified nucleotide. In some embodiments, the modified oligonucleotide comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1, 2, or 3 mismatches from any one of nucleic acid sequences of SEQ ID NOs: 433-742. In some embodiments, the nucleic acid sequence of the modified oligonucleotide is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of the nucleic acid sequences of SEQ ID NOs: 433-742. In some embodiments, the modified oligonucleotide consists of any one of nucleic acid sequences of SEQ ID NOs: 433-742. In some embodiments, the modified oligonucleotide comprises a nucleic acid sequence complementary to at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1, 2, or 3 mismatches to a target sequence in positions 29-48, 392-417, 460-479, 731-756, 772-807, 1073-1108, 1194-1276, 1270-1360, or 1388-1452 of SEQ ID NO: 1. In some embodiments, the modified oligonucleotide comprises a nucleic acid sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1, 2, or 3 mismatches from any one of the nucleobase sequences of SEQ ID NOs: 125, 146, 148-152, 158, 182-186, 189, 191, 193, 197, 203, 222-226, 241-260, 262-266, 271, 272, 274-281, 283, 286, 287, 300, 301, 311-319, 321, 322, 328-335, 344, 345, 347-351, 377-381, 390, 391, 395-397, 400-409, 412, 413, 415-420, or 422. In some embodiments, the modified oligonucleotide comprises a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of the nucleic acid sequences of SEQ ID NOs: 125, 146, 148-152, 158, 182-186, 189, 191, 193, 197, 203, 222-226, 241-260, 262-266, 271, 272, 274-281, 283, 286, 287, 300, 301, 311 -319, 321, 322, 328-335, 344, 345, 347-351, 377-381, 390, 391, 395-397, 400-409, 412, 413, 415-420, or 422. In some embodiments, the modified oligonucleotide comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13 , at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1, 2, or 3 mismatches from any one of nucleic acid sequences of SEQ ID NOs: 435, 456, 458-462, 468, 492-496, 499, 501, 503, 507, 513, 532-536, 551-570, 572-576, 581, 582, 584-591, 593, 596, 597, 610, 611, 621 -629, 631, 632, 638-645, 654, 655, 657-661, 687-691, 700, 701, 705-707, 710- 719, 722, 723, 725-730, or 732. In some embodiments, the nucleic acid sequence of the modified oligonucleotide is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of the nucleic acid sequences of SEQ ID NOs: 435, 456, 458- 462, 468, 492496, 499, 501, 503, 507, 513, 532-536, 551-570, 572-576, 581, 582, 584-591, 593, 596, 597, 610, 611, 621 -629, 631, 632, 638-645, 654, 655, 657-661, 687-691, 700, 701, 705-707,710-719, 722, 723, 725-730, or 732. In some embodiments, the modified oligonucleotide consists of any one of nucleic acid sequences of SEQ ID NOs: 435, 456, 458-462, 468, 492-496, 499, 501, 503, 507, 513, 532-536, 551 -570, 572-576, 581, 582, 584-591, 593, 596, 597, 610, 611, 621-629, 631, 632, 638-645, 654, 655, 657-661, 687-691, 700, 701, 705-707, 710-719, 722, 723, 725-730, or 732. In some embodiments, the modified oligonucleotide comprises a nucleic acid sequence complementary to at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13 , at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1 , 2, or 3 mismatches to a target sequence in positions 1270-1360, or 1388-1452 of SEQ ID NO: 1. In some embodiments, the modified oligonucleotide comprises a nucleic acid sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1 , 2, or 3 mismatches from any one of the nucleic acid sequences of SEQ ID NOs: 313, 316, 317, 319, 347-349, 402, 417, or 418. In some embodiments, the modified oligonucleotide comprises a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of the nucleic acid sequences of SEQ ID NOs: 313, 316, 317, 319, 347-349, 402, 417, or 418. In some embodiments, the modified oligonucleotide comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, atleast 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1 , 2, or 3 mismatches from any one of nucleic acid sequences of SEQ ID NOs: 623, 626, 627, 629, 657-659, 712, 727, or 728. In some embodiments, the nucleic acid sequence of the modified oligonucleotide is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of the nucleic acid sequences of SEQ ID NOs: 623, 626, 627, 629, 657-659, 712, 727, or 728. In some embodiments, the modified oligonucleotide consists of any one of nucleic acid sequences of SEQ ID NOs: 623, 626, 627, 629, 657-659, 712, 727, or 728.

[0004] Provided herein, in some embodiments, is a compound comprising a modified oligonucleotide. In some embodiments, the modified oligonucleotide consists of 12 to 30 linked nucleotides and comprising a nucleic acid sequence complementary to at least 8, at least 9, at least 10, at least 11, at least 12, atleast 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1, 2, or 3 mismatches from SEQ ID NOs: 5 or 6. In some embodiments, the modified oligonucleotide comprises atleast8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1, 2, or 3 mismatches from any one of nucleic acid sequences of SEQ ID NOs: 7-64. In some embodiments, the nucleic acid sequence of the modified oligonucleotide is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of the nucleic acid sequences of SEQ ID NOs: 7-64. In some embodiments, the modified oligonucleotide consists of 12-20 nucleotides in length. In some embodiments, the modified oligonucleotide comprises at least one modified internucleotide linkage, a modified base, or a modified sugar ring. In some embodiments, the at least one modified internucleotide linkage is a phosphorothioate internucleotide linkage. In some embodiments, all intemucleotide linkages in the modified oligonucleotide are phosphorothioate internucleotide linkages. In some embodiments, the modified sugar ring is a bicyclic sugar or a 2' modified sugar ring. In some embodiments, the 2' modified sugar ring comprises a 2'-O-methoxy ethyl sugar ring (2 ’-MOE), 2'- O-methyl sugar ring (2’-OMe), or 2’-fluoro sugar ring (2’-F). In some embodiments, the modified oligonucleotide comprises at least one locked nucleic acid (LNA). In some embodiments, the modified oligonucleotide comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, atleast 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1 , 2, or 3 mismatches from any one of nucleic acid sequences of SEQ ID NOs: 65-122. In some embodiments, the nucleic acid sequence of the modified oligonucleotide is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of the nucleic acid sequences of SEQ ID NOs: 65-122. In some embodiments, the modified oligonucleotide comprises at least 8, at least 9, at least 10, at least 11, atleast 12, at least 13, atleast 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1 , 2, or 3 mismatches from any one of nucleic acid sequences of SEQ ID NOs: 16-22, 32-36, 38-42, or 44. In some embodiments, the nucleic acid sequence of the modified oligonucleotide is at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of the nucleic acid sequences of SEQ ID NOs: 16-22, 32-36, 38-42, or 44. In some embodiments, the modified oligonucleotide comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least15, atleast 16, atleast 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1, 2, or 3 mismatches from any oneof SEQ ID NOs: 74-80, 90-94, 96-100, or 102. In some embodiments, the nucleic acid sequence of the modified oligonucleotide is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of nucleic acid sequences of SEQ ID NOs: 74-80, 90-94, 96-100, or 102. In some embodiments, the modified oligonucleotide consists of to any one of nucleic acid sequences of SEQ ID NOs: 74- 80, 90-94, 96-100, or 102.

[0005] Provided herein, in some embodiments, is a pharmaceutical composition comprising the compound of any one of the preceding compounds and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is formulated for parenteral administration.

[0006] Provided herein, in some embodiments, is a method of treating a neurological disease or disorder. In some embodiments, the method comprises administering a therapeutically effective amount of a compound or pharmaceutical composition of any one of the preceding compounds to a subject in need thereof. In some embodiments, the neurological disease or disorder comprises neuroinflammation or neurodegeneration. In some embodiments, the neurological disease or disorder comprises amyotrophic Lateral Sclerosis (ALS), frontotemporal dementia (FTD), Parkinson’s disease (PD), or Alzheimer’s disease (AD). In some embodiments, FTD comprises a hereditary disease. In some embodiments, FTD comprises amyotrophic lateral sclerosis (FTD- ALS), behavioral variant FTD (bvFTD), primary progressive aphasia (PPA), progressive supranuclear palsy or the corticobasal syndrome. In some embodiments, treating results in prevention, slows progression, or reverses the neurodegenerative disease or disorder.

[0007] Provided herein, in some embodiments, is a method of downregulating the expression of CD33 comprising administering a therapeutically effective amount of a compound or pharmaceutical composition of any one of the preceding compounds.

[0008] Provided herein, in some embodiments, is a method of modulating the expression of one or more CD33 isoforms. In some embodiments, the method comprises administering a therapeutically effective amount of a compound or pharmaceutical composition of any one of the preceding compounds. In some embodiments, the compound or the pharmaceutical composition induces exon skipping. In some embodiments, the compound or the pharmaceutical composition increases the relative ratio of CD33 mRNA in which exon 2 is absent compared to CD33 mRNA in which exon 2 is present. In some embodiments, the compound or the pharmaceutical composition increases the relative ratio of CD33 mRNA without exon 2 (e.g., SEQ ID NOs: 3 or4) relative to CD33 mRNA comprising exon 2. In some embodiments, the compound or the pharmaceutical composition is configured to bind an intron, an intron adjacent to exon 2, or a 5’ or 3’ flanking region of exon 2. In some embodiments, administering comprises parenteral administration. In some embodiments, parenteral administration comprises intrathecal or intravenous (IV) administration.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0010] FIG. 1A depicts a comparison of normalized CD33 RNA expression in ALS samples (right) versus non-neurological control (left). A significant difference of p<0.001 was found between the two populations. This indicates a higher RNA expression level in the post-mortem cervical spinal cord tissue of ALS samples than healthy controls. The y-axis is labeled RNA expression (normalized counts) from 0 to 12 and 2 unit intervals.

[0011] FIG. IB depicts a correlation between CD33 RNA levels in the cervical spinal cord of ALS patients and their survival (in months). A significant correlation of p<0.007 was shown that demonstrated higher CD33 RNA levels correlated with a shorter survival length. The x-axis is labeled survival (months) from 0 to 132 at 12 unit intervals, and the y-axis corresponds to mRNA levels and is labeled from 0 to 12 at 2 unit intervals.

[0012] FIG. 2A depicts the expression of CD33 in U937 cell line with CDS 3 gene knockout. Live U937 wild-type (WT) and CD33 knockout (CD33 KO) cells were stained with fluorescently labeled CD33 antibodies and analyzed by flow cytometry. Shown is CD33 expression on WT cells (left panel) and CD33 KO cells (right panel).

[0013] FIG. 2B depicts a series of graphs from RNA-seq profiling showing decrease of the indicated inflammatory markers in CD33 knockout U937 cells. CDS 3, IL1B, SPP1, CXCL1, and CHI3L2 expression was significantly lower in CD33 KO cells compared to WT cells.

[0014] FIG. 3A depicts a schematic of the alternative splicing isoform induced by splice switch oligonucleotide (SSO) regulation for exon 2. The SSO binds pre-mRNA. The mRNA is then processed into the exon inclusion isoform (left) or alternative splicing isoform (right, not containing exon 2) and expressed as the full length or truncated protein form.

[0015] FIG. 3B depicts results of an SSO screen to induce exon skipping. Selected SSOs were evaluated based on the relative molarity of the inclusion isoform. The x-axis is labeled SSOs; the y-axis is labeled relative molarity of inclusion iso from 0.0 to 1.5 at 0.25 unit intervals. Theselected SSO hits inducing more than 50% exon 2 skipping are marked as empty circles, the selected SSO hits inducing less than 50% but more than 20% exon 2 skipping are marked as half- full circles, and the SSOs inducing less than 20% exon 2 skipping are marked as filled circles.

[0016] FIG. 3C depicts an image of a representative electropherogram of samples after treatment with SSOs SO 1 -008 (SEQ ID NO: 75, lane 2), SO 1-010 (SEQ ID NO: 90, lane 3), S02-023 (SEQ ID NO: 99, lane 4), or non-treated (NT, lane 1). Lane one represents the ladder depicting bands for 50, 100, 200, 300, 400, 500, 700, and 1000 bp. The lower band near 300 is indicative of the exon-2 skipped isoform.

[0017] FIG. 3D depicts a change in CD33 protein isoform expression following a single dose treatment with 10 pM S02-023 (SEQ ID NO: 99) in microglia for 9 days. The left panel illustrates a schematic of the use of two anti-CD33 antibodies targeting different epitopes to detect the full-length long isoform and the truncated isoform. Induced pluripotent stem cells (iPSC)-derived differentiated microglia were stained with an anti-CD33 antibody recognizing both the full-length and the truncated isoform (FITC) and an antibody recognizing exclusively the long isoform (APC). FACS plots show the percentage of the truncated isoform in non-treated and S02-023 (SEQ ID NO: 99 treated microglia.

[0018] FIG. 4 A depicts a series of graphs from RNA-seq profiling showing decrease of the indicated inflammatory markers in iPSC-derived microglia treated with 10 pM S02-023 (SEQ ID NO: 99) for 9 days. The expression levels of SPP1, CHIU, CHI3L1 were significant downregulated in treated cells compared to non-treated cells.

[0019] FIG. 4B depicts a graph of uptake kinetics of pHrodo-labeled beta-amyloid aggregates by microglia during the indicated time course. iPSC-derived microglia treated with S02-023(SEQ ID NO: 99) exhibited enhanced phagocytosis of beta-amyloid aggregates compared to non-treated microglia (NT).

[0020] FIG. 5A depicts a series of graphs from RNA-seq profiling showing decrease of inflammatory and neurotoxic markers, and increase of homeostatic phagocytosis markers after treatment with 10 pM of the CD33 SSO S01-008 (SEQ ID NO: 75) (right) or NT control (left). The graphs are titled (left to right) IL1B, CI S, CYBB, SORL1, and CD36.

[0021] FIG. 5B depicts graphs (individual 1, 2, and 3) of 10 pM CD33 SSO S01-008 (SEQ ID NO: 75) treatment which resulted in increased phagocytic activity by human monocyte -derived microglial-like cells in vitro. The x-axis represents time from 0 to 24 hrs; the y-axis is labeled Relative cumulative phagocytosis (pHrodo, FI) from 0 to 1.

[0022] FIG. 6A depicts a schematic depicting an anti-sense gapmer oligonucleotide that recruits endogenous RNase-H to degrade the target CD33 transcript.

[0023] FIG. 6B depicts results of an ASO screen to induce CD33 transcript degradation. Phorbol 12-myristate 13 -acetate (PMA)-differentiated adherent U937 cells were treated with a single dose of 5 pM antisense oligonucleotides (ASO) for 72 hrs before the cells were harvested for mRNA isolation and qRT-PCR analysis to quantify CD33 transcript level using 3 non-overlapping Taqman probes for each sample. The x-axis is sequential ASOs; the y-axis is labeled CD33 transcript log2 fold-change from -3.5 to 2.0 at 0.5 unit intervals. The ASO hits inducing more than 75% transcript downregulation are marked as empty circles, the ASO hits inducing less than 75% but more than 50% transcript downregulation are marked as half -full circles, and the ASOs inducing less than 50% transcript downregulation are marked as filled circles.

[0024] FIG. 6C depicts sigmoidal dose-response curves of ASO hits. The dose-dependent activity of the selected hits was validated using qPCR to measure CD33 RNA levels in PMA- differentiated U937 cells treated with 8 different doses of the ASOs: 0.002 pM, 0.005 pM, 0.02 pM, 0.05 pM, 0.2 pM, 0.5 pM, 2 pM, and 5pM. The two gapmers (SEQ ID NO: 659 and SEQ ID NO: 727) were selected by curve fit analysis.

[0025] FIG. 6D depicts a graph of alteration in CD33 expression in microglia following a single treatment with 5pM gapmer ASO (SEQ ID NO: 659). CD33 staining was performed on the indicated days post treatment to monitor changes over time.

[0026] FIG. 7A depicts a series of graphs from RNA-seq profiling showing decrease of the indicated inflammatory markers in iPSC-derived microglia following a single dose treatment with 5pM gapmer ASO (SEQ ID NO: 659) or 5pM gapmer ASO (SEQ ID NO: 727) for 9 days. Significant downregulation was observed in the expression of CD33, SPP1 , CHI3L1, CHIT1, MMP9 in treated cells compared to non-treated cells.

[0027] FIG. 7B depicts graphs of inflammatory factor secretion by microglia treated with a gapmer ASO (SEQ ID NO: 659) and stimulated with TAR DNA-binding protein 43 (TDP-43). ELISA was used to measure the levels of IL1B and CHI3L1 secreted by microglia with or without 5 pM gapmer ASO (SEQ ID NO: 659) treatment, with or without the stimulation of with 5nM TDP-43 for 24 hours.

[0028] FIG. 7C depicts a graph of uptake kinetics of pHrodo-labeled beta-amyloid aggregates by iPSC-derived microglia over the indicated time course. Increased phagocytosis of pHrodo- labeled beta-amyloid aggregates was observed in microglia treated with gapmer ASOs (SEQ ID NO: 659 or SEQ ID NO: 727) compared to non-treated microglia (NT).

[0029] FIG. 7D depicts a graph of the measurement of neurofilament light (NFL) release into cell culture media. iPSC-derived motor neurons and microglia were co-cultured and stimulated with lOOng / m lipopolysaccharide (LPS). The co-culture was treated with either a single 5pM gapmer ASO (SEQ ID NO: 659) or control ASO, and NFL levels were assessed. Shown are NFLlevels in neurons from co-cultures treated with control ASO or a gapmer ASO (SEQ ID NO: 659).DETAILED DESCRIPTION

[0030] Provided herein are compositions and methods for modulating CD33 expression. In some instances, compositions and methods are used to treat a neurodegenerative disorder. In some instances, a neurodegenerative disorder comprises neuroinflammation or neuronal death (neurodegeneration). In some instances, modulation of CD33 results in prevention, slowing progression, or reversal of a neurodegenerative disorder. In some instances, CD33 expression is modulated by a composition comprising an antisense oligonucleotide (ASO) or a spliceswitching oligonucleotide (SSO).

[0031] In one aspect, provided herein are therapeutic modalities to augment or interfere with a cellular process that causes a disease or pathological symptoms. In some embodiments, the therapeutic modalities described herein are CD33 inhibitors. In some embodiments, a therapeutic modality, composition, or compound described herein includes an oligonucleotide-based therapeutics including, but not limited to, an antisense oligonucleotide or a splice-switching oligonucleotide. In some instances, a therapeutic modality, composition, or compound described herein is an antisense oligonucleotide or a splice-switching oligonucleotide. In some cases, the therapeutic modality, composition, or compound is a small molecule. As used herein, the terms “therapeutic modality”, “composition”, and “compound”, are used interchangeably.

[0032] Also provided herein, includes compositions and methods that facilitate the manufacture, storage, administration, and delivery of such a therapeutic modality, composition, or compound. In some embodiments, these modalities deliver oligonucleotide-based therapeutics to a therapeutically significant percentage of the affected cells in a manner that is both efficient and safe. The delivered oligonucleotide-based therapeutics can provide a beneficial function, for example, by modulating (e.g., blocking or inhibiting) activity or expression of an endogenous gene or by compensating a function of a missing endogenous gene. If these materials are appropriately delivered to a patient, they can potentially enhance a patient’s health and, in some instances, lead to a cure.Therapeutic Target - CD33

[0033] CD33 RNA expression levels are found upregulated in post-mortem tissue of amyotrophic lateral sclerosis (ALS) and ALS-FTD (clinical continuum of ALS and frontotemporal dementia (FTD)) patients and negatively correlate with the patient survival. Distribution of a common SNP rl 2459419 in the second coding exon of CD33 is co-inherited with an intergenic SNP rs3865444 that was initially identified to decrease the risk ofneurodegenerative diseases including Alzheimer’s disease (AD) and Parkinson’s disease (PD). The OT substitution in rl2459419 decreases inclusion of an exon encoding most of the extracellular domain of the receptor necessary for ligand binding and inhibition of phagocytosis. Without being bound by any specific theory, the splicing-dependent decrease of CD33 protein function, in some instances, improves the microglial phagocytic function. Yet, after chronic disease onset, the modest splicing-dependent protection of microglia is overridden by the elevated CD33 transcription which may become the dominant factor negatively affecting the survival.

[0034] Human CD33 gene is located on chromosome 19ql3.41 andthe Ensembl Gene ID for the human CD33 gene is ENSG00000105383. CD33 is expressed from the genome as a series of isoforms through alternative splicing. Table 1 provides two transcript variants of CD33 mRNA - SEQ ID NO: 1 (NM_001772.4) and SEQ ID NO: 2 (NM_001082618.2). The underlined region of SEQ ID NO:1 in Table 1 corresponds to exon 2. SEQ ID NOs: 3 and 4 represent splicing variants of CD33 which do not comprise exon 2.Table 1Therapeutic Compositions and Compounds

[0035] In some aspects, the therapeutic modality, composition, or compound modulates the expression, activity, or splicing of the CD33 mRNA transcript. In some aspects, the therapeutic modality, composition, or compound suppresses or reduces the expression or activity of the CD33 mRNA transcript. In some aspects, the therapeutic modality, composition, or compound suppress or enhances expression of a certain splicing variant of the CD33 mRNA transcript. In some aspects, the therapeutic modality, composition, or compound facilitates an exon skipping of the CD33 mRNA transcript.

[0036] In some aspects, the therapeutic modality, composition, or compound comprises an oligonucleotide. In some instances, the oligonucleotides comprise antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligomeric compounds that hybridize to at least a portion of the target nucleic acid. As such, these compounds may be introduced in the form of single -stranded, double-stranded, partially single-stranded, or circular oligomeric compounds. In some instances, certain antisense oligonucleotide is a splice switching oligonucleotide.

[0037] In some embodiments, oligonucleotides, particularly antisense oligonucleotide compounds, bind to target nucleic acid molecules (e.g., DNA or RNA) and modulate the expression and / or function of molecules encoded by the target nucleic acid molecules. The functions of DNA to be interfered with comprise, for example, replication and transcription. The functions of RNA to be interfered with comprise all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and / or catalytic activity which may be engaged in or facilitated by the RNA. The functions may be up -regulated or down-regulated (e.g., inhibited) depending on the functions desired. In certain instances, such compounds are useful as research tools, diagnostic reagents, and / or as therapeutic agents.

[0038] In some instances, the oligonucleotides are DNA-like oligonucleotides (e.g., DNA analogue). In some instances, the oligonucleotide are RNA-like oligonucleotides (e.g., RNA analogue). Certain DNA-like oligomeric compounds can reduce protein expression. Certain RNA-like compounds can inhibit protein expression in cells. Such RNA-like compounds function, at least in part, through the RNA-inducing silencing complex (RISC). RNA-like compounds can be single-stranded or double-stranded. In some instances, antisense oligonucleotide compounds can alter processing of pre-mRNA and to modulate non-coding RNA molecules. In certain instances, antisense oligonucleotide compounds can modulate protein expression by binding to a target messenger RNA (mRNA) encoding the protein.

[0039] In some embodiments, oligonucleotides include RNA, DNA, mimetic, chimera (e.g., DNA-RNA mixture, gapmer, mixmer, etc.), RNA analog or homolog thereof, DNA analog or homolog thereof), ribozymes, external guide sequence (EGS) oligonucleotides, siRNA oligonucleotides, single- or double-stranded RNA interference (RNAi) compounds, saRNA, aRNA, and other oligomeric compounds which hybridize to at least a portion of the target nucleic acid and modulate its function. In some instances, oligonucleotides comprise antisense oligonucleotides, which are DNA, RNA, DNA-like, RNA-like, DNA-RNA mixtures thereof, mimetics or chimera of DNA, RNA, DNA-like, and RNA-like molecules. In general, nucleic acids (including oligonucleotides) may be described as "DNA-like" (i.e., generally having one or more 2'-deoxy sugars and, generally, T rather than U bases) or "RNA-like" (i.e., generally having one or more 2'-hydroxyl or 2'-modified sugars and, generally U rather than T bases). Nucleic acid helices can adopt more than one type of structure, e.g., the A- and B-forms. In some cases, oligonucleotides which have B-form-like structure are "DNA-like" and those which have A- form-like structure are "RNA-like." In some (chimeric) embodiments, an antisense compound may contain both A- and B-form regions. Thus, the oligonucleotides can be composed of naturally occurring nucleotides, sugars and covalent intemucleotide (backbone) linkages as wellas oligonucleotides having non -naturally occurring portions which function similarly. Such modified or substituted oligonucleotides may have properties such as, for example, enhanced cellular uptake, enhanced affinity for a target nucleic acid, and / or increased stability in the presence of nucleases.

[0040] Once introduced to a system, the compounds (e.g., oligonucleotides) may elicit the action of one or more enzymes or structural proteins to effectuate cleavage or other modification of the target nucleic acid or may work via occupancy -based mechanisms.

[0041] The oligonucleotides can be single-stranded, double-stranded, circular or hairpin oligomeric compounds and can contain structural elements such as internal or terminal bulges, mismatches or loops. For example, antisense compounds may be linear, but can be joined or otherwise prepared to be circular and / or branched. The oligonucleotides can include constructs such as, for example, two strands hybridized to form a wholly or partially double -stranded compound or a single strand with sufficient self-complementarity to allow for hybridization and formation of a fully or partially double-stranded compound. The two strands can be linked internally leaving free 3' or 5' termini or can be linked to form a continuous hairpin structure or loop. The hairpin structure may contain an overhang on either the 5' or 3' terminus producing an extension of single stranded character. The double stranded compounds optionally can include overhangs on the ends of one or both of the strands. Further modifications can include conjugate groups attached to one of the termini, selected nucleotide positions, sugar positions or to one of the internucleotide linkages. In some cases, the two strands can be linked via a non -nucleic acid moiety or linker group. When formed from only one strand, dsRNA can take the form of a self - complementary hairpin-type molecule that doubles back on itself to form a duplex. Thus, the dsRNAs can be fully or partially double stranded. Specific modulation of gene expression can be achieved by stable expression of dsRNA hairpins in transgenic cell lines, however, in some embodiments, the gene expression or function is upregulated. When formed from two strands, or a single strand that takes the form of a self -complementary hairpin-type molecule doubled back on itself to form a duplex, the two strands (or duplex -forming regions of a single strand) are complementary RNA strands that base pair in Watson -Crick fashion. In some embodiments, oligonucleotides comprise at least one of: antisense RNA, antisense DNA, chimeric antisense oligonucleotides, antisense oligonucleotides comprising modified linkages, interference RNA (RNAi), short interfering RNA (siRNA), micro RNA (miRNA), small temporal RNA (stRNA), or short hairpin RNA (shRNA), small RNA-induced gene activation (RNAa), small activating RNAs (saRNAs), or combinations thereof.Antisense Oligonucleotide (ASO)

[0042] In one aspect, an antisense oligonucleotide (ASO) described herein (e.g., RNA, DNA, mimetic, chimera (e.g., DNA-RNA mixture, gapmer, mixmer, etc.), RNA analog or homolog thereof, DNA analog or homolog thereof) is specifically hybridizable to the target nucleic acid, and interferes with the normal function, activity, or utility of the target nucleic acid when binding of the compound to the target nucleic acid to cause a loss of activity. A sufficient degree of complementarity between the ASO molecule and the target nucleic acid is desired to avoid nonspecific binding of the antisense compound to non-target nucleic acid sequences to increase the efficacy of the treatment or accuracy of the in vivo or in vitro assays.

[0043] Targeting the target nucleic acid using an antisense compound to modulate the activity, expression, or function of the target nucleic acid can be a multistep process. In some cases, the process begins with the identification of a target nucleic acid whose function is to be modulated. This target nucleic acid may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In some embodiments, the target nucleic acid encodes CD33.

[0044] Targeting the target nucleic acid using an antisense compound to modulate the activity, expression, or function of the target nucleic acid, in some instances, can include determination of at least one target region, segment, or site within the target nucleic acid for the antisense oligonucleotide to bind such that the desired effect, e.g., modulation of expression, will result from. In some embodiments, "region" is a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic. Within regions of target nucleic acids are segments. In some embodiments, "segments" are smaller or sub -portions of regions within a target nucleic acid. In some embodiments, "sites," are positions within a target nucleic acid.

[0045] DNA-RNA and RNA-RNA hybridizations are important to many aspects of nucleic acid function, including DNA replication, transcription, and translation. Hybridization is also central to a variety of technologies that either detect a particular nucleic acid or alter its expression. In some instances, antisense oligonucleotides, for example, disrupt, inhibit, or modify gene expression by hybridizing to target RNA, thereby interfering with RNA splicing, transcription, and translation. Antisense DNA molecule can form DNA-RNA hybrids with the endogenous RNA molecule or fragments thereof to so serve as a substrate for digestion by ribonuclease H, an activity that is present in most cell types. Antisense oligonucleotide molecules can be delivered into cells, as is the case for oligodeoxynucleotides (ODNs), or they can be expressed from endogenous genes as RNA molecules.Double-stranded siRNA

[0046] Single stranded RNA sequences provided herein can hybridize with a second RNA strand to form a double stranded oligonucleotide. RNA interference or "RNAi" describes the observation that double-stranded RNAi (dsRNA) can block gene expression upon hybridization with the target RNA molecule. In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand. In one aspect of the disclosure, provided herein is a therapeutic mechanism utilizing RNAi approaches designed to inhibit translation of or reduce expression of CD33 mRNA by degrading CD33 mRNA. In one example, the RNAi based therapeutic modalities include siRNA targeting of CD33 resulting in RISC mediated mRNA cleavage and exonuclease degradation, and / or miRNA targeting resulting in inhibition of translation and / or degradation by exonucleases. RNAi can be mediated by dsRNA molecules that have sequencespecific homology to their target nucleic acid sequences. In certain embodiments, the mediators are 5-25 nucleotide "small interfering" RNA duplexes (siRNAs). The siRNAs are derived from the processing of dsRNA by an RNase enzyme known as Dicer. siRNA duplex products are recruited into a multi-protein siRNA complex termed RISC. Without wishing to be bound by any specific theory, a RISC is then believed to be guided to a target nucleic acid (suitably mRNA), where the siRNA duplex interacts in a sequence-specific way to mediate cleavage in a catalytic fashion. Small interfering RNAs that can be used in accordance with the present embodiments can be synthesized and used to inhibit translation of or degrade CD33 mRNA. In some embodiments, small interfering RNAs for use in the methods herein comprise between about 1 to about 50 nucleotides (nt). In examples of non-limiting embodiments, siRNAs can comprise about 5 to about 40 nt, about 5 to about 30 nt, about 10 to about 30 nt, about 15 to about 25 nt, or about 20-25 nucleotides. In some embodiments, provided are siRNA compounds that are dsRNA agents capable of inhibiting the expression of CD33.Ribozyme

[0047] Enzymatic nucleic acid molecules (e.g., ribozymes) are nucleic acid molecules capable of catalyzing one or more of a variety of reactions, including the ability to repeatedly cleave other separate nucleic acid molecules in a sequence-specific manner. Such enzymatic nucleic acid molecules can be used, for example, to target virtually any RNA transcript.

[0048] Because of their sequence-specificity, trans-cleaving enzymatic nucleic acid molecules show promise as therapeutic agents for treating human disease. Enzymatic nucleic acid molecules can be designed to cleave specific RNA targets within the background of cellular RNA. Such a cleavage event renders the mRNA non-functional and abrogates protein expression from that RNA. In this manner, synthesis of a protein associated with a disease state can be selectively inhibited.

[0049] In general, enzymatic nucleic acids with RNA cleaving activity act by first binding to a target RNA. Such binding occurs through the target binding portion of an enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.

[0050] Several approaches such as in vitro selection (evolution) strategies have been used to evolve new nucleic acid catalysts capable of catalyzing a variety of reactions, such as cleavage and ligation of phosphodiester linkages and amide linkages.

[0051] The development of ribozymes that are optimal for catalytic activity can be employed with the embodiments of the present disclosure. The hammerhead ribozyme, for example, functions with a catalytic rate (kcat) of about 1 min in the presence of saturating (10 mM) concentrations of Mg2+cofactor. An artificial "RNA ligase" ribozyme hasbeen shown to catalyze the corresponding self -modification reaction with a rate of about 100 min4. In addition, it is known that certain modified hammerhead ribozymes that have substrate binding arms made of DNA catalyze RNA cleavage with multiple turn-over rates that approach 100 min4. Finally, replacement of a specific residue within the catalytic core of the hammerhead with certain nucleotide analogues gives modified ribozymes that showas much as a 10-fold improvement in catalytic rate. These findings demonstrate that ribozymes can promote chemical transformations with catalytic rates that are significantly greater than those displayed in vitro by most natural self-cleaving ribozymes. It is then possible that the structures of certain self -cleaving ribozymes can be optimized to give maximal catalytic activity, or that entirely new RNA motifs can be made that display significantly faster rates for RNA phosphodiester cleavage.

[0052] In some instances, screening and / or selection of appropriate oligonucleotide modality can be facilitated by a computer program that automatically aligns nucleic acid sequences of the target nucleic acid and indicates regions of identity or homology. Such programs are used to compare nucleic acid sequences obtained, for example, by searching databases such as GenBank or by sequencing PCR products. Comparison of nucleic acid sequences from a range of specie s allows the selection of nucleic acid sequences that display an appropriate degree of identity between species. In the case of genes that have not been sequenced, Southern blots are performed to allow a determination of the degree of identity between genes in target species and other species. By performing Southern blots at varying degrees of stringency, it is possible to obtain anapproximate measure of identity. These procedures allow the selection of oligonucleotides that exhibit a high degree of complementarity to target nucleic acid sequences in a subject to be controlled and a lower degree of complementarity to corresponding nucleic acid sequences in other species. One skilled in the art will realize that there is considerable latitude in selecting appropriate regions of genes. In some embodiments, additional computational methods are applied to ensure the target sequences are specific in the human genome and will not result in off-target effects, to assure drug-like physicochemical properties of the oligonucleotides that are affectedby the combination of sequence and chemical composition, and to exclude sequences that would be predicted to be toxic.CD33 Targeting OligonucleotidesCD33 mRNA Targeting Regions

[0053] In some aspects, provided herein are compounds which modulate CD33 expression and / or activity. In certain aspect, the compounds target and modulate CD 33 mRNA expression and activity. In some embodiments, the composition comprises an oligonucleotide that inhibits the expression of CD33 mRNA. In some instances, the oligonucleotide comprises an antisense oligonucleotide (ASO). In some embodiments, the ASO comprises an oligonucleotide other than an siRNA. In some embodiments, the ASO is single stranded. In some embodiments, the ASO is designed to hybridize to a portion of CD33 RNA transcript via the binding, partially or wholly. In some instances, the ASO hybridizes to the portion of CD33 mRNA and downregulate the expression level of CD33 mRNA by RNase H-mediated degradation. In some embodiments, the composition comprises an oligonucleotide (splice switching oligos, SSOs) that modulates the relative expression level of one or more isoforms of CD33. In some instances, the SSOs modulate the exon skipping of one or more exons of CD33 mRNA. In some instances, the SSOs modulate the exon skipping of exon 2 of CD33 mRNA. In some instances, the composition comprises an oligonucleotide (SSO)that increases the relative ratio of CD33m (exon 2 skipped isoform) relative to an isoform which contains exon 2.

[0054] In some aspects, the oligonucleotide targets a coding region of the CD33 mRNA or the CD33 pre-mRNA. In some embodiments, the translation initiation codon is 5’ -AUG (in transcribed mRNA molecules; 5'-ATG in the corresponding DNA molecule), and the translation initiation codon is also referred to as the "AUG codon", the "start codon" or the "AUG start codon". A minority of genes have a translation initiation codon having the RNA sequence 5'- GUG, 5'-UUG or 5'-CUG; and 5'-AUA, 5'-ACG and 5'-CUG have been shown to function in vivo. Thus, in some cases, the terms "translation initiation codon" and "start codon" can encompass many codon sequences, even though the initiator amino acid in each instance istypically methionine (in eukaryotes) or formylmethionine (in prokaryotes). Eukaryotic and prokaryotic genes can have two or more alternative start codons, any one of which can be utilized fortranslation initiation in a particular cell type or tissue, or under a particular set of conditions. In some embodiments, "start codon" and "translation initiation codon" refer to the codon or codons that are used in vivo to initiate translation of an mRNA transcribed from a gene encoding Siglec-3 (CD33), regardless of the sequence(s) of such codons. A translation termination codon (or "stop codon") of a gene can have one of three sequences, i.e., 5'-UAA, 5’-UAG and 5'-UGA (the corresponding DNA sequences are 5’-TAA, 5'-TAG and 5’-TGA, respectively).

[0055] In some embodiments, "start codon region" and "translation initiation codon region" refer to a portion of an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from a translation initiation codon. Similarly, in some cases, the terms "stop codon region" and "translation termination codon region" refer to a portion of an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from a translation termination codon. Consequently, the "start codon region" (or "translation initiation codon region") and the "stop codon region" (or "translation termination codon region") are all regions that can be targeted effectively with the antisense compounds described herein.

[0056] The open reading frame (ORF) or "coding region", referring to the region between the translation initiation codon and the translation termination codon, is also a region which can be targeted effectively. In some embodiments, a targeted region is the intragenic region encompassing the translation initiation or termination codon of the open reading frame (ORF) of a gene.

[0057] Another target region includes the 5' untranslated region (5'-UTR), a portion of an mRNA in the 5' direction from the translation initiation codon, and thus includes nucleotides between the 5' cap site and the translation initiation codon of an mRNA (or corresponding nucleotides on the gene). Another exemplary target region includes the 3' untranslated region (3'-UTR), a portion of an mRNA in the 3' direction from the translation termination codon, and thus includes nucleotides between the translation termination codon and 3 ' end of an mRNA (or corresponding nucleotides on the gene). The 5' cap site of an mRNA comprises an N7-methylated guanosine residue joined to the 5' end of the mRNA via a 5' -5' triphosphate linkage. The 5' cap region of an mRNA is considered to include the 5' cap structure itself as well as the first 50 nucleotides adjacent to the cap site. Another target region is the 5' cap region.

[0058] In some embodiments, the antisense oligonucleotides bind to coding and / or non -coding regions of a target nucleic acid (e.g., mRNA) and modulate the expression and / or function of the target nucleic acid (e.g., mRNA). In some embodiments, the antisense oligonucleotides bind tothe complementary target nucleic acid (e.g., mRNA) and modulate the expression and / or function of the target nucleic acid.

[0059] In some instances, alternative RNA transcripts (or RNA isoforms) can be produced from the same genomic region of DNA. These alternative transcripts are generally known as "variants". More specifically, "pre-mRNA variants" are transcripts produced from the same genomic DNA that differ from other transcripts produced from the same genomic DNA in either their start or stop position and contain both intronic and exonic sequence. Upon excision of one or more exon or intron regions, or portions thereof during splicing, pre-mRNA variants produce smaller "mRNA variants". Consequently, mRNA variants are processed pre-mRNA variants and each unique pre-mRNA variant must always produce a unique mRNA variant as a result of splicing. These mRNA variants are also known as "alternative splice variants". If no splicing of the pre-mRNA variant occurs, then the pre-mRNA variant is identical to the mRNA variant.

[0060] Variants can be produced through the use of alternative signals to start or stop transcription. Pre-mRNAs and mRNAs can possess more than one start codon or stop codon. Variants that originate from a pre-mRNA or mRNA that use alternative start codons are known as "alternative start variants" of that pre-mRNA or mRNA. Those transcripts that use an alternative stop codon are known as "alternative stop variants" of that pre -mRNA or mRNA. One specific type of alternative stop variant is the "polyA variant" in which the multiple transcripts produced result from the alternative selection of one of the "polyA stop signals" by the transcription machinery, thereby producing transcripts that terminate at unique polyA sites. In some embodiments, the types of variants described herein are also embodiments of target nucleic acids.

[0061] In some embodiments, the oligonucleotides are specific for nucleic acids encoding CD33, which includes, without limitation, noncoding regions. In some instances, the nucleic acid encoding CD33 has a nucleic acid sequence of accession numb er NM_001772.4 (SEQ ID NO: 1) orNM_001082618.2 (SEQ ID NO: 2). In some instances, the nucleic acid encoding CD33 has a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to a nucleic acid sequence of accession number NM_001772.4 (SEQ ID NO: 1) or NM_001082618.2 (SEQ ID NO: 2). . The nucleic acid encoding CD33 comprises variants of CD33 mRNA (e.g., isoforms, splicing variants, homologs, etc.), mutations, single nucleotide polymorphism (SNP), noncoding sequences (e.g., introns, 5’ or 3 ’ UTRs, promoters, etc.), alleles, or fragments thereof.

[0062] In some embodiments, the oligonucleotides are complementary to or bind to nucleic acid sequences of CD33 transcripts and modulate expression and / or function of CD33 molecules. In some embodiments, oligonucleotides comprise sequences of at least 5 consecutive nucleotideswith no more than 1, 2, or 3 mismatches to modulate expression and / or function of CD33 molecules. In some embodiments, the polynucleotide targets comprise CD33, which includes family members thereof, variants of CD33, mutants of CD.3.3 (including SNPs), noncoding sequences of CD33, alleles of CD33, species variants, fragments, and the like.

[0063] Once one or more target regions, segments, or sites in the target nucleic acid (e.g., CD33 mRNA) have been identified, oligonucleotides (e.g., ASO) can be designed that are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect. In some embodiments, specific portion of the nucleic acids in the target nucleic acid (e.g., CD33 mRNA) is targeted by antisense oligonucleotides. Targeting an antisense oligonucleotide to a particular nucleic acid can be a multistep process. In some cases, the process comprises identification of a nucleic acid sequence whose function is to be modulated. This can be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state.

[0064] The target region or segments in the target nucleic acid (e.g., CD33 mRNA) can be combined with their respective complementary antisense compounds disclosed herein to form stabilized double-stranded (duplexed) oligonucleotides. Such double stranded oligonucleotide moieties can be degraded by enzymatic cleavage process (e.g., RNase H-mediated degradation), thereby the expression of the target nucleic acid can be modulated.

[0065] In some embodiments, the locations on the target nucleic acid to which the antisense compounds hybridize are at least 5 nucleotides in length. In some embodiments, target segments are 5-100 nucleotides in length and comprise a stretch of at least five (5) consecutive nucleotides selected from within the target segment. Target segments can include DNA or RNA sequences that comprise at least the 5 consecutive nucleotides from the 5' -terminus of one of the target segments (the remaining nucleotides being a consecutive stretch of the same DNA or RNA beginning immediately upstream of the 5'-terminus of the target segment and continuing until the DNA or RNA contains about 5 to about 100 nucleotides). In some embodiments, target segments are represented by DNA or RNA sequences that comprise at least the 5 consecutive nucleotides from the 3 '-terminus of one of the target segments (the remaining nucleotides being a consecutive stretch of the same DNA or RNA beginning immediately downstream of the 3' -terminus of the target segment and continuing until the DNA or RNA contains about 5 to about 100 nucleotides).

[0066] In some embodiments, a target segment can be employed in a screen for additional compounds that modulate the expression of CD33 mRNA or pre-mRNA. In some cases, "modulators" are those compounds that decrease or increase the expression of a nucleic acid molecule encoding CD33 and which comprise at least a 5 -nucleotide portion that is complementary to a target segment. In some cases, the screening method comprises the steps ofcontacting a target segment of a nucleic acid molecule encoding the sense or natural antisense nucleic acid sequence of CD 33 mRNA or pre-mRNA with one or more selected modulators, and selecting for one or more selected modulators which decrease or increase the expression of a nucleic acid molecule encoding CD33. Once it is shown that the selected modulator or modulators are capable of modulating (e.g., either decreasing or increasing) the expression of a nucleic acid molecule encoding CD33, the modulator can then be employed in further investigative studies of the function of CD33, or foruse as a research, diagnostic, or therapeutic agent.ASO Compound and Sequences

[0067] In some aspects, the oligonucleotide (e.g., ASO) binds to or targets CD33 mRNA or pre- mRNA. In some instances, the CD33 mRNA comprises a sequence of NM_001772.4 (SEQ ID NO: 1). In some instances, the CD 33 mRNA comprises at least a portion of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the oligonucleotides target one or more regions of the nucleic acid molecules sense and / or antisense of coding and / or non -coding (e.g., intron) sequences associated with CD33 and the sequences set forth as SEQ ID NOs: 1 and 2. In some cases, the oligonucleotides target (e.g., hybridize) overlapping regions of SEQ ID NOs: 1 and 2. In some cases, the oligonucleotides target (e.g., hybridize) overlapping regions of SEQ ID NOs: 5 and 6.

[0068] In some instances, provided herein are compounds (e.g., oligonucleotides) comprising a modified oligonucleotide that binds to (or hybridizes to) or targets at least a portion of SEQ ID NO: 1 or SEQ ID NO: 2. In some instances, the oligonucleotide comprises or consists of 12 to 30 linked nucleotides and having a nucleic acid sequence complementary to at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides of SEQ ID NO: 1 or SEQ ID NO: 2, with no more than 1, 2, 3 mismatches. In some instances, provided herein are compounds comprising a modified oligonucleotide consisting of 12 to 30 linked nucleotides and having a nucleic acid sequence complementary to at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1, 2, 3 mismatches corresponding to positions 1-21, 29-48, 94- 116, 137-187, 211-241, 263-282, 392-417, 416-435, 456-523, 460-479, 504-671, 652-671, 683- 721, 724-746, 730-753, 731-756, 770-819, 772-807, 857-876, 880-899, 1050-1138, 1073-1108, 1165-1184, 1189-1266, 1194-1276, 1250-1334, 1270-1360, 1285-1332, 1336-1360, 1340-1360, 1364-1383, 1388-1452, or 1370-1462 of SEQ ID NO: 1.

[0069] In some instances, the mismatches can be located at the 5’ end or 3 ’ end of the oligonucleotide (e.g., ASO). In some instances, one or more mismatches can be located at theinternal locations (e.g., between positions 3 -17) of the oligonucleotide (ASO) counted from the 5’ end. In some embodiments, the oligonucleotide (ASO) described herein include variants in which a different base is present at one or more of the nucleotide positions in the compound. For example, if the first nucleotide is an adenine, variants can be produced which contain thymidine, guanosine, cytidine or other natural or unnatural nucleotides at this position. This can be done at any of the positions of the antisense compound. These compounds are then tested using the methods described herein to determine their ability to inhibit expression of a target nucleic acid.

[0070] In some instances, the composition comprises an oligonucleotide that inhibits the expression of CD33, wherein the oligonucleotide comprises an ASO comprising about 12-30 nucleotides in length and comprising a nucleic acid sequence comprising about 12-20 contiguous nucleotides of any one of SEQ ID NOs: 123-432, with no more than 1, 2, or 3 mismatches. In some instances, the composition comprises an oligonucleotide that inhibits the expression of CD33, wherein the oligonucleotide comprises an ASO comprising about 12-30 nucleotides in length and comprising a nucleic acid sequence comprising about 12-20 contiguous nucleotides of any one of SEQ ID NOs: 123-432, with no more than 1, 2, or 3 mismatches; wherein the ASO comprises a modification comprising a modified nucleotide analogue or nonnatural nucleotide, and / or a modified internucleotide linkage. In some embodiments, the composition comprises an oligonucleotide that inhibits the expression of CD33, wherein the oligonucleotide comprises an ASO comprising a nucleic acid sequence comprising or consisting of a nucleic acid sequence of any one of SEQ ID NOs: 123-432, or a nucleic acid sequence thereof having 1 or 2 nucleotide substitutions, additions, or deletions. In some embodiments, the ASO comprises a nucleic acid sequence comprising or consisting of the sequence of any one of SEQ ID NOs: 123 -432, or a nucleic acid sequence thereof having 3 or 4 nucleotide substitutions, additions, or deletions. In some embodiments, the composition comprises an oligonucleotide that inhibits the expression of CD33, wherein the oligonucleotide comprises an ASO comprising a nucleotide sequence comprising or consisting of the nucleic acid sequence of any one of SEQ ID NOs: 123 -432. In some embodiments, compounds comprise a modified oligonucleotide consisting of 12 to 30 linked nucleotides and having a nucleic acid sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1, 2, or 3 mismatches from any of the nucleic acid sequences in Table 4.

[0071] In some instances, provided herein are compounds comprising a modified oligonucleotide consisting of 12 to 30 linked nucleotides and having a nucleic acid sequence complementary to at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1, 2, 3mismatches corresponding to positions 29-48, 392-417, 460-479, 731-756, 772-807, 1073-1108, 1194-1276, 1270-1360, 1388-1452, or 1370-1462 of SEQ ID NO: 1.

[0072] In some instances, the composition comprises an oligonucleotide that inhibits the expression of CD33 by about 50%, wherein the oligonucleotide comprises an ASO comprising about 12-30 nucleotides in length and comprising a nucleic acid sequence comprising about 12- 20 contiguous nucleotides of any one of SEQ ID NOs: 125, 146, 148-152, 158, 182-186, 189, 191, 193, 197, 203, 222-226, 241 -260, 262-266, 271, 272, 274-281, 283, 286, 287, 300, 301, 311-319, 321, 322, 328-335, 344, 345, 347-351, 377-381, 390, 391, 395-397, 400-409, 412, 413, 415-420, or 422, with no more than 1, 2, or 3 mismatches. In some instances, the composition comprises an oligonucleotide that inhibits the expression of CD33 by about 50%, wherein the oligonucleotide comprises an ASO comprising about 12-30 nucleotides in length and comprising a nucleic acid sequence comprising about 12-20 contiguous nucleotides of any one of SEQ ID NOs: 125, 146, 148-152, 158, 182-186, 189, 191, 193, 197, 203, 222-226, 241-260, 262-266, 271, 272, 274-281, 283, 286, 287, 300, 301, 311 -319, 321, 322, 328-335, 344, 345, 347-351, 377-381, 390, 391, 395-397, 400-409, 412, 413, 415-420, or 422, with no more than 1, 2, or 3 mismatches; wherein the ASO comprises a modification comprising a modified nucleotide analogue or nonnatural nucleotide, and / or a modified intemucleotide linkage. In some embodiments, the composition comprises an oligonucleotide that inhibits the expression of CD33 by about 50%, wherein the oligonucleotide comprises an ASO comprising a nucleic acid sequence comprising or consisting of a nucleic acid sequence of any one of SEQ ID NOs: 125, 146, 148-152, 158, 182-186, 189, 191, 193, 197, 203, 222-226, 241-260, 262-266, 271, 272, 274- 281, 283, 286, 287, 300, 301, 311 -319, 321, 322, 328-335, 344, 345, 347-351, 377-381, 390, 391, 395-397, 400-409, 412, 413, 415-420, or 422, or a nucleic acid sequence thereof having 1 or 2 nucleotide substitutions, additions, or deletions. In some embodiments, the ASO comprises a nucleic acid sequence comprising or consisting of the sequence of any one of SEQ ID NOs: 125, 146, 148-152, 158, 182-186, 189, 191, 193, 197, 203, 222-226, 241-260, 262-266, 271, 272, 274- 281, 283, 286, 287, 300, 301, 311 -319, 321, 322, 328-335, 344, 345, 347-351, 377-381, 390, 391, 395-397, 400-409, 412, 413, 415-420, or 422, or a nucleic acid sequence thereof having 3 or 4 nucleotide substitutions, additions, or deletions. In some embodiments, the composition comprises an oligonucleotide that inhibits the expression of CD33 by about 50%, wherein the oligonucleotide comprises an ASO comprising a nucleotide sequence comprising or consisting of the nucleic acid sequence of any one of SEQ ID NOs: 125, 146, 148-152, 158, 182-186, 189, 191, 193, 197, 203, 222-226, 241 -260, 262-266, 271, 272, 274-281, 283, 286, 287, 300, 301, 311-319, 321, 322, 328-335, 344, 345, 347-351, 377-381, 390, 391, 395-397, 400-409, 412, 413, 415-420, or 422.

[0073] In some instances, provided herein are compounds comprising a modified oligonucleotide consisting of 12 to 30 linked nucleotides and having a nucleic acid sequence complementary to at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1, 2, 3 mismatches corresponding to positions 1270-1360, 1388-1452, or 1370-1462 of SEQ ID NO: 1.

[0074] In some embodiments, the composition comprises an oligonucleotide that inhibits the expression of CD33 by about 75%, wherein the oligonucleotide comprises an ASO comprising about 14-30 nucleotides in length and comprising a nucleic acid sequence comprising about 12- 20 contiguous nucleotides of any one of SEQ ID NOs: 313, 316, 317, 319, 347-349, 402, 417, or 418, with no more than 1, 2, or 3 mismatches. In some embodiments, the composition comprises an oligonucleotide that inhibits the expression of CD33 by about 75%, wherein the oligonucleotide comprises an ASO comprising about 12-30 nucleotides in length and comprising a nucleic acid sequence comprising about 12-20 contiguous nucleotides of any one of SEQ ID NOs: 313, 316, 317, 319, 347-349, 402, 417, or 418, with no more than 1, 2, or 3 mismatches; wherein the ASO comprises a modification comprising a modified nucleotide analogue or nonnatural nucleotide, and / or a modified intemucleotide linkage. In some embodiments, the composition comprises an oligonucleotide that inhibits the expression of CD33 by about 75%, wherein the oligonucleotide comprises an ASO comprising a nucleic acid sequence comprising or consisting of a nucleic acid sequence of any one of SEQ ID NOs: 313, 316, 317, 319, 347-349, 402, 417, or 418, or a nucleic acid sequence thereof having 1 or 2 nucleotide substitutions, additions, or deletions. In some embodiments, the ASO comprises a nucleic acid sequence comprising or consisting of the nucleic acid sequence of any one of SEQ ID NOs: 313, 316, 317, 319, 347-349, 402, 417, or 418, or a nucleic acid sequence thereof having 3 or 4 nucleotide substitutions, additions, or deletions. In some embodiments, the composition comprises an oligonucleotide that inhibits the expression of CD33 by about 75%, wherein the oligonucleotide comprises an ASO comprising a nucleic acid sequence comprising or consisting of the nucleic acid sequence of any one of SEQ ID NOs: 313, 316, 317, 319, 347-349, 402, 417, or 418. In some embodiments, compounds comprise a modified oligonucleotide consisting of 12 to 30 linked nucleotides and having a nucleic acid sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1, 2, or 3 mismatches from the sequence of any one of SEQ ID NOs: 313, 316, 317, 319, 347-349, 402, 417, or 418.

[0075] In some embodiments, homology, sequence identity or complementarity, between the antisense oligonucleotide (ASO) and target is from about 50% to about 60%. In some embodiments, homology, sequence identity or complementarity, is from about 60% to about70%. In some embodiments, homology, sequence identity or complementarity, is from about 70% to about 80%. In some embodiments, homology, sequence identity or complementarity, is from about 80% to about 90%. In some embodiments, homology, sequence identity or complementarity, is about 90%, about 92%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100%.

[0076] Thus, in some instances, compounds (e.g., ASOs) comprises a modified oligonucleotide consisting of 12 to 30 linked nucleotides and having a nucleic acid sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or atleast20 consecutive nucleotides with no more than 1, 2, or 3 mismatches from any of the nucleic acid sequences of SEQ ID NOs: 123-432. In some embodiments, the nucleic acid sequence of the modified oligonucleotide is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% identity to any one of SEQ ID NOs: 123-432. In some embodiments, compounds comprise a modified oligonucleotide consisting of 12 to 30 linked nucleotides and having a nucleic acid sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1, 2, or 3 mismatches from any of the nucleic acid sequences of SEQ ID NOs: 125, 146, 148-152, 158, 182-186, 189, 191, 193, 197, 203, 222-226, 241-260, 262-266, 271, 272, 274-281, 283, 286, 287, 300, 301, 311 -319, 321, 322, 328-335, 344, 345, 347-351, 377-381, 390, 391, 395-397, 400-409, 412, 413, 415-420, or 422. In some embodiments, the nucleic acid sequence of the modified oligonucleotide is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 125, 146, 148-152, 158, 182-186, 189, 191, 193, 197, 203, 222-226, 241-260, 262-266, 271, 272, 274-281, 283, 286, 287, 300, 301, 311-319, 321, 322, 328-335, 344, 345, 347-351, 377-381, 390, 391, 395-397, 400-409, 412, 413, 415-420, or 422. In some embodiments, compounds comprise a modified oligonucleotide consisting of 12 to 30 linked nucleotides and having a nucleic acid sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, atleast 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1, 2, or 3 mismatches from any of the nucleic acid sequences of SEQ ID NOs: 313, 316, 317, 319, 347-349, 402, 417, or 418. In some embodiments, the nucleic acid sequence of the modified oligonucleotide is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 313, 316, 317, 319, 347-349, 402, 417, or 418.

[0077] In some embodiments, the ASO is about 10-30, 12-30, 12-30, 12-25, 12-20, 13-30, 13-25, 13-20, 14-30, 14-25, 14-20, 15-30, 15-25, or 15-20 nucleotides in length. In some embodiments, the ASO is at least about 10, 11, 12, 13, 14, 15, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length, or a range defined by any of the two aforementioned numbers. In some embodiments, the ASO is at least 6 nucleotides in length. In some embodiments, the ASO is at least 7 nucleotides in length, at least 8 nucleosides in length, or at least 9 nucleotides in length.

[0078] In some embodiments, the antisense oligonucleotide comprises from about 5 to about 80 nucleotides (i.e. from about 5 to about 80 linked nucleotides) in length. In some instances, the antisense oligonucleotide is a single-stranded molecule from 5 to about 80 nucleotides. In some embodiments, the antisense oligonucleotide has portions of 10 to 50 nucleotides in length. In some cases, this embodies oligonucleotides having antisense portions of 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, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length, or any range there within. In some embodiments, the antisense oligonucleotides are 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. In some instances, the antisense oligonucleotide is about 20 nucleotides in length.

[0079] In one aspect, an oligonucleotide described herein, e.g., ASO, comprises one or more modifications. Modifications in some instances comprise modifications to the sugar ring of the nucleotide, a backbone (e.g., internucleotide linkage), or a base.

[0080] In some embodiments, ASOs are chimeric oligonucleotides. In some embodiments, chimeric oligonucleotides or chimeras are oligonucleotides which contain two or more chemically distinct regions, each made up of at least one nucleotide. In some cases, these oligonucleotides contain at least one region of modified nucleotides that confers one or more beneficial properties (such as, for example, increased nuclease resistance, increased uptake into cells, increased binding affinity for the target) and a region that is a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of antisense modulation of gene expression. Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used, compared to phosphorothioate deoxy oligonucleotides hybridizing to the same target region. Cleavage of theRNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art. In some embodiments, a chimeric oligonucleotide comprises at least one region modified to increase target binding affinity, and, optionally, a region that acts as a substrate for RNase H. Affinity of an oligonucleotide for its target can be determined by measuring the thermal melting point (Tm) of an oligonucleotide target pair, which is the temperature at which the oligonucleotide and target dissociate; where dissociation is detected spectrophotometrically. The higher the Tm, the greater is the affinity of the oligonucleotide for the target.

[0081] Chimeric antisense compounds can be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleotides and / or oligonucleotides mimetics as described above. Such compounds have also been referred to in the art as hybrids, gapmers, or mixmers. In some instances, a modified oligonucleotide is a gapmer, which comprises wing segments (a 5' wing segment and a 3' wing segment), and a gap segments located between the 5’ and 3 ’ wing segments. In some instances, the gap segment consists of 1-20, 2-20, 5-20, 5-15, 5- 10, 10-20, or 10-15 linked deoxynucleotides (DNA molecules). In some instances, the 5' wing segment consists of 1 -20, 3-20, 4-20, 4-15, 4-10, 6-20, 8-15, or 10-15 linked nucleotides. In some instances, the 3' wing segment consists of 1 -20, 3-20, 4-20, 4-15, 4-10, 6-20, 8-15, or 10-15 linked nucleotides. In some instances, the 5’ and 3’ wing segments comprise one or more modified, nonnatural nucleotides. In some instances, the 5’ and 3 ’ wing segments comprises one or more modified RNA molecules. For example, the 5’ and 3 ’ wing segments comprises one or more locked nucleic acids (LN A). In another example, the 5’ and 3 ’ wing segments comprises one or more 2'-O-methoxyethyl (2'-M0E) modified RNA molecules. In certain instances, the gapmer comprises 5’ wing segment in which each of nucleotide in the 5’ wing segment is modified, nonnatural nucleotide. In certain instances, the gapmer comprises 3’ wing segment in which each of nucleotide in the 3’ wing segment is modified, nonnatural nucleotide. In certain instances, the gapmer comprises 5’ wing segment and 3’ wing segment in which each of nucleotide in the 5’ wing segment and 3’ wing segment is modified, nonnatural nucleotide. In certain instances, the gapmer comprises 5-10-5 structure, where 5 nucleotides at the 5’ end (5’ wing) and 5 nucleotides at the 3 ’ end (3 ’ wing) are 2'-M0E modified RNA molecules and 10 nucleotides between the 5’ and 3’ wings are DNA molecules.

[0082] In some instances, the ASO is a gapmer comprising the base nucleic acid sequence of any one of SEQ ID NOs: 123-432. In some instances, the gap segment is positioned between the 5' wing segment and the 3' wing segment. In some instances, the ASO is a gapmer comprising the nucleic acid sequence of any one of SEQ ID NOs: 433-742. In some instances, the ASO is a gapmer comprising the nucleic acid sequence of any one of SEQ ID NOs: 125, 146, 148-152,158, 182-186, 189, 191, 193, 197, 203, 222-226, 241-260, 262-266, 271, 272, 274-281, 283, 286, 287, 300, 301, 311 -319, 321, 322, 328-335, 344, 345, 347-351, 377-381, 390, 391, 395-397, 400- 409, 412, 413, 415-420, or 422. In some instances, the ASO is a gapmer comprising the nucleic acid sequence of any one of SEQ ID NOs: 435, 456, 458-462, 468, 492-496, 499, 501, 503, 507, 513, 532-536, 551 -570, 572-576, 581, 582, 584-591, 593, 596, 597, 610, 611, 621-629, 631, 632, 638-645, 654, 655, 657-661, 687-691, 700, 701, 705-707, 710-719, 722, 723, 725-730, or 732. In some instances, the ASO is a gapmer comprising the nucleic acid sequence of any one of SEQ ID NOs: 313, 316, 317, 319, 347-349, 402, 417, or 418. In some instances, the ASO is a gapmer comprising the nucleic acid sequence of any one of SEQ ID NOs: 623, 626, 627, 629, 657-659, 712, 727, or 728.

[0083] In some embodiments, CD33 transcript expression or function can be modulated by antisense oligonucleotides which are identified and expanded, using for example, PCR, hybridization, etc., based on the sequence set forth as SEQ ID NOs: 123-432, and the like. In some embodiments, expression or function is down -regulated as compared to a control. In some embodiments, oligonucleotides comprise nucleic acid sequences set forth as SEQ ID NOs: 433- 742, including antisense oligonucleotide sequences which are identified and expanded, using for example, PCR, hybridization etc. These oligonucleotides can comprise one or more modified nucleotides, shorter or longer fragments, modified bonds and the like. Examples of modified bonds or intemucleotide linkages comprise phosphorothioate, phosphorodithioate or the like. In some embodiments, the nucleotides comprise a phosphorus derivative. The phosphoru s derivative (or modified phosphate group) which can be attached to the sugar or sugar analog moiety in the modified oligonucleotides, may be a monophosphate, diphosphate, triphosphate, alkylphosphate, alkanephosphate, phosphorothioate or the like.

[0084] In some embodiments, one or more antisense oligonucleotides can be fused or combined together to form a single molecule. For example, the oligonucleotide comprises a first ASO targeting a first target nucleic acid and a second ASO targeted to a second target nucleic acid. For example, the first target can be CD33 mRNA, and the second target can be a region from another nucleotide sequence. In another example, the first target can be a first region within the CD33 mRNA or CD 33 pre-mRNA, and the second target can be a second region within the CD 33 mRNA or CD33 pre-mRNA. In some instances, the oligonucleotide includes a ASO targeting a portion of CD33 mRNA or CD33 pre-mRNA and an SSO targeting another portion of CD33 mRNA or CD33 pre-mRNA. Numerous examples of antisense compounds are illustrated herein, and others can be selected from among suitable compounds known in the art. Two or more combined compounds can be used together or sequentially.-SO-Splice Switching Oligonucleotide (SSO)

[0085] Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as "introns," which are excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as "exons" and are spliced together to form a continuous mRNA sequence. In some embodiments, targeting splice sites, i.e., intronexon junctions or exon-intron junctions, is useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular splice product is implicated in disease. An aberrant fusion junction due to rearrangement or deletion is another embodiment of a target site. mRNA transcripts produced via the process of splicing of two (or more) mRNAs from different gene sources are known as "fusion transcripts". Introns can be effectively targeted using antisense oligonucleotide hybridizing or targeting, for example, DNA or pre-mRNA.

[0086] CD33 can be alternatively spliced to skip exon 2. Exon 2-skipped isoforms such as CD33m in some cases are neuro protective. As such, in some aspects, an oligonucleotide targeting CD33 mRNA (or pre-mRNA) can modulate the alternative splicing of CD33 mRNA (or pre- mRNA) such that certain isoform (alternatively spliced product) of CD33 mRNA is expressed more than another isoform of CD33 mRNA. In some aspects, the oligonucleotide targeting CD33 mRNA (or pre-mRNA) can modulate the ratio between one isoform of the CD33 mRNA and another isoform of the CD33 mRNA. In some instances, the oligonucleotide is a Splice Switching Oligonucleotide (SSO) that induces exon skipping of the CD33 mRNA. In some instances, the SSO induces the skipping of Exon 2 of the CD33 mRNA such that the expression of the CD33 mRNA without exon 2 can be induced and increased.

[0087] In some aspects, SSOs bind to (hybridizes or targets) an intron. In some instances, antisense oligonucleotides bind to an intron of the CD33 mRNA (encoded by NCBI reference NM 001772.4). In some instances, SSOs bind to an intron adjacent to exon 2 of the CD33 mRNA. In some instances, SSOs bind to a 5’ flanking intron adjacent to exon 2 of the CD33 mRNA. In some instances, antisense oligonucleotides bind to a 3 ’ flanking intron adjacent to exon 2 of the CD33 mRNA. In some instances, SSOs bind to a region within exon 2 of CD33 mRNA. In some instances, SSOs bind to the 5’ exon -intron junction of exon 2 of CD33 mRNA. In some instances, SSOs bind to the 3’ exon-intron junction of exon 2 of CD33 mRNA. In some instances, SSOs bind to the region at least within 5, 10, 15, 20, 25, 30 nucleotides (upstream or downstream) from the exon-intron junction of exon 2 of CD33 mRNA. In some instances, SSO binds to about 5, 10, 12, 15, 17, 18, 19, 20, 21, 22, 24, 25, 26, 27, 28, 29, or 30 nucleotides of any one of SEQ ID NOs: 5-6. In some instances, antisense oligonucleotides bind to about 5 -30, 5-20 5-15, 10-30, 10-25, 10-20, 10-15, 12-30, 15-30, 15-25, 20-30, 20-25, or 25-30 nucleotides of orwithin any one of SEQ ID NOs: 5-6. In some instances, SSOs which bind to adjacent introns of exon 2 result in a decreased expression of isoforms which comprise exon 2.

[0088] In some embodiments, homology, sequence identity or complementarity, between the SSO and target sequence in CD33 mRNA is from about 40% to about 60%. In some embodiments, homology, sequence identity or complementarity, is from about 60% to about 70%. In some embodiments, homology, sequence identity or complementarity, is from about 70% to about 80%. In some embodiments, homology, sequence identity or complementarity, is from about 80% to about 90%. In some embodiments, homology, sequence identity or complementarity, is about 90%, about 92%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100%.

[0089] In some aspects, the SSO comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides from any one of SEQ ID NOs: 7-64, with no more than 1, 2, or 3 mismatches. In some instances, the nucleic acid sequence of the SSO is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 7-64.

[0090] In some embodiments, compounds comprise a modified oligonucleotide consisting of 12 to 30 linked nucleotides and having a nucleic acid sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1, 2, or 3 mismatches from any of the nucleic acid sequences of Table 2.

[0091] In some embodiments, the SSO increases the relative ratio of CD33 mRNA in which exon 2 is absent compared to CD33 mRNA in which exon 2 is present and results in a decreased percent of relative inclusion of exon 2. In some embodiments, the compound results in a decreased percent of relative inclusion of exon 2, with at most 75% wherein the compound comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, atleast 19, or at least20 consecutive nucleotides from any one of SEQ ID NOS: 16-22, 32-36, 38-42, or 44, with no morethan 1, 2, or 3 mismatches from. In some embodiments, the nucleic acid sequence of the modified oligonucleotide is at least 80%, at least 81%, atleast 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOS: 16-22, 32-36, 38-42, or 44.

[0092] In one aspect, an oligonucleotide described herein, e.g., SSO, comprises one or more modifications. Modifications in some instances comprise modifications to the sugar ring of the nucleotide, a backbone (e.g., internucleotide linkage), or a base. In some instances, the SSO comprises one or more 2'-O-methoxyethyl (2'-M0E) modified nucleotides. In some instances, at least 80%, 85%. 90%, 95%, or 100% of the nucleotides in the SSO are 2'-M0E modified nucleotides.

[0093] In some embodiments, CD33 transcript expression or function can be modulated by antisense oligonucleotides which are identified and expanded, using for example, PCR, hybridization etc., based on the sequence set forth as SEQ ID NOs: 7-64, and the like. In some embodiments, expression or function is down -regulated (e.g., mRNA comprising exon 2) as compared to a control. In some embodiments, oligonucleotides comprise nucleic acid sequences set forth as SEQ ID NOs: 65-122, including antisense sequences which are identified and expanded, using for example, PCR, hybridization etc. These oligonucleotides can comprise one or more modified nucleotides, shorter or longer fragments, modified bonds and the like. Examples of modified bonds or intemucleotide linkages comprise phosphorothioate, phosphorodithioate or the like. In some embodiments, the nucleotides comprise a phosphorus derivative. The phosphorus derivative (or modified phosphate group) which can be attached to the sugar or sugar analog moiety in the modified oligonucleotides, may be a monophosphate, diphosphate, triphosphate, alkylphosphate, alkanephosphate, phosphorothioate or the like.Nucleotide modifications for ASO and SSO

[0094] In some embodiments, the oligonucleotide (ASO or SSO) is modified to enhance nuclease resistance. Cells contain a variety of exo- and endo-nucleases which can degrade nucleic acids. Nuclease resistance can be measured by incubating oligonucleotides with cellular extracts or isolated nuclease solutions and measuring the extent of intact oligonucleotide remaining over time, e.g., by gel electrophoresis. Oligonucleotides which have been modified to enhance their nuclease resistance can survive intact for a longer time than unmodified oligonucleotides. In some embodiments, the oligonucleotide comprises at least one phosphorothioate modification. In some cases, oligonucleotide modifications which enhance target binding affinity are also, independently, able to enhance nuclease re sistance. Enemas and intramuscular, intravitreal, intrathecal injections have been used for the administration of a variety of oligonucleotides with and without phosphorothioate bonds.

[0095] In certain embodiment, the antisense oligonucleotides, such as nucleic acid molecules set forth in SEQ ID NOs: 7-64 and 123-432, comprise one or more substitutions or modifications. In some embodiments, the nucleotides are substituted with locked nucleic acids (LNA) or 2’-M0Emodified nucleotide. In some instances, a modified oligonucleotide comprises any one of SEQ ID NOs: 65-122 and 433-742.

[0096] In some embodiments, a modified oligonucleotide comprises at least 8, at least 9, at least 10, at least 11, atleast 12, at least 13, atleast 14, atleast 15, atleast 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1, 2, or 3 mismatches from any one of nucleic acid sequences of SEQ ID NOs: 433-742.

[0097] In some embodiments, the nucleic acid sequence of the modified oligonucleotide is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of the nucleic acid sequences of SEQ ID NOs: 433-742.

[0098] In some embodiments, the modified oligonucleotide consists of any one of nucleic acid sequences of SEQ ID NOs: 433-742.

[0099] In some embodiments, a modified oligonucleotide comprises a nucleic acid sequence comprising at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1, 2, or 3 mismatches from any one of the nucleobase sequences of SEQ ID NOs: 435, 456, 458-462, 468, 492-496, 499, 501, 503, 507, 513, 532-536, 551-570, 572-576, 581, 582, 584- 591, 593, 596, 597, 610, 611, 621 -629, 631, 632, 638-645, 654, 655, 657-661, 687-691, 700, 701, 705-707, 710-719, 722, 723, 725-730, or 732.

[0100] In some embodiments, the nucleic acid sequence of the modified oligonucleotide is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of the nucleic acid sequences of SEQ ID NOs: 435, 456, 458-462, 468, 492-496, 499, 501, 503, 507, 513, 532-536, 551 -570, 572-576, 581, 582, 584-591, 593, 596, 597, 610, 611, 621-629, 631, 632, 638-645, 654, 655, 657-661, 687-691, 700, 701, 705-707, 710-719, 722, 723, 725-730, or 732.

[0101] In some embodiments, the modified oligonucleotide consists of any one of nucleic acid sequences of SEQ ID NOs: 435, 456, 458-462, 468, 492-496, 499, 501, 503, 507, 513, 532-536, 551-570, 572-576, 581, 582, 584-591, 593, 596, 597, 610, 611, 621 -629, 631, 632, 638-645, 654, 655, 657-661, 687-691, 700, 701, 705-707, 710-719, 722, 723, 725-730, or 732.

[0102] In some embodiments, a modified oligonucleotide comprises at least 8, at least 9, at least 10, at least 11, atleast 12, at least 13, atleast 14, atleast 15, atleast 16, at least 17, at least 18, atleast 19, or at least 20 consecutive nucleotides with no more than 1, 2, or 3 mismatches from any one of nucleic acid sequences of SEQ ID NOs: 623, 626, 627, 629, 657-659, 712, 727, or 728.

[0103] In some embodiments, the nucleic acid sequence of the modified oligonucleotide is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of the nucleic acid sequences of SEQ ID NOs: 623, 626, 627, 629, 657-659, 712, 727, or 728.

[0104] In some embodiments, the modified oligonucleotide consists of any one of nucleic acid sequences of SEQ ID NOs: 623, 626, 627, 629, 657-659, 712, 727, or 728.

[0105] In some embodiments, a modified oligonucleotide comprises at least 8, at least 9, at least 10, at least 11, atleast 12, at least 13 , atleast 14, atleast 15, atleast 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotideswith no more than 1, 2, or 3 mismatches from any one of nucleic acid sequences of SEQ ID NOs: 65-122.

[0106] In some embodiments, the nucleic acid sequence of the modified oligonucleotide is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of the nucleic acid sequences of SEQ ID NOs: 65-122.

[0107] In some embodiments, a modified oligonucleotide comprises at least 8, at least 9, at least 10, at least 11, at least 12, atleast 13, at least 14, atleast 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotideswith no more than 1, 2, or 3 mismatches from any one of SEQ ID NOs: 74-80, 90-94, 96-100, or 102.

[0108] In some embodiments, the nucleic acid sequence of the modified oligonucleotide is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of nucleic acid sequences of SEQ ID NOs: 74-80, 90-94, 96-100, or 102.

[0109] In some embodiments, the modified oligonucleotide consists of to any one of nucleic acid sequences of SEQ ID NOs: 74-80, 90-94, 96-100, or 102.Sugar modification

[0110] There are a variety of nucleotide mimics wherein the ribose or deoxyribose is modified to increase affinity for target and / or increase nuclease resistance. Modifications to all five positions of the ribose ring have been made; however, the modifications of the 2’ position of ribose havebeen the most studied. In some embodiments, an oligonucleotide comprises a nucleotide analogue that alters the structure of ribose.

[0111] In some embodiments, modifications such as the use of LNA monomers are used to enhance the potency, specificity and duration of action and broaden the routes of administration of oligonucleotides. This can be achieved by substituting some of the monomers in the current oligonucleotides by LNA monomers. The LNA modified oligonucleotide can have a size similar to the parent compound or can be larger or smaller. In some embodiments, LNA-modified oligonucleotides contain less than about 70%, less than about 60%, or less than about 50% LNA monomers, and that their sizes are between about 5 and25 nucleotides, orbetween about 12 and 20 nucleotides.

[0112] In some embodiments, the region of the oligonucleotide which is modified comprises at least one nucleotide modified at the 2' position of the sugar, e.g., a 2'-O-alkyl, 2,-O-alkyl-O-alkyl or 2' -fluoro-modified nucleotide. In some embodiments, RNA modifications include 2'-fluoro, 2'- amino and 2' O-methyl modifications on the ribose of pyrimidines, abasic residues or an inverted base at the 3' end of the RNA. Such oligonucleotides can have a higher Tm (i.e., higher target binding affinity) than 2' -deoxy oligonucleotides against a given target. The effect of such increased affinity canbe to enhance RNAi oligonucleotide inhibition of gene expression. RNase H is a cellular endonuclease that cleaves the RNA strand of RNA:DNA duplexes; activation of this enzyme therefore results in cleavage of the RNA target, and thus in some cases can enhance the efficiency of RNAi inhibition. Cleavage of the RNA target can be routinely demonstrated by gel electrophoresis.

[0113] In some instances, oligonucleotides comprise one or more substituted sugar moieties. In some embodiments, oligonucleotides comprise one of the following at the 2' position: OH, SH, SCH3, F, OCN, OCH3JO(CH2)nCH3, O(CH2)nNH2or O(CH2)nCH3where n is from 1 to about 10; Ci to Cio lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF3; OCF3; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH3; SO2CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; poly alkylamino; substituted silyl; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. A non-limiting exemplary modification includes 2' -meth oxy ethoxy [2’- OCH2CH2OCH3, also known as 2'-O-(2 -methoxyethyl)]. Other modifications include 2' -methoxy (2'-OCH3), 2'-propoxy (2'-OCH2CH2CH3) and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the oligonucleotide, e.g., the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of 5' terminal nucleotide. Oligonucleotides can also have sugarmimetics such as cyclobutyls or morpholinyl in place of the pentofuranosyl group. Modifications of the hydroxyl group at the 2’ position of ribose have been used to mimic the structure of the ribose ring while inhibiting ribonucleases that require the 2’ -OH group for hydrolysis of RNA. 2’-O-Methyl ribonucleic acids are naturally occurring nucleotides and have been shown to increase binding affinity to RNA itself while being resistant to ribonuclease. 2’ -O-Methyl groups can be extensively substituted into RNAi triggers, and were the first nucleotide analogues used in “antagomirs.” 2’-O-Methoxyethyl (MOE) modification was designed to mimic the ribonuclease resistance of O-methyl, attenuate protein-oligonucleotide interactions and have increased affinity for RNA.

[0114] Fluorine is highly electronegative, and 2’ -deoxy-2’ -fluoro (2’-F) analogues of nucleotides adopt C3’-endo conformations characteristic of the sugars in RNA helices. In some embodiments, an oligonucleotide comprises a base modification at the 2’ position.

[0115] Alkoxy substituents at the 4’ position of 2 ’deoxyribose mimic the conformation of ribose. In some embodiments, an oligonucleotide comprises a base modification at the 4’ position.

[0116] There are a variety of ribose derivatives that lock the carbohydrate ring into the 3’ endo conformation by the formation of bicyclic structures with a bridge between the 2’ oxygen and the 4’ position. The original bicyclic structure has a methylene bridging group and is termed locked nucleic acids (LNAs). The bicyclic structure “locks” the ribose into its preferred 3’ endo conformation and increases base pairing affinity. It has been shown that incorporation of LNAs into a DNA duplex can increase melting points up to 8° C per LNA. Subsequently, a variety of bicyclic nucleotides have been developed such as Bridged Nucleic Acids (BNAs), Ethyl-bridged (ENAs), constrained ethyl (cEt) nucleic acids and tricyclic structures with varying affinity for target sites. LNAs can be incorporated into antagomirs, splice blocking oligonucleotides, either strand of an RNAi duplex; however, like other 3 ’ endo conformers, LNAs are not substrates for RNase H. On the other end of the stability spectrum from bicyclic nucleic acids are acyclic nucleic acids, most commonly called unlocked nucleic acids, which destabilizes hybridization to the target. In some embodiments, an oligonucleotide comprises a bicyclic modification.

[0117] Phosphorodiamidate Morpholino oligomer (PMOs) use a hydrolytically stable, uncharged phosphorodiamidate functional group. Peptide nucleic acids (PNAs) are based upon the amide functional group. In some embodiments, an oligonucleotide comprises a PNA.Backbone modifications (modified internucleotide linkage)

[0118] In some aspects, oligonucleotides (ASO, SSO) disclosed herein include modified backbones, for example, phosphorothioates modified internucleotide linkages, phosphorodithioates modified internucleotide linkages phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclicintersugar linkages. In some embodiments, oligonucleotides comprise phosphorothioate backbones, or heteroatom backbones, e.g., CH2NHOCH2, CH2N(CH3)OCH2[known as a methylene(methylimino) or MM backbone], CH2ON(CH3)CH2, CH2N(CH3)N(CH3)CH2and / or ON(CH3)CH2CH2backbones, wherein the native phosphodiester backbone is represented as O-P- O-CH2. In some embodiments, oligonucleotides have a polyamide backbone, by which the nucleotides being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone. In some embodiments, a modified oligonucleotide backbone comprises, but is not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising 3 'alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 3 ' -amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3 -5' linkages, 2-5 ' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleotide units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2. Various salts, mixed salts and free acid forms are also included. In some instances, a PS bond creates a new stereocenter in the nucleotide and when synthesized under standard achiral conditions creates diastereomeric mixtures of Rp and Sp at the phosphorous atom.

[0119] In some embodiments, modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleotide linkages, mixed heteroatom and alkyl or cycloalkyl internucleotide linkages, or one or more short chain heteroatomic or heterocyclic internucleotide linkages. These comprise those having morpholino linkages (formed in part from the sugar portion of a nucleotide); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S, and CH2component parts. In some instances, the modified internucleotide linkage (or backbone) is uncharged one, such as phosphorodiamidate morpholino oligomer (PMO), peptide nucleic acid (PNA), phosphotriesters, and phosphonates. It has been postulated that the uncharged analogues are not only nuclease resistant, but can also be more membrane permeable; however, the size and hydrophilicity of uncharged oligonucleotides still preclude their passive diffusion across membranes.

[0120] In some embodiments, both the sugar and the internucleotide linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound,an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar -backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleotides are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.Base modification

[0121] In some embodiments, the oligonucleotides comprise a base (often referred to in the art simply as "base") modifications or substitutions. In some embodiments, unmodified or natural nucleotides include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U). In some embodiments, a nucleotide is present in an oligonucleotide herein to increase the binding affinity of the oligonucleotide to its target. By way of non -limiting example, the nucleotide can comprise 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6, and 0-6 substituted purines, comprising 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5 -methylcytosine substitutions can increase nucleic acid duplex stability, e.g., by 0.6-1.2°C. In some cases, 5- methylcytosine substitutions are combined with 2'-O-methoxyethyl sugar modifications.

[0122] In some instances, base modifications are designed to increase base pairing. For example, G-clamp, which is a cytidine mimic is designed to have increased affinity for guanosine bases due to hydrogen bonding through an aminoethyl group. C-5 propynyl pyrimidines are known to form more stable duplexes; however, they appear to be more toxic as well. In some embodiments, an oligonucleotide comprises a base modification at the 1 ’ position.

[0123] In some embodiments, modified nucleotides include nucleotides found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6 -methyladenine, 5 -Me pyrimidines, particularly 5 -methylcytosine (also referred to as 5 -methyl-2'-deoxy cytosine and often referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, as well as synthetic nucleotides, e.g., 2 -aminoadenine, 2-(methylamino)adenine, 2- (imidazolylalkyl)adenine, 2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines, 2-thiouracil, 2-thiothymine, 5- bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7- deazaguanine, N6-(6-aminohexyl)adenine and 2,6-diaminopurine. In some cases, a universal base, e.g., inosine, can be included. In some cases, the oligonucleotide comprises a 5-Me-C substitution, where this substitution can increase nucleic acid duplex stability, e.g., by 0.6-1 ,2°C. Modified nucleotides comprise other synthetic and natural nucleotides such as 5 -methylcytosine (5-me-C), 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2 -aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2 -propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2 -thiocytosine, 5-halouracil and cytosine, 5- propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5 -uracil (pseudo-uracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8 -hydroxyl and other 8 -substituted adenines and guanines, 5-halo particularly 5-bromo, 5 -trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylquanine and 7 -methyladenine, 8 -azaguanine and 8 -azaadenine, 7- deazagnanine and 7-deazaadenine and 3 -deazaguanine and 3 -deazaadenine.

[0124] In some embodiments, it is not necessary for all positions in a given oligonucleotide to be uniformly modified. For example, in some cases, more than one of the modifications described herein can be incorporated in a single oligonucleotide or even within a single nucleotide ofthe oligonucleotide.

[0125] The oligonucleotides described herein can be made through solid phase synthesis. Equipment for such synthesis is sold by several vendors including Applied Biosystems. Any other means for such synthesis can also be employed. In some cases, oligonucleotides comprising phosphorothioates and alkylated derivatives are prepared. Similar techniques and commercially available modified amidites and controlled-pore glass (CPG) products such as biotin, fluorescein, acridine or psoralen-modified amidites and / or CPG (available from Glen Research, Sterling VA) can be used to synthesize fluorescently labeled, biotinylated or other modified oligonucleotides such as cholesterol-modified oligonucleotides.Double-Stranded siRNAs

[0126] In some instances, the oligonucleotide is a double-stranded RNA (dsRNA) that targets CD33 mRNA and downregulate the expression of the CD33 mRNA. In some instances, the oligonucleotide is a dsRNA having a sense strand (passenger strand) and an antisense strand (guide strand). In some instances, the oligonucleotide is a siRNA having a sense strand (passenger strand) and an antisense strand (guide strand). In some embodiments, each strand of the dsRNA or siRNA comprises from 12-30 nucleotides in length. In some cases, each strand is between 14-30 nucleotides in length, 17-30 nucleotides in length, 25-30 nucleotides in length, 27-30 nucleotides in length, 17-23 nucleotides in length, 17-21 nucleotides in length, 17-19 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length, or 21 -23 nucleotides in length. In some embodiments, the sense strand and antisense strand form a duplex dsRNA. The duplex region of a dsRNA agent can be 12-30 nucleotide pairs in length. For example, the duplex region can be between 14 -30 nucleotide pairs in length, 17-30 nucleotide pairs in length, 25-30 nucleotides in length, 27-30 nucleotide pairs in length, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs in length, 17- 19 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs in length, 21 -25 nucleotide pairs in length, or 21 -23 nucleotide pairs in length. In an example, the duplex region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, and 27. In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 30 nucleotides.

[0127] In some embodiments, the oligonucleotide is a dsRNA or siRNA having a sense strand (passenger strand) and an antisense strand (guide strand). In some embodiments, the antisense strand of the dsRNA or siRNA comprises or consists of a nucleic acid sequence of any one of SEQ ID NOs: 123-432. In some embodiments, the antisense strand of the dsRNA or siRNA comprises an oligonucleotide consisting of 12 to 30 linked nucleotides and having a nucleic acid sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1, 2, or 3 mismatches from any one of the nucleic acid sequences of SEQ ID NOs: 123-432. In some embodiments, the nucleic acid sequence of the antisense strand of the dsRNA or siRNA is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 123 -432.

[0128] In some embodiments, the oligonucleotide is a dsRNA or siRNA, having a sense strand (passenger strand) and an antisense strand (guide strand). In some embodiments, the sense strand of the dsRNA or siRNA comprises or consists of a nucleic acid sequence that is complementary to any one of SEQ ID NOs: 123-432. In some embodiments, the sense strand of the dsRNA or siRNA comprises an oligonucleotide consisting of 12 to 30 linked nucleotides and having a nucleic acid sequence complementary to at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1, 2, or 3 mismatches from any of the nucleic acid sequences of SEQ ID NOs: 123-432. In some embodiments, the nucleic acid sequence of the sense strand of the dsRNA or siRNA is complementary at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 123-432.

[0129] In some embodiments, the dsRNA or siRNA comprises one or more overhang regions and / or capping groups at the 3 ’-end, or 5 ’-end, or both ends of a strand. The overhang can be 1 -6 nucleotides in length, for instance 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5 nucleotides in length, 1 -4 nucleotides in length, 2-4 nucleotides in length, 1 -3 nucleotides in length, 2-3 nucleotides in length, or 1 -2 nucleotides in length. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. The overhang can form a mismatch with the target mRNA or it can be complementaryto the gene sequences being targeted or can be other sequence. The first and second strands can also be joined, e.g., by additional bases to form a hairpin, or by other non -base linkers. In some embodiments, the nucleotides in the overhang region of the dsRNA agent are each independently a modified or unmodified nucleotide including, but no limited to 2'-sugar modified, such as, 2’- fluoro, 2'-O-methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine (Teo), 2'-O- methoxy ethyladenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo), 2’-O- methyoxyethyl (MOE) and any combinations thereof. For example, TT can be an overhang sequence for either end on either strand. The overhang can form a mismatch with the target mRNA or it can be complementary to the gene sequences being targeted or can be other sequence. In some embodiments, the 5'- or 3'- overhangs at the sense strand, antisense strand or both strands of the dsRNA or siRNA can be phosphorylated. In some embodiments, the overhang region contains two nucleotides having a phosphorothioate between the two nucleotides, where the two nucleotides can be the same or different. In some embodiments, the overhang is present at the 3 ’-end of the sense strand, antisense strand or both strands. In some embodiments, this 3 ’ - overhang is present in the antisense strand. In some embodiments, this 3 ’ -overhang is present in the sense strand. The dsRNA can comprise a blunt end, located at the 5’-end of the antisense strand (or the 3 ’-end of the sense strand) or vice versa. In some cases, the antisense strand of the dsRNA has a nucleotide overhang at the 3 ’-end, and the 5’-end is blunt. While not bound by theory, the asymmetric blunt end at the 5 ’ -end of the antisense strand and 3 ’ -end overhang of the antisense strand favor the guide strand loading into RISC process. In some embodiments, the dsRNA or siRNA can also have two blunt ends, at both ends of the dsRNA or siRNA duplex.

[0130] In some embodiments, stabilization of synthetic siRNA against rapid nuclease degradation is a prerequisite for in vivo and therapeutic applications. This can be achieved using a variety of stabilization chemistries previously developed for other nucleic acid drugs, such as ribozymes and antisense molecules. These include chemical modifications to the native 2'-OH group in the ribose sugar backbone, such as 2'-O-methyl (2'-0Me) and 2'-fluoro (2'-F) substitutions that can be readily introduced into siRNA as 2'-modified nucleotides during RNA synthesis. In some embodiments, chemically stabilized siRNA comprises a 2'-0Me, 2'-F, 2'- deoxy, or “locked nucleic acid” (LN A). Such modifications can be designed to retain functional RNAi activity. In some embodiments, the modification is tolerated only in certain ill -defined positional or sequence-related contexts. In some embodiments, the introduction of chemical modifications to native siRNA duplexes can have a negative impact on RNAi activity. Therefore, in some cases, the design of chemically modified siRNA can require a stochastic screening approach to identify duplexes that retain potent gene silencing activity. In some embodiments, inhibition of cleavage of the sense strand impairs the endonucleolytic cleavage of target mRNA.In some embodiments, incorporation of a 2'-0-Me ribose to the Ago2 cleavage site in the sense strand inhibits RNAi in HeLa cells. In some embodiments, for phosphorothioate modifications, cleavage of the sense strand is required for efficient RNAi. In some embodiments, a siRNA duplex comprises 2'-F modified residues, among other sites and modifications, also at the Ago2 cleavage site, where compatible silencing is achieved as compared to the unmodified siRNAs. In some cases, this modification is not motif specific, e.g., one modification includes 2'-F modifications on all pyrimidines on both sense and antisense strands as long as pyrimidine residue is present. In some embodiments, specific motif modification at the cleavage site of sense strand does not have an effect on gene silencing activity. In some embodiments, a siRNA duplex comprises two 2'-F modified residues at the Ago2 cleavage site on the sense or antisense strand. In some cases, the modification is sequence specific, e.g., for each particular strand, all pyrimidines or all purines, are modified. In some embodiments, a siRNA duplex comprises alternative modifications by 2'-0Me or various combinations of 2'-F, 2'-0Me and phosphorothioate modifications to stabilize siRNA in serum. In some cases, the residues at the cleavage site of the antisense strand are not modified with 2'-0Me in order to increase the stability of the siRNA.

[0131] In some embodiments, every nucleotide in the sense strand and antisense strand of dsRNA or siRNA, including the nucleotides that are part of the motifs, is modified. Each nucleotide can be modified with the same or different modification which can include one or more alteration of one or both of the non-linking phosphate oxygens and / or of one or more of the linking phosphate oxygens; alteration of a constituent of the ribose sugar, e.g., of the 2' hydroxyl on the ribose sugar; wholesale replacement of the phosphate moiety with "dephospho" linkers; modification or replacement of a naturally occurring base; and / or replacement or modification of the ribose-phosphate backbone. In some embodiments, the modified siRNA comprises modified nucleotides including, but not limited to, 2'-0Me nucleotides, 2'-deoxy-2'-fluoro (2'-F) nucleotides, 2 '-deoxy nucleotides, 2 '-O-(2 -meth oxy ethyl) (MOE) nucleotides, locked nucleic acid (LNA) nucleotides, and mixtures thereof. In some embodiments, the modified siRNA comprises 2'-0Me nucleotides (e.g., 2'-0Me purine and / or pyrimidine nucleotides) such as, for example, 2'-OMe-guanosine nucleotides, 2'-OMe-uridine nucleotides, 2'-OMe-adenosine nucleotides, 2'-OMe-cytosine nucleotides, and mixtures thereof. In certain instances, the modified siRNA does not comprise 2'-OMe-cytosine nucleotides. In some embodiments, the modified siRNA comprises a hairpin loop structure. In some embodiments, the modified siRNA comprises 3 ' overhangs of one, two, three, four, or more nucleotides on one or both sides of the double-stranded region, or can lack overhangs (i.e., haveblunt ends). In some cases, the modified siRNA has 3 ' overhangs of two nucleotides on each side of the double-stranded region. In certaininstances, the 3 ' overhang on the antisense strand has complementarity to the target sequence and the 3 ' overhang on the sense strand has complementarity to the complementary strand of the target sequence. In certain instances, the 3 ' overhangs do not have complementarity to the target sequence or the complementary strand thereof. In some embodiments, the 3' overhangs comprise one, two, three, four, or more nucleotides such as 2'-deoxy(2'H) nucleotides. In some cases, the 3 ' overhangs comprise deoxythymidine (dT) nucleotides. In some embodiments, the modified siRNA comprises from about 1% to about 100% (e.g., about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) modified nucleotides in the double-stranded region of the siRNA duplex. In some embodiments, less than about 30% (e.g., less than about 30%, 25%, 20%, 15%, 10%, or 5%) or from about 1% to about 30% (e.g., from about l%-30%, 5%-30%, 10%-30%, 15%-30%, 20%-30%, or 25%- 30%) of the nucleotides in the double -stranded region comprise modified nucleotides.

[0132] In some embodiments, the modified siRNA does not comprise phosphate backbone modifications, e.g., in the sense and / or antisense strand of the double -stranded region. In some embodiments, the modified siRNA does not comprise 2'-deoxy nucleotides, e.g., in the sense and / or antisense strand of the double-stranded region. In certain instances, the nucleotide at the 3 '-end of the double-stranded region in the sense and / or antisense strand is not a modified nucleotide. In certain instances, the nucleotides near the 3 '-end (e.g., within one, two, three, or four nucleotides of the 3 '-end) of the double-stranded region in the sense and / or antisense strand are not modified nucleotides.

[0133] As nucleic acids are polymers of subunits, in some cases, a modification occurs at a position which is repeated within a nucleic acid, e.g., a modification of a base, or a phosphate moiety, or a non-linking O of a phosphate moiety. In some cases, the modification occurs at all of the subject positions in the nucleic acid. In some cases, the modification does not occur at all of the subject positions in the nucleic acid. By way of a non -limiting, example, a modification can only occur at a 3 ’ or 5 ’ terminal position, can only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. A modification can occur in a double strand region, a single strand region, or in both. A modification can occur only in the double strand region of an RNA or can only occur in a single strand region of an RNA. For example, a phosphorothioate modification at a non-linking O position can only occur at one or both termini, can only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, or can occur in double strand and single strand regions, particularly at termini. The 5' end or ends can be phosphorylated.

[0134] In some cases, particular bases are included in overhangs, e.g., modified nucleotides or nucleotide surrogates, in single strand overhangs, e.g., in a 5' or 3' overhang, or in both. This canenhance stability of the composition. For example, purine nucleotides are included in overhangs. In some embodiments, all or some of the bases in a 3' or 5' overhang can be modified, e.g., with a modification described herein. Modifications can include, e.g., the use of modifications at the 2' position of the ribose sugar, e.g., the use of deoxyribonucleotides, 2' -deoxy -2' -fluoro (2'-F) or 2'- O-methyl modified instead of the ribosugar of the nucleotide, and modifications in the phosphate group, e.g., phosphorothioate modifications. In some cases, overhangs need not be homologous with the target sequence.

[0135] In some embodiments, each residue of an oligonucleotide described herein is independently modified with LNA, HNA, CeNA, 2'-methoxy ethyl, 2'-O-methyl, 2'-O-ally 1, 2'-C- allyl, 2'-deoxy, or 2'-fluoro. The strands can contain more than one modification. In some embodiments, each residue of an oligonucleotide described herein is independently modified with 2'-O-methyl or 2' -fluoro. In some embodiments, at least two different modifications are present on an oligonucleotide described herein. Those two modifications can be the 2'-O-methyl or 2' -fluoro modifications, or others. In some embodiments, an oligonucleotide described herein each comprise two differently modified nucleotides selected from 2'-O-methyl or 2'-fluoro. In some embodiments, each residue of an oligonucleotide described herein is independently modified with 2'-O-methyl nucleotide, 2' -deoxy fluoro nucleotide, 2 ’-0 -methoxy ethyl (MOE), 2’- O-N-methylacetamido (2'-0-NMA) nucleotide, a 2'-O-dimethylaminoethoxyethyl (2'-O- DMAEOE) nucleotide, 2'-O-aminopropyl (2'-O-AP) nucleotide, or 2'-ara-F nucleotide.

[0136] For RNAi duplexes, recognition by RISC requires RNA-like 3 ’-endo nucleotides and some patterns of RNA analogues. It was observed that a pattern of alternating 2’ -O-methyl groups provides stability against nucleases, but not all permutations of alternating 2’ -O-methyl are active RNAi agents. The fact that one can remove all 2’-hydroxy groups with alternating 2’- fluoro and 2’-O-methyl groupsto produce duplexes that are resistant to nucleases and active in RNAi suggests the 2’ -hydroxy group is not absolutely required for activity, but that some sites in the RNAi duplex are sensitive to the added steric bulk of the methyl group. In some embodiments, an oligonucleotide comprises one or more of the aforementioned patterns.

[0137] In some instances, one or both strands of dsRNA or siRNA disclosed herein include modified backbones, for example, phosphorothioates modified intemucleotide linkages, phosphorodithioates modified internucleotide linkages, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages, or short chain heteroatomic or heterocyclic intersugar linkages. In some embodiments, oligonucleotides comprise phosphorothioate backbones, or heteroatom backbones, e.g., CH2NHOCH2, CH2N(CH3)OCH2[known as a methylene(methylimino) or MM backbone], CH2ON(CH3)CH2, CH2N(CH3)N(CH3)CH2and / or ON(CH3)CH2CH2backbones, wherein the native phosphodiester backbone is represented as O-P-O-CH2. In some embodiments, oligonucleotides have a polyamide backbone, by which the nucleotides being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone. In some embodiments, a modified oligonucleotide backbone comprises, but is not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising 3 'alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 3 ' -amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3 -5' linkages, 2-5 ' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleotide units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2. Various salts, mixed salts and free acid forms are also included. In some instances, a PS bond creates a new stereocenter in the nucleotide and when synthesized under standard achiral conditions creates diastereomeric mixtures of Rp and Sp at the phosphorous atom.Conjugates

[0138] In some embodiments, oligonucleotides comprise a group conjugated- via covalent bonds- that prolongs circulation, provides targeting to tissues and / or facilitates intracellular delivery. Thus, in some embodiments, the oligonucleotide is conjugated with another moiety, e.g., an abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, and / or polyhydrocarbon compounds. In some cases, these molecules are linked to one or more of any nucleotides at one or more positions on the sugar, base or phosphate group (intemucleotide linkage).

[0139] In certain embodiments, the oligonucleotide (e.g., modified oligonucleotide) comprises a carrier system, e.g., to deliver the modified oligonucleotide into a cell of a mammal. Non- limiting examples of carrier systems suitable for use herein include nucleic acid -lipid particles, liposomes, micelles, virosomes, nucleic acid complexes, and mixtures thereof. In certain instances, the modified oligonucleotide molecule is complexed with a lipid such as a cationic lipid to form a lipoplex. In certain instances, the modified oligonucleotide molecule is complexed with a polymer such as a cationic polymer (e.g., polyethylenimine (PEI)) to form a polyplex. The modified oligonucleotide molecule can also be complexed with cyclodextrin or a polymer thereof. In some cases, the modified oligonucleotide molecule is encapsulated in a nucleic acid- lipid particle.

[0140] In some embodiments, the oligonucleotide is chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. These moieties or conjugatescan include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Exemplary conjugate groupsinclude intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, poly ethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Exemplary conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhe, Jamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, include groups that improve uptake, enhance resistance to degradation, and / or strengthen sequence-specific hybridization with the target nucleic acid. Groups that enhance the pharmacokinetic properties, include groups that improve uptake, distribution, metabolism or excretion of the compounds provided herein. Conjugate moieties include, but are not limited to, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5 -tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O- hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or Adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino -carbonyloxycholesterol moiety. Oligonucleotides can also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)- pranoprofen, carprofen, dansylsarcosine, 2,3, 5 -triiodobenzoic acid, flufenamic acid, folinic acid, a benzthiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. In some embodiments, a moiety comprises, but is not limited to, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac- glycero-3-H-phosphonate, a poly amine or a polyethylene glycol chain, or Adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino -carbonyl-t oxycholesterol moiety.

[0141] In some embodiments, an oligonucleotide is conjugated to prolong circulation, e.g., conjugation to polyethylene glycol (PEG), which can prevent clearance by two mechanisms: the increase in molecular weight above threshold for renal clearance and the prevention of nonspecific interactions with extracellular surfacesand serum components. PEG can be incorporated into nucleic acid delivery vehicles by attachment to components that non-covalently associate with the nucleic acids, e.g., PEGylated lipids and polymers; however, directPEG conjugation can be employed to increase nucleic acid circulation times, decrease nonspecific interactions and alter biodistribution; although in some cases, the targeting is passive and the potency of the nucleic acid can be compromised as PEG MW increases.

[0142] Another class of molecules that can be conjugated in order to increases circulation times is the attachment of lipophilic groups such as cholesterol or other lipophilic moiety with >12carbons which interact with serum components such as albumen and lipoproteins thereby increasing circulation times and passive accumulation in the liver. It should be remembered that extensive PS modification increases circulation times through associations with serum components, with roughly 10 PS groups required for serum binding.

[0143] In some embodiments, the oligonucleotide is chemically linked to one or more moieties or conjugates which enhance the activity or cellular uptake of the oligonucleotide. Such moieties include, but are not limited to, lipid moieties such as a cholesterol moiety, a cholesteryl moiety, an aliphatic chain(e.g., dodecandiol or undecyl residues, a polyamine or a polyethylene glycol chain, and adamantane acetic acid.Therapeutic targets and methods of use

[0144] In some embodiments, a composition disclosed herein (e.g., antisense oligonucleotides, splice-switching oligonucleotides) targets CD33, including, without limitation, sense and or antisense noncoding and / or coding sequences associated with CD33 (e.g., CD33 mRNA). In some embodiments, reference to a “composition” refers to an antisense oligonucleotide or spliceswitching oligonucleotide (e.g., CD33 modulator).

[0145] In some embodiments, provided herein are methods for inhibiting the action or expression of a natural transcript by using antisense oligonucleotide(s) targeting a region of the natural transcript. It is also contemplated herein that inhibition of the natural transcript activity or expression can be achieved by siRNA, ribozymes and small molecules, which are considered to be within the scope of this disclosure. The method comprises the step of administering the antisense oligonucleotide in an amount sufficient to a subject or contacting the antisense oligonucleotide to a cell to inhibit expression or activity of the target gene.

[0146] In some embodiments, an oligonucleotide targets a natural sequence of CD33 polynucleotides (for example, nucleic acid sequences set forth in SEQ ID NOs: 1 or 2) and any variants, alleles, homologs, mutants, derivatives, fragments and complementary sequences thereto.

[0147] In some embodiments, a composition comprises one or more antisense oligonucleotides which bind to CD33 mRNA. In some embodiments, the oligonucleotides comprise one or more modified or substituted nucleotides. In some embodiments, the oligonucleotides comprise one or more modified intemucleotide linkage (backbone). In some embodiments, the modified nucleotides comprise phosphorothioate modified internucleotide linkage, methylphosphonate modified internucleotide linkage, peptide nucleic acids, 2'-O-methyl, 2’-O-methoxyethyl, 2'- fluoro- modified nucleotide, or carbon, methylene modified nucleotide, locked nucleic acid (LNA) molecule, UNA (unlocked / open chain nucleic acids), morpholinyl sugar surrogates (e.g.,phosphorodiamidates morpholino oligonucleotide (PMOs)). In some embodiments, the modified nucleotides are locked nucleic acid molecules, including a-L-LNA.

[0148] In some embodiments, the oligonucleotides are administered to a patient intranasally, subcutaneously, intramuscularly, intravenously or intraperitoneally. In some embodiments, the oligonucleotides are administered in a pharmaceutical composition. In some exemplary embodiments, a treatment regimen comprises administering the antisense compounds at least once to patient; however, this treatment can be modified to include multiple doses over a period of time. The treatment can be combined with one or more other types of therapies. In some embodiments, the oligonucleotides are encapsulated in a liposome or attached to a carrier molecule (e.g., cholesterol, TAT peptide). The compositions described herein can be used to treat a patient suffering from a disease or disorder. In some instances, the disease or disorder comprises a neurodegenerative disorder. In some instances, a neurodegenerative disorder comprises neuroinflammation or neuronal death. In some embodiments, a pharmaceutical composition comprises an oligonucleotide (e.g., ASO or SSO) described herein. In some instances, the disease is treated by modulating CD33 expression (e.g., reducing CD33 or modulating the ratio of isoforms).

[0149] Conditions treated by compositions provided herein include, but are not limited to amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), ALS with FTD (FTD- ALS), behavioral variant FTD (bvFTD), primary progressive aphasia (PPA), progressive supranuclear palsy, corticobasal syndrome, Parkinson’s disease (PD), or Alzheimer’s disease (AD). In some instances, the disease comprises a hereditary disease. In some instances, the disease comprises an inflammatory disease.

[0150] Another aspect relates to a method of modulating the expression of a target gene in a cell, comprising providing to the cell an antisense oligonucleotide or splice-switching oligonucleotides described herein. In some embodiments, the target gene is CD33. In some embodiments, modulation of CD33 by one or more antisense oligonucleotides or spliceswitching oligonucleotides is achieved by administering antisense oligonucleotides or spliceswitching oligonucleotides to a patient in need thereof, to prevent or treat a neurological disease or disorder which benefits from modulation of CD33 expression, function, or activity, as compared to a normal control.

[0151] In some embodiments, provided is a method of modulating function and / or expression of an CD33 polynucleotide in mammalian cells or tissues in vivo or in vitro by contacting the cells or tissues with an antisense oligonucleotide of 5 to 30 nucleotides in length, wherein the oligonucleotide has at least 50% sequence identity to a reverse complement of a polynucleotide comprising 5 to 30 consecutive nucleotides with no more than 1, 2, or 3 mismatches withinnucleotides 1-21, 29-48, 94-116, 137-187, 211-241, 263-282, 392-417, 416-435, 456-523, 460- 479, 504-671, 652-671, 683-721, 724-746, 730-753, 731-756, 770-819, 772-807, 857-876, 880- 899, 1050-1138, 1073-1108, 1165-1184, 1189-1266, 1194-1276, 1250-1334, 1270-1360, 1285- 1332, 1336-1360, 1340-1360, 1364-1383, 1388-1452, or 1370-1462 of SEQ ID NO: 1, and any variants, alleles, homologs, mutants, derivatives, fragments and complementary sequences thereby modulating function and / or expression of the CD33 polynucleotide in mammalian cells or tissues in vivo or in vitro.

[0152] In some embodiments, a composition described herein (e.g., ASO) is used in methods for silencing expression of a target sequence. In one aspect, provided herein are in vitro and in vivo methods for treatment of a disease or disorder in a mammal by downregulating or silencing the transcription and / or translation of a target gene of interest. In some embodiments, provided are methods for introducing a composition that silences expression (e.g., mRNA and / or protein levels) of a target sequence into a cell by contacting the cell with an oligonucleotide described herein. In some embodiments, provided are methods for in vivo delivery of an oligonucleotide that silences expression of a target sequence by administering to a mammal an oligonucleotide described herein. Administration of the oligonucleotide can be done by any route known in the art, such as, e.g., oral, intranasal, intravenous, intraperitoneal, intramuscular, intra -articular, intralesional, intratracheal, subcutaneous, or intradermal. In some cases, the oligonucleotide is modified.

[0153] In some embodiments, a composition described herein (e.g., SSO) is used in methods for modulating expression of a target sequence. In one aspect, provided herein are in vitro and in vivo methods for treatment of a disease or disorder in a mammal by controlling splicing of the transcription and / or translation of a target gene of interest. In some instance modulating gene expression comprises increasing or decreasing the amount of specific mRNA / protein isoforms. In some instances, modulating gene expression comprises decreasing harmful isoforms and increasing beneficial isoforms. In some instances, SSOs modulate exon inclusion in pre -mRNA. In some embodiments, provided are methods for introducing a composition that modulates expression (e.g., mRNA and / or protein levels) of a target sequence into a cell by contacting the cell with an oligonucleotide described herein. In some embodiments, provided are methods for in vivo delivery of an oligonucleotide that modulates expression of a target sequence by administering to a mammal an oligonucleotide described herein. Administration of the oligonucleotide can be by any route known in the art, such as, e.g., oral, intranasal, intravenous, intraperitoneal, intramuscular, intra-articular, intralesional, intratracheal, subcutaneous, or intradermal. In some cases, the oligonucleotide is modified.

[0154] Methods described herein can comprise administration of a therapeutically -effective amount. In some instances, a therapeutically -effective amount comprises an amount of a compound, material, or composition comprising a compound herein which is effective for producing some desired therapeutic effect in at least a sub -population of cells in an animal at a reasonable benefit / risk ratio applicable to any medical treatment.

[0155] Drug discovery: The compounds described herein can also be applied in the areas of drug discovery and target validation. In some embodiments, the compounds and target segments identified herein are useful in drug discovery efforts to elucidate relationships that exist b etween CD33 mRNAs or transcripts and a disease state, phenotype, or condition. These methods include detecting or modulating CD33 mRNAs or transcripts comprising contacting a sample, tissue, cell, or organism with the compounds described herein, measuring the nucleic acid or protein level of CD 33 mRNAs or transcripts and / or a related phenotypic or chemical endpoint at some time after treatment, and optionally comparing the measured value to a non-treated sample or sample treated with a further compound described herein. These methods can also be performed in parallel or in combination with other experiments to determine the function of unknown genes for the process of target validation or to determine the validity of a particular gene product as a target for treatment or prevention of a particular disease, condition, or phenotype. Assessing upregulation or down-regulation of gene expression: Transfer of an exogenous nucleic acid into a host cell or organism can be assessed by directly detecting the presence of the nucleic acid in the cell or organism. Such detection can be achieved by several methods well known in the art. For example, the presence of the exogenous nucleic acid can be detected by Southern blot or by a polymerase chain reaction (PCR) technique using primers that specifically amplify nucleotide sequences associated with the nucleic acid. Expression of the exogenous nucleic acids can also be measured using conventional methods including gene expression analysis. For instance, mRNA produced from an exogenous nucleic acid can be detected and quantified using a Northern blot and reverse transcription PCR (RT-PCR).

[0156] Expression of RNA from the exogenous nucleic acid can also be detected by measuring an enzymatic activity or a reporter protein activity. For example, antisense modulatory activity can be measured indirectly as a decrease or increase in target nucleic acid expression as an indication that the exogenous nucleic acid is producing the effector RNA. Based on sequence conservation, primers can be designed and used to amplify coding regions of the target genes. Initially, the most highly expressed coding region from each gene can be used to build a model control gene, although any coding or non-coding region can be used. Each control gene is assembled by inserting each coding region between a reporter coding region and its poly(A) signal. These plasmids would produce an mRNA with a reporter gene in the upstream portion ofthe gene and a potential ASO or SSO target in the 3' non-coding region. The effectiveness of individual oligonucleotides would be assayed by modulation of the reporter gene. Reporter genes useful in the methods herein include acetohydroxy acid synthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), horseradish peroxidase (HRP), luciferase (Lac), nopaline synthase (NOS), octopine synthase (OCS), and derivatives thereof. Multiple selectable markers are available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, and tetracycline. Methods to determine modulation of a reporter gene are well known in the art, and include, but are not limited to, fluorometric methods (e.g., fluorescence spectroscopy, Fluorescence Activated Cell Sorting (FACS), fluorescence microscopy), and antibiotic resistance determination.

[0157] In some embodiments, CD33 expression (e.g., mRNA or protein) in a sample (e.g., cells or tissues in vivo or in vitro) treated using an ASO or SSO described herein is evaluated by comparison with CD33 expression in a control sample. For example, expression of the protein or nucleic acid can be compared using methods known to those of skill in the art with that in a mock-treated or untreated sample. Alternatively, comparison with a sample treated with a control ASO or SSO (e.g., one having an altered or different sequence) can be made depending on the information desired. In some embodiments, a difference in the expression of the CD33 protein or nucleic acid in a treated vs. an untreated sample can be compared with the difference in expression of a different nucleic acid (including any standard deemed appropriate by the researcher, e.g., a housekeeping gene) in a treated sample vs. an untreated sample. CD33 protein and mRNA expression can be assayed using methods known to those of skill in the art and described elsewhere herein. For example, immunoassays such as the ELISA can be used to measure protein levels. CD33 ELISA assay kits are available commercially.

[0158] Observed differences can be expressed as desired, e.g., in the form of a ratio or fraction, for use in a comparison with a control. In some embodiments, the level of CD33 mRNA or protein, in a sample treated with an ASO or SSO described herein, is increased or decreased by about 1.25 -fold to about 10-fold or more relative to an untreated sample or a sample treated with a control oligonucleotide. In some embodiments, the level of CD33 mRNA or protein is increased or decreased by at least about 1.25-fold, at least about 1 .3 -fold, at least about 1 .4-fold, at least about 1.5 -fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5 -fold, at least about 5 -fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5 -fold, at least about 7-fold, at least about 7.5 -fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, or at least about 10-fold or more.

[0159] Observed differences can be expressed as a percentage of downregulation. In some embodiments, the level of CD33 mRNA or protein, in a sample treated with an ASO or SSO described herein results in a percentage of downregulation of at least 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or at least 95 percent. In some embodiments, the level of CD33 mRNA or protein, in a sample treated with an ASO or SSO described herein results in a percentage of downregulation of 1-95, 2-90, 5-90, 5-85, 5-80, 10-90, 10-80, 10-70, 20-80, 20-70, 30-80, 30-70, 40-90, 40-80, 50-60, 50-90, 50-95, 60-90, 60-95, or 70-95 percent.

[0160] Observed differences can be expressed as a percent transcript level (i.e., the inverse of percentage of downregulation). In some embodiments, the level of CD33 mRNA or protein, in a sample treated with an ASO or SSO described herein results in a percent transcript level of no more than 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or no more than 95 percent. In some embodiments, the level of CD 33 mRNA or protein, in a sample treated with an ASO or SSO described herein results in a percent transcript level of 1-95, 2-90, 5-90, 5-85, 5-80, 10-90, 10-80, 10-70, 20-80, 20-70, 30-80, 30-70, 40-90, 40-80, 50-60, 50-90, 50-95, 60-90, 60-95, or 70-95 percent.

[0161] Observed differences in splice-switching can be expressed as desired, e.g., in the form of a ratio or fraction, for use in a comparison with control. In some embodiments, the level of a specific CD33 isoform encoding for mRNA or protein, in a sample treated with an SSO described herein, is increased or decreased by about 1.25 -fold to about 10-fold or more relative to an untreated sample or a sample treated with a control oligonucleotide. In some embodiments, the level of CD33 mRNA or protein isoform is increased or decreased by at least about 1.25 -fold, at least about 1.3 -fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7 -fold, at least about 1.8-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5 -fold, at least about 4 -fold, at least about 4.5-fold, at least about 5- fold, at least about 5.5 -fold, at least about 6-fold, at least about 6.5 -fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, or at least about 10-fold or more.

[0162] In some instances, splice switching can be defined as a percentage of relative inclusion (e.g., of an exon such as exon 2). In some embodiments, the level of CD33 mRNA or protein, in a sample treated with an SSO described herein results in a relative inclusion percentage of no more than 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80 or no more than 90 percent. In some embodiments, the level of CD33 mRNA or protein, in a sample treated with an SSO describedherein results in a relative inclusion percentage of 1-95, 2-90, 5-90, 5-85, 5-80, 10-90, 10-80, 10- 70, 20-80, 20-70, 20-50, 30-80, 30-70, 30-50, 40-90, 40-80, 40-70, 40-60, 50-60, 50-70, 60-95, 70-95, or 80-95 percent. In some instances, splice switching can be defined as a percentage of relative inclusion inhibition (i.e., the inverse of relative inclusion). In some embodiments, the level of CD33 mRNA or protein, in a sample treated with an SSO described herein results in a relative inclusion percentage of at least 1, 2, 5, 10, 15, 20, 25 , 30, 35, 40, 50, 60, 70, 80 or at least 90 percent. In some embodiments, the level of CD33 mRNA or protein, in a sample treated with an SSO described herein results in a relative inclusion percentage of 1 -95, 2-90, 5-90, 5-85, 5-80, 10-90, 10-80, 10-70, 20-80, 20-70, 20-50, 30-80, 30-70, 30-50, 40-90, 40-80, 40-70, 40-60, or 50-60 percent.Formulations, dosing, and administrationPharmaceutical compositions

[0163] The antisense oligonucleotides and splice -switching oligonucleotides described herein can be formulated for pharmaceutical use. In some aspects, pharmaceutically acceptable compositions comprise a therapeutically -effective amount of one or more of the compounds in any of the embodiments herein, taken alone or formulated together with one or more pharmaceutically acceptable carriers (additives), excipient and / or diluents. In some embodiments, a pharmaceutically acceptable composition provided herein comprises a base nucleic acid sequence of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1, 2, or 3 mismatches from any of the nucleic acid sequences of SEQ ID NOs: 7-64 or 123-432. In some embodiments, a pharmaceutically acceptable composition provided herein comprises a base nucleic acid sequence of atleast 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 , or at least 20 consecutive nucleotides with no more than 1, 2, or 3 mismatches from any of the nucleic acid sequences of SEQ ID NOs: 16-22, 32-36, 38-42, or 44. In some embodiments, a pharmaceutically acceptable composition provided herein comprises a base nucleic acid sequence of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1, 2, or 3 mismatches from any of the nucleic acid sequences of SEQ ID NOs: 125, 146, 148-152, 158, 182-186, 189, 191, 193, 197, 203, 222-226, 241-260, 262-266, 271, 272, 274-281, 283, 286, 287, 300, 301, 311-319, 321, 322, 328-335, 344, 345, 347-351, 377-381, 390, 391, 395-397, 400-409, 412, 413, 415-420, or 422. In some embodiments, a pharmaceutically acceptable composition provided herein comprises a base nucleic acid sequence of at least 8, atleast 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1, 2, or 3 mismatches from any of the nucleic acid sequences of SEQ ID NOs: 313, 316, 317, 319, 347- 349, 402, 417, or 418. In some embodiments, a pharmaceutically acceptable composition provided herein comprises a compound comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1 , 2, or 3 mismatches from any of the nucleic acid sequences of SEQ ID NOs: 65-122 or 433-742. In some embodiments, a pharmaceutically acceptable composition provided herein comprises a compound comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1, 2, or 3 mismatches from any of the nucleic acid sequences of SEQ ID NOs: 74-80, 90-94, 96-100, or 102. In some embodiments, a pharmaceutically acceptable composition provided herein comprises a compound comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1, 2, or 3 mismatches from any of the nucleic acid sequences of SEQ ID NOs: 435, 456, 458-462, 468, 492-496, 499, 501, 503, 507, 513, 532-536, 551-570, 572- 576, 581, 582, 584-591, 593, 596, 597, 610, 611, 621 -629, 631, 632, 638-645, 654, 655, 657-661, 687-691, 700, 701, 705-707, 710-719, 722, 723, 725-730, or 732. In some embodiments, a pharmaceutically acceptable composition provided herein comprises a compound comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1, 2, or 3 mismatches from any of the nucleic acid sequences of SEQ ID NOs: 623, 626, 627, 629, 657-659, 712, 727, or 728. In some instances, the nucleic acid sequence of a modified oligonucleotide present in a pharmaceutically acceptable composition is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 65-122 or 433-742.

[0164] The pharmaceutical compositions can be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (3) topical application, for example, as a cream, ointment, or a controlled- release patch or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8) nasally. Delivery using subcutaneous or intravenous methods can be particularly advantageous. In some instances, a pharmaceutical composition is formulated for parenteral administration.

[0165] In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. In some cases, this can be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, can depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

[0166] Methods described herein comprise compositions and treatments which are pharmaceutically acceptable. In some instances, pharmaceutically acceptable refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0167] Methods and compositions described herein comprise a pharmaceutically-acceptable carrier. In some instances, a pharmaceutically-acceptable carrier comprises pharmaceutically- acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically - acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium state, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen -free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and / or poly anhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; and (22) other non -toxic compatible substances employed in pharmaceutical formulations.

[0168] In certain embodiments, a formulation comprises an excipient selected from the group consisting of cyclodextrins, celluloses, liposomes, micelle forming agents, e.g., bile acids, and polymeric carriers, e.g., polyesters and polyanhydrides; and a compound described herein. In certain embodiments, an aforementioned formulation renders orally bioavailable a compound described herein.

[0169] The formulations can conveniently be presented in unit dosage form and can be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 0.1 percent to about 99 percent of active ingredient, from about 5 percent to about 70 percent, or from about 10 percent to about 30 percent.

[0170] An agent preparation can be formulated in combination with another agent, e.g., another therapeutic agent or an agent that stabilizes an ASO, SSO or siRNA, e.g., a protein that complexes with an ASO, SSO, or siRNA to form an iRNP. Still other agents include chelators, e.g., EDTA (e.g., to remove divalent cations such as Mg2+), salts, RNase inhibitors (e.g., a broad specificity RNase inhibitor such as RNAsin) and so forth.

[0171] Methods of preparing these formulations or compositions include the step of bringing into association a compound described herein with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound described herein with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0172] The compounds described herein can be formulated for administration in any convenient way for use in human or veterinary medicine, by analogy with other pharmaceuticals.

[0173] ASO, SSO or siRNA can be produced in a cell in vivo, e.g., from exogenous DNA templates that are delivered into the cell. For example, the DNA templates can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (U.S. Pat. No. 5,328,470), or by stereotactic injection (see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91 :3054 -3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector inan acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. The DNA templates, for example, can include two transcription units, one that produces a transcript that includes the top strand of a dsRNA agent and one that produces a transcript that includes the bottom strand of a dsRNA agent. When the templates are transcribed, the dsRNA agent is produced, and processed into siRNA agent fragments that mediate gene silencing.Definitions

[0174] All genes, gene names, and gene products disclosed herein are intended to correspond to homologs from any species for which the compositions and methods disclosed herein are applicable. Thus, the terms include, but are not limited to genes and gene products from humans and mice. It is understood that when a gene or gene product from a particular species is disclosed, this disclosure is intended to be exemplary only, and is not to be interpreted as a limitation unless the context in which it appears clearly indicates. Thus, for example, for the genes disclosed herein, which in some embodiments relate to mammalian nucleic acid and amino acid sequences are intended to encompass homologous and / or orthologous genes and gene products from other animals including, but not limited to other mammals, fish, amphibians, reptiles, and birds. In an embodiment, the genes or nucleic acid sequences are human.

[0175] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "including", "includes", "having", "has", "with", or variants thereof are used in either the detailed description and or the claims, such terms are intended to be inclusive in a manner similar to the term "comprising."

[0176] The term "about" or "approximately" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, per the practice in the art. In some cases, "about" can mean a range of up to 20%, up to 10%, up to 5%, and up to 1% of a given value. In some cases, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, within 5-fold, or within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term "about" meaning within an acceptable error range for the particular value should be assumed.

[0177] In some embodiments, "mRNA" means the presently known mRNA transcript(s) of a targeted gene, and any further transcripts which can be elucidated.

[0178] In some embodiments, "antisense oligonucleotides" or "antisense compound" is meant an RNA or DNA molecule that binds to another RNA or DNA (target RNA, DNA). For example, if it is an RNA oligonucleotide it binds to another RNA target by means of RNA-RNA interactions and alters the activity of the target RNA. An antisense oligonucleotide can upregulate or downregulate expression and / or function of a particular polynucleotide. In some cases, an antisense oligonucleotide includes any foreign RNA or DNA molecule which is useful from a therapeutic, diagnostic, or other viewpoint. Such molecules include, for example, antisense RNA or DNA molecules, interference RNA (RNAi), micro-RNA, decoy RNA molecules, siRNA, enzymatic RNA, therapeutic editing RNA and agonist and antagonist RNA, antisense oligomeric compounds, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligomeric compounds that hybridize to at least a portion of the target nucleic acid. In some cases, these compounds can be introduced in the form of single-stranded, double-stranded, partially single-stranded, or circular oligomeric compounds.

[0179] In some embodiments, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. The term “oligonucleotide” also includes linear or circular oligomers of natural and / or modified monomers or linkages, including deoxyribonucleotides, ribonucleotides, substituted and alpha-anomeric forms thereof, peptide nucleic acids (PNA), locked nucleic acids (LNA), phosphorothioate, methylphosphonate, and the like. Oligonucleotides are capable of specifically binding to a target polynucleotide by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type of base pairing, Hoogsteen or reverse Hoogsteen types of base pairing, or the like.

[0180] In some embodiments, "dsRNA", "siRNA", and "iRNA agent" are used interchangeably to agents that can mediate silencing of a target RNA, e.g., mRNA, e.g., a transcript of a gene that encodes a protein. Such a gene can also be referred to as a target gene. In some cases, the RNA to be silenced is an endogenous gene or a pathogen gene. In addition, RNAs other than mRNA, e.g, tRNAs and viral RNAs, can also be targeted.

[0181] In some embodiments, "CD33" and "SIGLEC-3" are inclusive of all family members, mutants, isoforms, alleles, fragments, species, coding and noncoding sequences, and sense and antisense polynucleotide strands of CD33.

[0182] In some embodiments, "CD33" and " SIGLEC-3", are considered the same as in the literature and are used interchangeably in the present application.

[0183] In some embodiments, "oligonucleotide specific for" or "oligonucleotide which targets" refers to an oligonucleotide having a sequence (i) capable of forming a stable complex with a portion of the targeted gene, and / or (ii) capable of forming a stable duplex with a portion of amRNA transcript of the targeted gene. Stability of the complexes and duplexes can be determined by theoretical calculations and / or in vitro assays.

[0184] In some embodiments “parenteral” administration means administration through injection (e.g., bolus injection) or infusion. Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, or intracranial administration, e.g., intrathecal or intracerebroventricular administration.

[0185] The term "treatment" is intended to encompass also prophylaxis, therapy and cure. The patient receiving this treatment is any animal in need, including primates, in particular humans, and other mammals such as equines, cattle, swine and sheep; and poultry and pets in general.

[0186] In some embodiments, "target nucleic acid" encompasses DNA, RNA (e.g., pre-mRNA, mRNA) transcribed from such DNA, and also cDNA derived from such RNA, coding, non coding sequences, sense, and antisense polynucleotides. The specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid. This modulation of function of a target nucleic acid by compounds, which specifically hybridize to it, is generally referred to as "antisense". The functions of DNA to be interfered include, for example, replication and transcription. The functions of RNA to be interfered, include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which can be engaged in or facilitated by the RNA. The overall effect of such interference with target nucleic acid function is modulation of the expression of an encoded product or oligonucleotides. In some instances, the effect of interference is the modulation of alternative splicing to control expression of one or more isoforms.

[0187] In some embodiments, "monomers" indicates monomers linked by phosphodiester bonds or analogs thereof to form oligonucleotides ranging in size from a few monomeric units, e.g., from about 3-4, to about several hundreds of monomeric units. Analogs of phosphodiester linkages include: phosphorothioate, phosphorodithioate, methylphosphonates, phosphoroselenoate, phosphoramidate, and the like.

[0188] In some embodiments, "nucleotide" includes naturally occurring nucleotides as well as non-naturally occurring, or non-canonical nucleotides. It should be clear to the person skilled in the art that various nucleotides which previously have been considered "non-naturally occurring" have subsequently been found in nature. Thus, "nucleotides" includes not only the known purine and pyrimidine heterocycles-containing molecules, but also heterocyclic analogues and tautomers thereof. Illustrative examples of other types of nucleotides are molecules containing adenine, guanine, thymine, cytosine, uracil, purine, xanthine, 2 -aminopurine, 8-oxo-N6-methyladenine, 7-deazaxanthine, 7-deazaguanine, N4,N4-ethanocytosin, N6,N6-ethano-2,6-diaminopurine, 5- methylcytosine, 5-(C3-C6)-alkynylcytosine, 5 -fluorouracil, 5 -bromouracil, pseudoisocytosine, 2- hydroxy-5-methyl-4-triazolopyridine, isocytosine, isoguanin, inosine and the "non -naturally occurring" nucleotides described in U.S. Pat No. 5,432,272 or US2020 / 0318122. The term "nucleotide" is intended to cover every and all of these examples as well as analogues and tautomers thereof. In some cases, nucleotide refers to an adenine, guanine, thymine, cytosine, or uracil, which are considered the naturally occurring nucleotides in relation to therapeutic and diagnostic application in humans. Nucleotides include the natural 2' -deoxy and 2'-hydroxyl sugars, as well as their analogs.

[0189] In some embodiments, an "analog" in reference to a nucleotide includes a synthetic nucleotide having modified basemoieties and / or modified sugar moieties. Such analogs include synthetic nucleotides designed to enhance binding properties, e.g., duplex or triplex stability, specificity, or the like.

[0190] In some embodiments, "hybridization" refers to the pairing of substantially complementary strands of oligomeric compounds. One mechanism of pairing involves hydrogen bonding, which can be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleotides) of the strands of oligomeric compounds. For example, adenine and thymine are complementary nucleotides which pair through the formation of hydrogen bonds. Hybridization can occur under varying circumstances.

[0191] In some embodiments, "specifically hybridizable" and "complementary" are terms which are used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between a compound herein and a target RNA molecule.

[0192] In some embodiments, an antisense compound is "specifically hybridizable" when binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid to cause a modulation of function and / or activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and under conditions in which assays are performed in the case of in vitro assays.

[0193] In some embodiments, specific binding requires a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of assays or therapeutic treatment, or in the case of in vitro assays, under conditions in which the assays are performed. The non-target sequences can differby atleast 5, 6, 7, 8, 9, or atleast 10 nucleotides.

[0194] In some embodiments, "complementary," refers to the capacity for precise pairing between two nucleotides on one or two oligomeric strands. For example, if a nucleotide at a certain position of an antisense compound is capable of hydrogen bonding with a nucleotide at a certain position of a target nucleic acid, said target nucleic acid being a DNA, RNA, or oligonucleotide molecule, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be a complementary position. The oligomeric compound and the further DNA, RNA, or oligonucleotide molecule are complementary to each other when a sufficient number of complementary positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. Thus, "specifically hybridizable" and "complementary" are terms can be used to indicate a sufficient degree of precise pairing or complementarity over a sufficient number of nucleotides such that stable and specific binding occurs between the oligomeric compound and a target nucleic acid.

[0195] In some embodiments, an antisense oligonucleotide is "sufficiently complementary" to a target RNA, e.g., a target mRNA, such that the antisense oligonucleotide agent silences production of protein encoded by the target mRNA. In some embodiments, an antisense oligonucleotide agent is "exactly complementary" to a target RNA, e.g., the target RNA and the antisense oligonucleotide anneal, for example to form a hybrid made exclusively of Watson- Crick base pairs in the region of exact complementarity. A "sufficiently complementary" target RNA can include an internal region (e.g., of at least 10 nucleotides) that is exactly complementary to a target RNA. Moreover, in some embodiments, the antisense oligonucleotide specifically discriminates a single-nucleotide difference. In some such cases, the antisense oligonucleotide only mediates RNAi if exact complementary is found in the region (e.g., within 7 nucleotides of) the single-nucleotide difference. In some embodiments, an "oligonucleotide" is a nucleic acid molecule (RNA or DNA) having, for example, a length less than 100, 200, 300, or 400 nucleotides.

[0196] In some embodiments, "mediates RNAi" refers to the ability to silence, in a sequence specific manner, a target RNA. While not wishing to be bound by theory, it is believed that silencing uses the RNAi machinery or process, and a guide RNA, e.g., a siRNA agent of 21 to 23 nucleotides.

[0197] It is understood in the art that the sequence of an oligomeric compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. Moreover, an oligonucleotide can hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure, mismatch or hairpin structure). In some embodiments, the oligomeric compounds disclosed herein comprise at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about90%, or at least about 95%, or at least about 99% sequence complementarity to a target region within the target nucleic acid sequence to which they are targeted. In a non -limiting example, an antisense compound in which 18 of 20 nucleotides of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining non -complementary nucleotides can be clustered or interspersed with complementary nucleotides and need not be contiguous to each other or to complementary nucleotides. As such, an antisense compound which is 18 nucleotides in length having 4 (four) non-complementary nucleotides which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present disclosure. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art. Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman.

[0198] In some embodiments, "Thermal Melting Point (Tm)" refers to the temperature, under defined ionic strength, pH, and nucleic acid concentration, at which 50% of the oligonucleotides complementary to the target sequence hybridize to the target sequence at equilibrium. Typically, stringent conditions will be those in which the salt concentration is at least about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short oligonucleotides (e.g., 10 to 50 nucleotide). Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide.

[0199] In some embodiments, "modulation" means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene. In some instances, modulation comprises alternative splicing of various isoforms during expression of a gene.

[0200] In some embodiments, "variant", when used in the context of a polynucleotide sequence, can encompass a polynucleotide sequence related to a wild type gene. This can also include, for example, "allelic," "splice," "species," or "polymorphic" variants. A splice variant can have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing. The corresponding polypeptide can possess additional functional domains or an absence of domains. Species variants are polynucleotide sequences that vary from one species to another. Of particular utility can be variants of wild type gene products. Variants can result from at least one mutation in the nucleic acid sequence and can result in altered mRNAs or in polypeptides whose structure orfunction can or cannot be altered. Any given natural or recombinant gene can have none, one, or many allelic forms. Common mutational changes that give rise to variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes can occur alone, or in combination with the others, one or more times in a given sequence.

[0201] In some embodiments, polypeptides generally have significant amino acid identity relative to each other. A polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants also can encompass "single nucleotide polymorphisms" (SNPs,) or single base mutations in which the polynucleotide sequence varies by one base. The presence of SNPs can be indicative of, for example, a certain population with a propensity for a disease state, that is susceptibility versus resistance.

[0202] Derivative polynucleotides include nucleic acids subjected to chemical modification, for example, replacement of hydrogenby an alkyl, acyl, or amino group. Derivatives, e.g., derivative oligonucleotides, can comprise non-naturally-occurring portions, such as altered sugar moieties or inter-sugar linkages. Exemplary among these are phosphorothioate and other sulfur containing species which are known in the art. Derivative nucleic acids can also contain labels, including radionucleotides, enzymes, fluorescent agents, chemiluminescent agents, chromogenic agents, substrates, co factors, inhibitors, magnetic particles, and the like.

[0203] In some embodiments, a "derivative" polypeptide or peptide is one that is modified, for example, by glycosylation, pegylation, phosphorylation, sulfation, reduction / alkylation, acylation, chemical coupling, or mild formalin treatment. A derivative can also be modified to contain a detectable label, either directly or indirectly, including, but not limited to, a radioisotope, fluorescent, and enzyme label.

[0204] In some embodiments, the term "animal" or "patient" includes, for example, any of the following: humans, sheep, elks, deer, mule deer, minks, mammals, monkeys, horses, cattle, pigs, goats, dogs, cats, rats, mice, birds, chicken, reptiles, fish, insects and arachnids.

[0205] In some embodiments, "mammal" includes warm blooded mammals that are typically under medical care (e.g., humans and domesticated animals). Examples include feline, canine, equine, bovine, and human, as well as just human.

[0206] In some embodiments, "treating" or "treatment" covers the treatment of a disease-state in a mammal, and includes: (a) preventing the disease-state from occurring in a mammal, in particular, when such mammal is predisposed to the disease-state but has not yet been diagnosed as having it; (b) inhibiting the disease-state, e.g., arresting it development; and / or (c) relieving the disease-state, e.g., causing regression of the disease state until a desired endpoint is reached. Treating also includes the amelioration of a symptom of a disease (e.g., lessen the pain ordiscomfort), wherein such amelioration can or cannot be directly affecting the disease (e.g., cause, transmission, expression, etc.).

[0207] In some embodiments, "carbohydrate" refers to a compound which is either a carbohydrate per se made up of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom; or a compound having as a part thereof a carbohydrate moiety made up of one or more monosaccharide units each having at least six carbon atoms (which can be linear, branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. Representative carbohydrates include the sugars (mono-, di-, tri- and oligosaccharides containing from about 4-9 monosaccharide units), and polysaccharides such as starches, glycogen, cellulose and polysaccharide gums. Specific monosaccharides include C5and above (preferably C5-C8) sugars; di- and trisaccharides include sugars having two or three monosaccharide units (preferably C5- C8).

[0208] In some embodiments, the term "halo" refers to any radical of fluorine, chlorine, bromine or iodine.

[0209] In some embodiments, the term "alkyl" refers to saturated and unsaturated non-aromatic hydrocarbon chains that can be a straight chain or branched chain, containing the indicated number of carbon atoms (these include without limitation propyl, allyl, or propargyl), which can be optionally inserted with N, O, or S. For example, Ci-Cio indicates that the group can have from 1 to 10 (inclusive) carbon atoms in it. The term "alkoxy" refers to an -O-alkyl radical. The term "alkylene" refers to a divalent alkyl (i.e., -R-). The term "alkylenedioxo" refers to a divalent species of the structure -O-R-O-, in which R represents an alkylene. The term "aminoalkyl" refers to an alkyl substituted with an amino. The term "mercapto" refers to an -SH radical. The term "thioalkoxy" refers to an -S-alkyl radical.

[0210] In some embodiments, the term "aryl" refers to a 6-carbon monocyclic or 10-carbon bicyclic aromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring can be substituted by a substituent. Examples of aryl groups include phenyl, naphthyl and the like. In some embodiments, the term "arylalkyl" or the term "aralkyl" refers to alkyl substituted with an aryl. In some embodiments, the term "arylalkoxy" refers to an alkoxy substituted with aryl.

[0211] In some embodiments, the term "cycloalkyl" as employed herein includes saturated and partially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons, for example, 3 to 8 carbons, and, for example, 3 to 6 carbons, wherein the cycloalkyl group additionally can be optionally substituted. Cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

[0212] In some embodiments, the term "heteroaryl" refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 1 1-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1 -3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3, or 4 atoms of each ring can be substituted by a substituent. Examples of heteroaryl groups include pyridyl, furyl or furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolyl, thiazolyl, and the like. The term "heteroarylalkyl" or the term "heteroaralkyl" refers to an alkyl substituted with a heteroaryl. The term "heteroarylalkoxy" refers to an alkoxy substituted with heteroaryl.

[0213] In some embodiments, the term "heterocyclyl" refers to a nonaromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1 -3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1 , 2 or 3 atoms of each ring can be substituted by a substituent. Examples of heterocyclyl groups include trizolyl, tetrazolyl, piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, and the like.

[0214] In some embodiments, the term "oxo" refers to an oxygen atom, which forms a carbonyl when attached to carbon, an N-oxide when attached to nitrogen, and a sulfoxide or sulfone when attached to sulfur.

[0215] In some embodiments, the term "acyl" refers to an alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent, any of which can be further substituted by substituents.

[0216] In some embodiments, the term "substituted" refers to the replacement of one or more hydrogen radicals in a given structure with the radical of a specified substituent including, but not limited to: halo, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, thiol, alkylthio, arylthio, alky, thioalkyl, arylthioalkyl, alkylsulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl, alkoxy, aryloxy, aralkoxy, aminocarbonyl, alkylamino carbonyl, arylaminocarbonyl, alkoxy carbonyl, aryloxy carbonyl, haloalkyl, amino, trifluoromethyl, cyano, nitro, alkylamino, aryl amino, alkylaminoalkyl, arylaminoalkyl, aminoalkylamino, hydroxy, alkoxyalkyl, carboxyalkyl, alkoxycarbonylalkyl, aminocarbonylalkyl, acyl, aralkoxycarbonyl, carboxylic acid, sulfonic acid, sulfonyl, phosphoric acid, aryl, heteroaryl, heterocyclic, and aliphatic. It is understood that the substituent can be further substituted.

[0217] Some embodiments refer to nucleic acid sequence information. It is contemplated that in some embodiments, thymine (T) can be interchanged with uracil (U), or vice versa. For example,some sequences in the sequence listing can recite Ts, but these can be replaced with Us in some embodiments. In some oligonucleotides with nucleic acid sequences that include uracil, the uracil can be replaced with thymine. Similarly, in some oligonucleotides with nucleic acid sequences that include thymine, the thymine can be replaced with uracil. In some embodiments, an oligonucleotide such as an siRNA comprises or consists of RNA. In some embodiments, the oligonucleotide can comprise or consist of DNA. For example, an ASO can include DNA.EXAMPLESExample 1: Increased CD33 expression level is associated with decreased survival in amyotrophic lateral sclerosis

[0218] Levels of CD33 RNA expression were measured by RNA-seq in post-mortem cervical spinal cord samples from 94 amyotrophic lateral sclerosis (ALS) patients. The RNA expression levels were compared against CD33 RNA expression levels obtained from post-mortem cervical spinal cord samples from 34 non -neurological controls. The raw RNA-seq data was obtained from The Target ALS Human Postmortem Tissue Core, New York Genome Center for Genomics of Neurodegenerative Disease, the Amyotrophic Lateral Sclerosis Association and the Tow Foundation. The relative RNA expression levels among ALS patients were then compared against the patient survival of the donor. It was found that the CD33 expression was induced in the ALS patients, and it was negatively correlated with the patient survival (FIGs. 1 A-1B).Example 2: Depletion of CD33 expression by genetic knockout in U937 cells results in decreased expression of inflammatory factors

[0219] To test whether CD33 depletion modulates inflammatory responses, CD 33 gene knockout (KO) myeloid cell line was generated, and expression levels of inflammatory factors were assessed. Briefly, U937 cell line with CD33 gene KO was generated by insertion of stop codons and a frameshift mutation into the second coding exon of the gene using CRISPR / Cas9 genome engineering. This approach aimed to destabilize CD33 mRNA through nonsense-mediated decay and completely disrupt full-length CD33 protein expression. Knockout efficiency was confirmed by measuring CD33 surface expression via flow cytometry (FIG. 2A).

[0220] To evaluate inflammatory factor expression associated with ALS and neuroinflammation in the presence or absence of CD33, RNA-seq was performed. The data showed that CD33 deficient myeloid cells expressed lower levels of inflammatory factors, such as IL1B, CXCL1, SPP1, CHI3L2, and MMP9, compared to wild-type (WT) cells (FIG. 2B). These findings suggest that downregulation of CD33 may reduce neuroinflammation.Example 3: Screening splice-switching oligos for inhibition of full-length CD33 expression in microglia

[0221] Splice-switching oligos (SSOs) were designed to induce exon skipping in CD33 mRNA (Table 2). Binding of an SSO to a target pre-mRNA transcript modulated exon inclusion and resulted in alternative splicing isoform expression. FIG. 3A shows a schematic of the alternative splicing isoform induced by SSO regulation. The SSO binds pre-mRNA, then the mRNA is then processed into the exon inclusion isoform (left) or alternative splicing isoform (right) and expressed as the full length or truncated protein form (FIG. 3A). SSOs were prepared and tested in differentiated microglia (iMG) derived from induced human pluripotent stem cells (iPSC) (Table 3). The efficacies of >30 selected SSOs were measured by the change in the relative molarity of the inclusion isoform. SSOs that resulted in >20% increase in exclusion (a decreased relative molarity of the inclusion isoform) were selected as SSOs to be further tested (FIG. 3B).

[0222] Tapestation electrophoretic analysis of CD33 RNA splicing pattern was conducted by using PCR of the target exons 1-3 of CD33 from microglia treated with several SSOs selected from the screening (S01-008 (SEQ ID NO: 75), S01-010 (SEQ ID NO: 90), S02-023 (SEQ ID NO: 99)) (FIG. 3C). The change in CD33 protein isoform ratio was observed using flow cytometric of microglia treated with S02-023 for 9 days and stained with CD33 antibody recognizing both the full-length and the truncated isoform (FITC) and an antibody recognizing exclusively the long isoform (APC) (FIG. 3D). About 67.4% of truncated isoform was detected in microglia treated with S02-023, compared to about 19.7% in non-treated microglia.Table 2. CD33 SSO SequencesTable 3. CD33 SSO ResultsExample 4: Treatment with CD33 SSO S02-023 decreased expression of select inflammatory markers and enhanced kinetics of phagocytosis by microglia

[0223] RNA-seq analysis of iPSC-derived microglia (iMG) treated with 10 pM S02-023 (SSO) for 9 days showed decreased level of expression for several inflammatory markers associated with neuroinflammation in ALS and other neurodegenerative diseases (FIG. 4A). Live fluorescence imaging of the microglia treated with S02-023 showed elevated uptake kinetics of pHrodo-labeled beta-amyloid aggregates relative to non-treated microglia (NT) (FIG. 4B).Example 5: Functional characterization of SSO S01-008 in microglia

[0224] The selected SSOs from Example 3 were tested in cell assays to evaluate their ability to promote the phagocytic function using a panel of microglia derived from healthy individuals or patients with neurodegenerative disease. RNA-seq analysis of iPSC-derived microglia pre-treatedfor 24 hrs with 10 pM SSO SO 1-008 revealed global change of their expression profile towards less inflammatory phenotype that maintains homeostatic phagocytic function as suggested by the decreased level of EL1B, CIS, and CYBB expression and increased SORL1, and CD 36 expression (FIG. 5A). Cumulative phagocytosis analysis of monocyte -derived microglial like cells from 3 individuals pre-treated for 24hrs with 5 pM SSO SO 1-008 revealed increase phagocytic activity of pHrodo-labeled particles coated with E. coli (FIG. 5B).Example 6: In vitro screening to identify ASO hits

[0225] Binding of a single stranded antisense oligonucleotide (ASO) to a transcript resulted in enzymatic cleavage of the target RNA by endogenously expressed RNase H that recognizes RNA::DNA dimers (FIG. 6A). In order to evaluate the modulation of CD33 expression, ASOs targeting variable regions of CD33 mRNA were generated. The ASOs used in this example has a gapmer structure - the first and last five bases of the ASO were composed of RNA while the central bases are DNA. Further chemical modifications of the ASOs in the primary screen included phosphorothioate backbone modifications and2’-O-methoxyethyl modifications (Table 4).

[0226] Screening of the ASOs was performed on PMA-differentiated U937 cells that express the CD33 transcript at high level. After mRNA extraction from the treated cells, a one-step qPCR was performed in 384-well plates to quantify relative transcript abundance (Table 5). Three validated CD33 Taqman assays (and a housekeeping gene Taqman) were used for each sample to account for expression of multiple transcript isoforms, and to avoid sequence-specific interference from the ASOs by excluding datapoints when the ASO overlaps with one of the Taqman assays. Results are shown in FIG. 6B.

[0227] Dose-dependent activity of the selected ASOs was validated using qPCR of CDS 3 RNA expression levels in PMA-differentiated U937 cells treated with 8 different doses of the ASOs. Based on a fit curve analysis two ASOs (SEQ ID NO: 659 and SEQ ID NO:727) were selected for further testing and analysis (FIG. 6C). CD33 protein level continuously decreased over 12 days in iPSC-derived microglia treated once with 5pM gapmer ASO (SEQ ID NO: 659) as determined by a Meso Scale Discovery (MSD) immunoassay with a pair of CD33 -specific antibodies (FIG. 6D). The experiment was performed in 3 biological replicates.Table 4. CD33 ASO sequences (target transcript ENST00000262262)Table 5. CD33 ASO ResultsExample 7: Treatment with CD33 ASOs decreased baseline expression of select inflammatory markers, protected from secretion of inflammatory factors induced by TDP- 43, enhanced kinetics of phagocytosis, and rescued microglial neurotoxicity

[0228] RNA-seq analysis of iPSC-derived microglia treated with 5 pM ASOs (SEQ ID NO: 659 and SEQ ID NO: 727) for 9 days showed decreased expression of CD33 and inflammatory markers associated with neuroinflammation in ALS and other neurodegenerative diseases (FIG. 7A). Moreover, ELISA analysis of IL1B and CHI3L1 secretion by microglia with (5 pM ASO) or without (NT) ASO (SEQ ID NO: 659) treatment and stimulated with 5nM TDP-43 for 24 hrs (TDP-43) showed protection from inflammatory activation upon CD33 ASO treatment (FIG. 7B).

[0229] Live fluorescence imaging of the microglia treated one with 5pM ASO (SEQ ID NO: 659) and ASO (SEQ ID NO: 727) showed significantly elevated uptake kinetics of pHrodo- labeled beta-amyloid aggregates relative to non-treated microglia (NT) (FIG. 7C). Neurofilament light (NfL) release into cell culture media was measured in co -cultures of iPSC-derived motor neurons and microglia upon lOOng / ml lipopolysaccharide (LPS) inflammatory induction with or without a single 5pM ASO (SEQ ID NO: 659) treatment. Non -targeting ASO was used as a control (ctrl ASO). Treatment with 5pM ASO (SEQ ID NO: 659) protected motor neurons and led to lowering of NFL release (FIG. 7D).Example 8: Treatment of Amyotrophic Lateral Sclerosis (ALS)

[0230] ASOs or SSOs from any one of Examples 1 -4 are administered by parenteral injection (e.g., intrathecal injection, IV) to treat a patient symptomatic of ALS, or a pre -symptomatic patient with familial ALS.Example 9: Treatment of Frontotemporal Dementia (FTP)

[0231] ASOs or SSOs from any one of Examples 1 -4 are administered by parenteral injection (e.g., intrathecal injection, IV) to treat a patient symptomatic of FTD, including but not limited to patients having FTD with amyotrophic lateral sclerosis (FTD-ALS), behavioral variant FTD (bvFTD), primary progressive aphasia (PPA), progressive supranuclear palsy or the corticobasal syndrome.Example 10: Treatment of Parkinson’s Disease (PD)

[0232] ASOs or SSOs from any one of Examples 1 -4 are administered by parenteral injection (e.g., intrathecal injection, IV) to treat a patient symptomatic of Parkinson’s disease (PD).Example 11: Treatment of Alzheimer’s disease (AD)

[0233] ASOs or SSOs from any one of Examples 1 -4 are administered by parenteral injection (e.g., intrathecal injection, IV) to treat a patient symptomatic of Alzheimer’s disease.

[0234] While preferred embodiments of the present disclosure have been shown and described herein, it will be understood by those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While embodiments have been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments described herein can be employed in practicing the invention. It is therefore contemplated that theinvention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMSWHAT IS CLAIMED IS:1 . A compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleotides and comprising a nucleic acid sequence complementary to at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1, 2, or 3 mismatches from any one of SEQ ID NOs: 1 or 2.

2. The compound of claim 1, wherein the modified oligonucleotide comprises a nucleic acid sequence complementary to at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, atleast 15, at least 16, atleast 17, at least 18, at least 19, or atleast20 consecutive nucleotides with no more than 1, 2, or 3 mismatches to a target sequence in positions 1270-1360, 1370-1462, 1-21, 29-48, 94-116, 137-187, 211-241, 263-282, 392-417, 416-435, 456-523, 460- 479, 504-671, 652-671, 683-721, 724-746, 730-753, 731-756, 770-819, 772-807, 857-876, 880- 899, 1050-1138, 1073-1108, 1165-1184, 1189-1266, 1194-1276, 1250-1334, 1285-1332, 1336- 1360, 1340-1360, 1364-1383, or 1388-1452 of SEQ ID NO: 1.

3. The compound of claim 1 or 2, wherein the modified oligonucleotide comprises at least 8, at least 9, atleast 10, atleast 11, atleast 12, at least 13, atleast 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1, 2, or 3 mismatches from any one of nucleic acid sequences of SEQ ID NOs: 123-432.

4. The compound of any one of claims 1 -3, wherein the nucleic acid sequence of the modified oligonucleotide is at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of the nucleic acid sequences of SEQ ID NOs: 123-432.

5. The compound of any one of claims 1-4, wherein the modified oligonucleotide is a single-stranded modified oligonucleotide.

6. The compound of any one of claims 1 -5, wherein the modified oligonucleotide comprises a gapmer.

7. The compound of claim 6, wherein the modified oligonucleotide comprises: a gap segment consisting of 5-15 linked deoxynucleotides; a 5’ wing segment consisting of 4-10 linked nucleotides; and a 3 ’ wing segment consisting of 4-10 linked nucleotides; wherein the gap segment is positioned between the 5 ’ wing segment and the 3 ’ wing segment, and wherein each nucleotide of 5’ and 3’ wing segment comprises at least one 2’- modified nucleotide.

8. The compound of claim 7, wherein the at least one 2’-modified nucleotide comprises a 2’-O-methoxyethyl (MOE) modified nucleotide.

9. The compound of any one of claims 1 -8, wherein the modified oligonucleotide comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1, 2, or 3 mismatches from any one of nucleic acid sequences of SEQ ID NOs: 433-742.

10. The compound of any one of claims 1 -9, wherein the nucleic acid sequence of the modified oligonucleotide is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of the nucleic acid sequences of SEQ ID NOs: 433-742.11 . The compound of any one of claims 1 -10, wherein the modified oligonucleotide consists of any one of nucleic acid sequences of SEQ ID NOs: 433-742.

12. The compound of claim 1-11, wherein the modified oligonucleotide comprises a nucleic acid sequence complementary to at least 8, at least 9, at least 10, at least 11, at least 12, at least13. at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1, 2, or 3 mismatches to a target sequence in positions 1270-1360, 1370-1462, 29-48, 392-417, 460-479, 731-756, 772-807, 1073-1108, 1194-1276, or 1388-1452 of SEQ ID NO: 1.

13. The compound of any one of claims 1 -12, wherein the modified oligonucleotide comprises a nucleic acid sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1, 2, or 3 mismatches from any one of the nucleobase sequences of SEQ ID NOs: 125, 146, 148-152, 158, 182-186, 189, 191, 193, 197, 203, 222-226, 241-260, 262-266, 271, 272, 274-281, 283, 286, 287, 300, 301, 311 -319, 321, 322, 328-335, 344, 345, 347-351, 377-381, 390, 391, 395-397, 400-409, 412, 413, 415-420, or 422.

14. The compound of any one of claims 1 -13, wherein the modified oligonucleotide comprises a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of the nucleic acid sequences of SEQ ID NOs: 125, 146, 148-152, 158, 182-186, 189, 191, 193, 197, 203, 222-226, 241-260, 262-266, 271, 272, 274-281, 283, 286, 287, 300, 301, 311 -319, 321, 322, 328-335, 344, 345, 347-351, 377-381, 390, 391, 395-397, 400-409, 412, 413, 415-420, or 422.

15. The compound of any one of claims 1 -14, wherein the modified oligonucleotide comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least15. at least 16, atleast 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1, 2, or 3 mismatches from any one of nucleic acid sequences of SEQ ID NOs: 435, 456, 458-462, 468, 492-496, 499, 501, 503, 507, 513, 532-536, 551-570, 572-576, 581, 582, 584- 591, 593, 596, 597, 610, 611, 621 -629, 631, 632, 638-645, 654, 655, 657-661, 687-691, 700, 701, 705-707, 710-719, 722, 723, 725-730, or 732.

16. The compound of any one of claims 1 -15, wherein the nucleic acid sequence of the modified oligonucleotide is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of the nucleic acid sequences of SEQ ID NOs: 435, 456, 458-462, 468, 492-496, 499, 501, 503, 507, 513, 532-536, 551-570, 572-576, 581, 582, 584-591, 593, 596, 597, 610, 611, 621 -629, 631, 632, 638-645, 654, 655, 657-661, 687-691, 700, 701, 705-707, 710-719, 722, 723, 725-730, or 732.

17. The compound of any one of claims 1 -16, wherein the modified oligonucleotide consists of any one of nucleic acid sequences of SEQ ID NOs: 435, 456, 458-462, 468, 492-496, 499, 501, 503, 507, 513, 532-536, 551 -570, 572-576, 581, 582, 584-591, 593, 596, 597, 610, 611, 621-629, 631, 632, 638-645, 654, 655, 657-661, 687-691, 700, 701, 705-707, 710-719, 722, 723, 725-730, or 732.

18. The compound of claim 1-17, wherein the modified oligonucleotide comprises a nucleic acid sequence complementary to at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, atleast 15, at least 16, atleast 17, atleast 18, atleast 19, or at least 20 consecutive nucleotides with no more than 1, 2, or 3 mismatches to a target sequence in positions 1270-1360, 1370-1462, or 1388-1452 of SEQ ID NO: 1.

19. The compound of any one of claims 1 -18, wherein the modified oligonucleotide comprises a nucleic acid sequence comprising at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, atleast 14, at least 15, atleast 16, atleast 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1, 2, or 3 mismatches from any one of the nucleic acid sequences of SEQ ID NOs: 313, 316, 317, 319, 347-349, 402, 417, or 418.

20. The compound of any one of claims 1 -19, wherein the modified oligonucleotide comprises a nucleic acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identity to any one of the nucleic acid sequences of SEQ ID NOs: 313, 316, 317, 319, 347-349, 402, 417, or 418.

21. The compound of any one of claims 1 -20, wherein the modified oligonucleotide comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1, 2, or 3 mismatches from any one of nucleic acid sequences of SEQ ID NOs: 623,626, 627, 629, 657-659, 712, 727, or 728.

22. The compound of any one of claims 1 -21, wherein the nucleic acid sequence of the modified oligonucleotide is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of the nucleic acid sequences of SEQ ID NOs: 623, 626,627, 629, 657-659, 712, 727, or 728.

23. The compound of any one of claims 1 -22, wherein the modified oligonucleotide consists of any one of nucleic acid sequences of SEQ ID NOs: 623, 626, 627, 629, 657-659, 712, 727, or 728.

24. A compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleotides and comprising a nucleic acid sequence complementary to at least 8, at least 9, at least 10, at least 11, at least 12, atleast 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1, 2, or 3 mismatches from SEQ ID NOs: 5 or 6.

25. The compound of claim 24, wherein the modified oligonucleotide comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1, 2, or 3 mismatches from any one of nucleic acid sequences of SEQ ID NOs: 7-64.

26. The compound of claim 24 or claim 25, wherein the nucleic acid sequence of the modified oligonucleotide is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of the nucleic acid sequences of SEQ ID NOs: 7-64.

27. The compound of any one of claim 24-26, wherein the modified oligonucleotide consists of 12-20 nucleotides in length.

28. The compound of any one of claims 24-27, wherein the modified oligonucleotide comprises at least one modified internucleotide linkage, a modified base, or a modified sugar ring.

29. The compound of claim 28, wherein the at least one modified internucleotide linkage is a phosphorothioate internucleotide linkage.

30. The compound of claim 29, wherein all internucleotide linkages in the modified oligonucleotide are phosphorothioate intemucleotide linkages.31 . The compound of any one of claims 28-30, wherein the modified sugar ring is a bicyclic sugar or a 2' modified sugar ring.

32. The compound of claim 31, wherein the 2' modified sugar ring comprises a 2'-O- methoxy ethyl sugar ring (2 ’-MOE), 2'-O-methyl sugar ring (2 ’-OMe), or 2’-fluoro sugar ring (2’- F).

33. The compound of any one of claims 28-32, the modified oligonucleotide comprises at least one locked nucleic acid (LNA).

34. The compound of any one of claims 24-33, wherein the modified oligonucleotide comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1, 2, or 3 mismatches from any one of nucleic acid sequences of SEQ ID NOs: 65-122.

35. The compound of any one of claims 24-34, wherein the nucleic acid sequence of the modified oligonucleotide is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of the nucleic acid sequences of SEQ ID NOs: 65-122.

36. The compound of any one of claims 24-35, wherein the modified oligonucleotide comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1, 2, or 3 mismatches from any one of nucleic acid sequences of SEQ ID NOs: 16-22, 32-36, 38-42, or 44.

37. The compound of any one of claims 24-36, wherein the nucleic acid sequence of the modified oligonucleotide is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of the nucleic acid sequences of SEQ ID NOs: 16-22, 32- 36, 38-42, or 44.

38. The compound of claim any one of claims 24-37, wherein the modified oligonucleotide comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, or at least 20 consecutive nucleotides with no more than 1, 2, or 3 mismatches from any one of SEQ ID NOs: 74-80, 90-94, 96-100, or 102.

39. The compound of any one of claims 24-38, wherein the nucleic acid sequence of the modified oligonucleotide is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of nucleic acid sequences of SEQ ID NOs: 74-80, 90-94, 96-100, or 102.

40. The compound of any one of claims 24-39, wherein the modified oligonucleotide consists of to any one of nucleic acid sequences of SEQ ID NOs: 74-80, 90-94, 96-100, or 102.41 . A pharmaceutical composition comprising the compound of any one of the preceding claims and a pharmaceutically acceptable excipient.

42. The pharmaceutical composition of claim 41, wherein the pharmaceutical composition is formulated for parenteral administration.

43. A method of treating a neurological disease or disorder comprising administering a therapeutically effective amount of a compound or pharmaceutical composition of any one of the preceding claims to a subject in need thereof.

44. The method of claim 43, wherein the neurological disease or disorder comprises neuroinflammation or neurodegeneration.

45. The method of claim 43, wherein the neurological disease or disorder comprises amyotrophic Lateral Sclerosis (ALS), frontotemporal dementia (FTD), Parkinson’s disease (PD), or Alzheimer’s disease (AD).

46. The method of claim 45, wherein FTD comprises a hereditary disease.

47. The method of claim 45, wherein FTD comprises amyotrophic lateral sclerosis (FTD- ALS), behavioral variant FTD (bvFTD), primary progressive aphasia (PPA), progressive supranuclear palsy or the corticobasal syndrome.

48. The method of claim 43, wherein treating results in prevention, slows progression, or reverses the neurodegenerative disease or disorder.

49. A method of downregulating the expression of CD33 comprising administering a therapeutically effective amount of a compound or pharmaceutical composition of any one of claims 1-23, and 41-42.

50. A method of modulating the expression of one or more CD33 isoforms comprising administering a therapeutically effective amount of a compound or pharmaceutical composition of any one of claims 24-40 and 41-42.51 . The method of claim 50, wherein the compound or the pharmaceutical composition induces exon skipping.

52. The method of claim 51, wherein the compound or the pharmaceutical composition increases the relative ratio of CD33 mRNA in which exon 2 is absent compared to CD33 mRNA in which exon 2 is present.

53. The method of claim 51, wherein the compound or the pharmaceutical composition increases the relative ratio of CD33 mRNA without exon2 (e.g., SEQ ID NOs: 3 or 4) relative to CD33 mRNA comprising exon 2.

54. The method of claim 51, wherein the compound or the pharmaceutical composition is configured to bind an intron, an intron adjacent to exon 2, or a 5’ or 3’ flanking region of exon 2.

55. The method of any one of claims 43-54, wherein administering comprises parenteral administration.

56. The method of claim 55, wherein parenteral administration comprises intrathecal or intravenous (IV) administration.

Images