Gene regulation using antisense oligonucleotides (ASOS)

Abstract

Disclosed herein are a method and composition for increasing TP53 gene expression to restore tumor suppressor function in conditions such as Li-Fraumeni Syndrome and cancer. The method comprises administering two antisense oligonucleotides targeting distinct splicing-regulatory elements within the TP53 gene to modulate splicing of an internal exon, thereby synergistically increasing TP53 mRNA and p53 protein levels. Also disclosed herein is a composition comprising the antisense oligonucleotides with chemical modifications and delivery systems for therapeutic applications.

Description

Attorney’s Docket No. 701586-000159WOPTGENE REGULATION USING ANTISENSE OLIGONUCLEOTIDES (ASOs)CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U. S. C. § 119 of U. S. Provisional Application No. 63 / 729,102 filed December 6, 2024, the contents of which are incorporated herein by reference in their entirety.GOVERNMENT SUPPORT

[0002] This invention was made with government support under Grant No. R35GM147254 awarded by the National Institutes of Health. The government has certain rights in the invention.TECHNICAL FIELD

[0003] The present disclosure relates to biotechnology, specifically to therapeutic methods for upregulating gene expression using antisense oligonucleotides to restore tumor suppressor function in conditions such as Li-Fraumeni Syndrome and cancer.BACKGROUND

[0004] TP53 is crucial for regulating the cell cycle and inducing apoptosis in response to DNA damage. Haploinsufficiency of TP53 results in reduced p53 protein levels, weakening the cellular’s ability to respond to genotoxic stress. Cells with diminished p53 activity are less likely to undergo apoptosis, allowing the survival and proliferation of cells with oncogenic mutations. TP53 mutations are linked to Li-Fraumeni Syndrome (LFS), a hereditary cancer syndrome with a prevalence of about 1 in 5,000 families. LFS patients are predisposed to a wide range of tumors due to the early loss of p53 function, which promotes cellular proliferation and tumor development.

[0005] Haploinsufficiency in TPS 3 is associated with increased cancer risk due to compromised DNA repair and cell cycle regulation, genomic instability leading to the accumulation of genetic mutations and chromosomal abnormalities, early onset of cancer as these patients often develop cancers at a younger ages, and resistance to therapy given that p53-deficient tumors are often resistant to radiation therapy due to impaired DNA repair mechanisms.

[0006] Disclosed herein is a novel therapeutic approach to upregulate the expression of TP53 through splicing modulation. By restoring normal tumor suppressor function, this invention improves patient outcomes and prevent the onset of cancer in LFS syndrome patients.SUMMARY14925-2218-9179 1701586-000159WOPTAttorney’s Docket No. 701586-000159WOPT

[0007] One aspect disclosed herein provides a synthetic antisense oligonucleotide that binds a region of a TP53 RNA transcript.

[0008] In one embodiment of this or any aspect, the TP53 RNA transcript is a TP53 pre-mRNA transcript.

[0009] In one embodiment of this or any aspect, binding modulates splicing of an internal exon of the TP53 transcript.

[0010] In one embodiment of this or any aspect, the modulation increases TP53 mRNA levels and p53 protein levels.

[0011] In one embodiment of this or any aspect, TP53 mRNA levels and p53 protein levels are increased by at least 10% as compared to an appropriate control.

[0012] In one embodiment of this or any aspect, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2. or wherein the antisense oligonucleotide comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0013] Another aspect disclosed herein provides a nucleic acid vector comprising a synthetic antisense oligonucleotide disclosed herein.

[0014] In one embodiment of this or any aspect, the synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 1, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 1.

[0015] In one embodiment of this or any aspect, the synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 2, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 2.

[0016] Another aspect disclosed herein provides a nucleic acid vector comprising a synthetic antisense oligonucleotide comprising a nucleotide sequence of SEQ ID NO: 1, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 1.

[0017] Another aspect disclosed herein provides a nucleic acid vector comprising a synthetic antisense oligonucleotides comprising a nucleotide sequence of SEQ ID NO: 2, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 2.

[0018] Another aspect disclosed herein provides a composition comprising at least one synthetic antisense oligonucleotides or any of the nucleic acid vectors disclosed herein.

[0019] In one embodiment of this or any aspect, the composition comprises two distinct synthetic antisense oligonucleotides, or a nucleic acid vector comprising two distinct synthetic antisense oligonucleotides, or at least two nucleic acid vectors, each of the at least two nucleic acid vectors comprising at least one of: a synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 1, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID24925-2218-9179 1701586-000159WOPTAttorney’s Docket No. 701586-000159WOPTNO: 1; and a synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 2, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 2.

[0020] In one embodiment of this or any aspect, at least one of the two distinct synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 1, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 1.

[0021] In one embodiment of this or any aspect, at least one of the two distinct synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 2, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 2.

[0022] Another aspect disclosed herein provides a composition comprising: a synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 1, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 1; and a synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 2, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 2; or a nucleic acid vector comprising a synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 1, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 1; and a synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 2, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 2; or at least two nucleic acid vectors, each of the at least two nucleic acid vectors expressing at least one of: a synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 1, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 1; and a synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 2, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 2.

[0023] Another aspect disclosed herein provides a method for increasing TP53 gene expression in a cell, the method comprising: contacting a cell with any of the synthetic antisense oligonucleotides, nucleic acid vectors, or compositions of disclosed herein.

[0024] In one embodiment of this or any aspect, the cell is a mammalian cell. In one embodiment of this or any aspect, the mammalian cell is a human cell.

[0025] In one embodiment of this or any aspect, contacting is in vitro, in vivo, or ex vivo. In one embodiment of this or any aspect, contacting is performed via liposomal delivery, nanoparticle delivery, or viral delivery system. In one embodiment of this or any aspect, the cell has reduced p53 protein activity as compared to an appropriate control.

[0026] Another aspect disclosed herein provides a method for increasing TP53 gene in a subject in need thereof, the method comprising administering to the subject in need thereof any of the synthetic antisense oligonucleotides, nucleic acid vectors, or compositions disclosed herein.34925-2218-9179 1701586-000159WOPTAttorney’s Docket No. 701586-000159WOPT

[0027] In one embodiment of this or any aspect, administration is local administration or systemic administration.

[0028] In one embodiment of this or any aspect, the subject in need thereof has reduced p53 protein activity as compared to an appropriate control. In one embodiment of this or any aspect, the subject in need thereof has been diagnosed as having, or is at risk of having a disease or disorder caused by reduced p53 protein activity.

[0029] In one embodiment of this or any aspect, the disease or disorder caused by reduced p53 protein activity is Li-Fraumeni syndrome or a cancer associated with TP53 haploinsufficiency.

[0030] In one embodiment of this or any aspect, administration is performed via liposomal delivery, nanoparticle deliver}’, or viral delivery system.

[0031] Another aspect disclosed herein provides a method for increasing TP53 gene in a subject in need thereof, the method comprising administering to the subject in need thereof a first synthetic antisense oligonucleotides comprising a nucleotide sequence of SEQ ID NO: 1, or comprising a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 1; and a second synthetic antisense oligonucleotides comprising a nucleotide sequence of SEQ ID NO: 2, or comprising a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 2

[0032] In one embodiment of this or any aspect, each of the first and second synthetic antisense oligonucleotide is comprised in different nucleic acid vectors. In one embodiment of this or any aspect, each of the first and second synthetic antisense oligonucleotide are administered in a single composition. In one embodiment of this or any aspect, each of the first and second synthetic antisense oligonucleotide are administered in different compositions. In one embodiment of this or any aspect, different compositions are administered at substantially the same time, or at different times

[0033] Definitions

[0034] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific tenns used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.

[0035] Methods and compositions described herein require that TP53 gene levels are increased. As used herein, “tumor protein p53” (TP53) refers to a gene that encodes a tumor suppressor protein containing transcriptional activation, DNA binding, and oligomerization domains. The encoded protein (p53)44925-2218-9179 1701586-000159WOPTAttorney’s Docket No. 701586-000159WOPTresponds to diverse cellular stresses to regulate expression of target genes, thereby inducing cell cycle arrest, apoptosis, senescence, DNA repair, or changes in metabolism. Sequences for TP53, also known as P53, BCC7, LFS1, BMFS5, and TRP53, are known for a number of species, e.g., human TP53 (NCBI Gene ID: 7157) polypeptide (e.g., NCBI Ref Seq NP_ 000537.3) and mRNA (e.g., NCBI Ref Seq NM 000546.6). TP53 can refer to human TP53, including naturally occurring variants, molecules, and alleles thereof. TP53 refers to the mammalian TP53 of, e.g., mouse, rat. rabbit, dog, cat, cow. horse, pig, and the like.

[0036] As used herein, ’‘synergistic increase” or ‘’synergistic upregulation” refers to a combined effect of administering two ASOs that exceeds the sum of their individual effects when administered alone at equivalent total oligonucleotide dose and under comparable conditions. For claim interpretation, a “synergistic increase” is satisfied if the measured TP53 mRNA or p53 protein level after dual-ASO administration is at least 1.5-fold higher than the best single-ASO condition and greater than tire arithmetic sum of each single ASO’s individual increases relative to control. Quantification may be performed by RT-qPCR for mRNA and immunoblot or ELISA for protein.

[0037] ‘ ‘Modulate splicing” means to change the splicing pattern of a pre-mRNA, including promoting inclusion of an exon, promoting skipping of an exon, altering usage of alternative 5' or 3' splice sites, or modifying intron retention. “Promote inclusion” means increasing the proportion of transcripts that contain the specified exon relative to a control.

[0038] “Capable of binding” describes an ASO that hybridizes to its target RNA region under standard stringency conditions typical for antisense therapeutics (e.g., physiological salt, 37°C), producing a stable duplex sufficient to exert the intended functional effect. Binding capability can be demonstrated by in vitro hybridization assays, melting temperature (Tm) measurement, or target engagement assays.

[0039] “Capable of modulating splicing” describes an ASO that, upon hybridization to a target splicing-regulatory element, produces a measurable change in exon inclusion or exclusion in the pre-mRNA relative to a control. Evidence of modulation may include RT-PCR, RT-qPCR, RNA-seq, or reporter assays.

[0040] ‘ ‘Nucleic acid vector” refers to an engineered nucleic acid construct capable of delivering, maintaining, or expressing one or more nucleic acid sequences in a host cell, including plasmid DNA, viral genomes, replicons, and self-amplifying RNA constructs.

[0041] As used herein, the term “internal exon” refers to any exon other than the first or last exon of the gene (i.e. an exon flanked by introns).

[0042] The terms “increase”, “enhance”, or “activate” are all used herein to mean an increase by a reproducible statistically significant amount. In some embodiments, the terms “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase54925-2218-9179 1701586-000159WOPTAttorney’s Docket No. 701586-000159WOPTof at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, a 20 fold increase, a 30 fold increase, a 40 fold increase, a 50 fold increase, a 6 fold increase, a 75 fold increase, a 100 fold increase, etc. or any increase between 2-fold and 10-fold or greater as compared to an appropriate control. In the context of a marker, an “increase” is a reproducible statistically significant increase in such level.

[0043] As used herein, an “appropriate control” refers to an untreated, otherwise identical cell or population (e.g., a patient who was not administered an ASO described herein, or was administered by only a subset of ASOs described herein, as compared to a non-control patient).

[0044] The term “statistically significant" or “significantly" refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.

[0045] As used herein the term "comprising" or "comprises" is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the method or composition, yet open to the inclusion of unspecified elements, whether essential or not.

[0046] As used herein the tenn "consisting essentially of refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment. The term "consisting of refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0047] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Fig. 1 presents exemplary data showing relative mRNA levels of p53 in MCF7 cells following treatment with ASOs. Cells were electroporated with 5 pM of each ASO. Corresponding control cells were electroporated with 10 pM scrambled ASO. Total TP53 expression is measured by RT-qPCR.

[0049] Fig. 2 presents exemplary data showing protein levels of p53 in MCF7 cells in the presence of Camptothecin. Cells were electroporated with treating ASOs or a scrambled ASO as explained above.64925-2218-9179 1701586-000159WOPTAttorney’s Docket No. 701586-000159WOPT

[0050] Fig. 3 presents exemplary data showing relative proliferation rate of cells at 0, 24, and 48 hours following ASO treatment. Cells were harvested, counted, and plated into a 96-well plate for the MTT assay.

[0051] Fig. 4 presents exemplary data showing representative images of Transwell migration assays showing that the combination of the two ASOs prevented cell migration in HeLa cells treated with Camptothecin.

[0052] Fig. 5 presents exemplary data showing representative images of MCF7 cells invasion assays shown in lOx microscope images taken at two different time intervals (24 and 48 hours) following ASO treatment. P < 0.01; *P < 0.001; **P < 0.001, determined by Fisher’s exact test.DETAILED DESCRIPTION

[0053] RNA splicing is a frequently regulated process that varies between tissues and species. Although 95% of multi-exon human genes undergo alternative splicing, transcript isoform differences across human tissues are heavily driven by alternative transcription start and termination sites, which are present in more than half of human genes. Tire processing of RNA transcripts from mammalian genes often occurs nearby in time and space to their synthesis, creating opportunities for functional connections between transcription and splicing. Several links between splicing and transcription are known, and both transcription rate and chromatin structure can influence splicing outcomes in some cases. Early studies suggest that both spliceosome assembly and catalysis of splicing occur in a co-transcriptional manner and it has been recently shown that splicing, transcription initiation, and termination can be coordinated. Splicing can impact transcription elongation rates and in yeast tire presence of an intron can generate a transcriptional checkpoint that is associated with pre-spliceosome fomiation. Furthermore, recruitment of the spliceosome complex can stimulate transcription initiation by enhancing preinitiation complex assembly, and inhibition of splicing can reduce levels of histone 3 lysine 4 trimethyl (H3K4me3), a chromatin mark associated with active transcription. There is evidence that adding an intron to an otherwise intron-less gene often boosts gene expression in plants, animals, and fungi; the mechanisms are not fully understood but impacts on transcription, nuclear export, mRNA stability, and / or translation have been noted.

[0054] The present disclosure provides methods for activating the transcription of the TP53 gene. The results herein found that activation of a splice site with antisense oligonucleotides (ASOs) enhances the splicing of an internal exon and results in an increase in transcription the TP53 gene from nearby upstream promoters. Also disclosed herein are methods for increasing transcription and expression of the TP53 gene via ASOs described herein results in increased p53 activity, and provides a therapeutic benefit in subjects having a disease or disorder caused by reduced p53 activity.74925-2218-9179 1701586-000159WOPTAttorney’s Docket No. 701586-000159WOPTSynthetic Antisense Oligonucleotides

[0055] Thus, in some aspects, the present disclosure relates to the use of oligonucleotides such as ASOs that bind a region of a TP53 RNA transcript. ASOs are nucleotide sequences that bind to target sequences on pre-mRNA via Watson-Crick base-pairing.

[0056] In one embodiment, the ASO comprises a nucleotide sequence of SEQ ID NO: 1. In one embodiment, the ASO consists of a nucleotide sequence of SEQ ID NO: 1. In one embodiment, the ASO consists essentially of a nucleotide sequence of SEQ ID NO: 1.

[0057] In one embodiment, the ASO comprises a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 1. In one embodiment, the ASO consists of a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%. 95%. 96%. 97%. 98%, 99% or more identical to SEQ ID NO: 1. In one embodiment, the ASO consists essentially of a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 1.

[0058] / 52MOErG / / i2MOErG / / i2MOErC / / i2MOErT / / i2MOErA / / i2MOErG / / i2MOErG / / i2MOErC / / i2M OErT / / i2MOErA / / i2MOErA / / i2MOErG / / i2MOErC / / i2MOErT / / i2MOErA / / i2MOErT / / i2MOErG / / i2MOE rA / / i2MOErT / / i2MOErG / / i2MOErT / / i2MOErT / / i2MOErC / / i2MOErC / / 32MOErT / (SEQ ID NO: 1)

[0059] In one embodiment, the ASO comprises a nucleotide sequence of SEQ ID NO: 2. In one embodiment, the ASO consists of a nucleotide sequence of SEQ ID NO: 2. In one embodiment, the ASO consists essentially of a nucleotide sequence of SEQ ID NO: 2.

[0060] In one embodiment, the ASO comprises a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%. 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 2. In one embodiment, the ASO consists of a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%. 95%. 96%. 97%. 98%. 99% or more identical to SEQ ID NO: 2. In one embodiment, the ASO consists essentially of a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 2.

[0061] / 52MOErA / / i2MOErC / / i2MOErA / / i2MOErA / / i2MOErT / / i2MOErT / / i2MOErT / / i2MOErT / / i2M OErC / / i2MOErT / / i2MOErT / / i2MOErT / / i2MOErT / / i2MOErT / / i2MOErG / / i2MOErA / / i2MOErA / / i2MOE rA / / i2MOErG / / i2MOErC / / i2MOErT / / i2MOErG / / i2MOErG / / i2MOErT / / i2MOErC / / i2MOErT / / i2MOErG / / i2MOErG / / 32MOErT / b (SEQ ID NO: 2)

[0062] In some embodiments, the region of complementarity of an oligonucleotide is complementary with at least 8 to 15, 8 to 30, 8 to 40, or 10 to 50, or 5 to 50, or 5 to 40 bases, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21. 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,84925-2218-9179 1701586-000159WOPTAttorney’s Docket No. 701586-000159WOPT41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 consecutive nucleotides of atarget. In some embodiments, the region of complementarity is complementary with at least 8 consecutive nucleotides of a target.

[0063] Complementary, as the term is used in the art, refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at a corresponding position of a target RNA, then the nucleotide of the oligonucleotide and the nucleotide of the target RNA are complementary to each other at that position. The oligonucleotide and target RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides that can hydrogen bond with each other through their bases. Tirus, "complementary" is a term which is used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and target nucleic acid. For example, if a base at one position of an oligonucleotide is capable of hydrogen bonding with a base at the corresponding position of atarget. then the bases are considered to be complementary to each other at that position. 100% complementarity is not required.

[0064] An oligonucleotide may be at least 80% complementary to (optionally one of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to) the consecutive nucleotides of atarget. In some embodiments an oligonucleotide may contain 1, 2 or 3 base mismatches compared to the portion of the consecutive nucleotides of the target. In some embodiments an oligonucleotide may have up to 3 mismatches over 15 bases, or up to 2 mismatches over 10 bases.

[0065] In some embodiments, an oligonucleotide is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80 or more nucleotides in length. In some embodiments, the oligonucleotide is 8 to 50, 10 to 30, 15 to 30 or 8 to 80 nucleotides in length.

[0066] Base pairings may include both canonical Watson-Crick base pairing and non-Watson-Crick base pairing (e.g., Wobble base pairing and Hoogsteen base pairing). It is understood that for complementary base pairings, adenosine-type bases (A) are complementary to thymidine-type bases (T) or uracil-type bases (U), that cytosine-type bases (C) are complementary’ to guanosine-type bases (G), and that universal bases such as 3 -nitropyrrole or 5 -nitroindole can hybridize to and are considered complementary to any A, C, U, or T. Inosine (I) has also been considered in the art to be a universal base and is considered complementary to any A, C, U or T.

[0067] It is understood that any reference to uses of oligonucleotides or other molecules throughout the description contemplates use of the oligonucleotides or other molecules in preparation of a pharmaceutical composition or medicament for use in the treatment of condition or a disease associated with decreased levels or activity of a protein.

[0068] In some embodiments, for tire 5' end, oligonucleotides may be used that are fully / partly complementary to 10-60 nts of the target exon sequence 5' end. In some embodiments, all nucleotides of94925-2218-9179 1701586-000159WOPTAttorney’s Docket No. 701586-000159WOPTan oligonucleotide may be complementary to the 5' end of a target exon sequence, with or without few nucleotide overhangs that may or may not be complementary to a sequence immediately adjacent to the 5' end of the target exon sequence. In some embodiments, for the 3' end, oligonucleotides may be fully or partly complementary to 10-60 nts of the target exon sequence 3' end. In some embodiments, all nucleotides of an oligonucleotide may be complementary to the 3' end of a target exon sequence, with or without few nucleotide overhangs that may or may not be complementary to a sequence immediately adjacent to the 3' end of the target exon sequence.

[0069] In some embodiments, the oligonucleotide comprises a region of complementarity that is complementary with tire target exon sequence (e.g., with at least 5 contiguous nucleotides) at a position that begins within 100 nucleotides, within 50 nucleotides, within 30 nucleotides, within 20 nucleotides, within 10 nucleotides or within 5 nucleotides of the 5'-end and / or 3'-end of the target exon sequence. In some embodiments, an oligonucleotide comprises a region of complementarity that is complementary with the target exon sequence (e.g., with at least 5 contiguous nucleotides of the target exon sequence) at a position that begins at the 5 '-end and / or 3 '-end of the target exon sequence.

[0070] In some embodiments, oligonucleotides are provided with chemistries suitable for deli ven. hybridization and stability within cells to target splicing. Furthennore, in some embodiments, oligonucleotide chemistries are provided that are useful for controlling the pharmacokinetics, biodistribution, bioavailability and / or efficacy of the oligonucleotides. Accordingly, oligonucleotides described herein may be modified, e.g., comprise a modified sugar moiety, a modified intemucleoside linkage, a modified nucleotide and / or combinations thereof. Any of the oligonucleotides disclosed herein may be linked to one or more other oligonucleotides disclosed herein by a linker, e.g., a cleavable linker.

[0071] In some embodiments, the ASO is a morpholino oligonucleotide. Morpholino SSOs are a type of antisense oligonucleotide in which the DNA bases are attached to a backbone of methylenemorpholine rings linked through phosphorodiamidate groups. Non-limiting examples of other SSOs that can be applied to the methods of the present disclosure include phosphorothioate oligos, 2'-O-methyl (2'-OMe) oligos and 2'-O-methoxyethyl (2'MOE) oligos. Oligonucleotides of the invention can be stabilized against nucleolytic degradation such as by the incorporation of a modification, e.g., a nucleotide modification. For example, nucleic acid sequences of tire invention include a phosphorothioate at least the first, second, or third intemucleotide linkage at the 5' or 3' end of the nucleotide sequence. As another example, the nucleic acid sequence can include a 2'-modified nucleotide, e.g., a 2'-deoxy, 2’-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2'-0-M0E), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-0 — N-methylacetamido (2'-0-NMA). As another example, the nucleic acid sequence can include at least one 2'-O-methyl-modified nucleotide, and in some embodiments, all of the104925-2218-9179 1701586-000159WOPTAttorney’s Docket No. 701586-000159WOPTnucleotides include a 2'-O-methyl modification. In some embodiments, the nucleic acids are “locked,” i.e., comprise nucleic acid analogues in which the ribose ring is “locked” by a methylene bridge connecting the 2'-0 atom and the 4'-C atom.

[0072] Any of the modified chemistries or formats of oligonucleotides described herein can be combined with each other, and that one, two. three, four, five, or more different types of modifications can be included within the same molecule.

[0073] In some embodiments, the oligonucleotide may comprise at least one ribonucleotide, at least one deoxyribonucleotide, and / or at least one bridged nucleotide. In some embodiments, the oligonucleotide may comprise a bridged nucleotide, such as a locked nucleic acid (LNA) nucleotide, a constrained ethyl (cEt) nucleotide, or an ethylene bridged nucleic acid (ENA) nucleotide. Examples of such nucleotides are disclosed herein and known in the art. In some embodiments, the oligonucleotides can comprise any combination of the modifications disclosed herein. Examples of these ASOs are provided in Havens and Hastings, Nucleic Acids Research 44(14): 6549-6563, 2016, the relevant disclosures of which are herein incorporated by reference for the purpose and subject matter referenced herein.

[0074] The oligonucleotide may comprise deoxyribonucleotides flanked by at least one bridged nucleotide (e.g., a LNA nucleotide, cEt nucleotide, ENA nucleotide) on each of the 5' and 3' ends of the deoxyribonucleotides. Tire oligonucleotide may comprise deoxyribonucleotides flanked by 1, 2, 3, 4, 5, 6,7, 8 or more bridged nucleotides (e.g., LNA nucleotides, cEt nucleotides, ENA nucleotides) on each of the 5' and 3' ends of the deoxyribonucleotides. The 3' position of the oligonucleotide may have a 3' hydroxyl group.

[0075] The 3′ position of the oligonucleotide may have a 3′ thiophosphate. The oligonucleotide may be conjugated with a label. For example, the oligonucleotide may be conjugated with a biotin moiety, cholesterol, Vitamin A, folate, sigma receptor ligands, aptamers, peptides, such as CPP, hydrophobic molecules, such as lipids, ASGPR or dynamic polyconjugates and variants thereof at its 5' or 3' end. The oligonucleotide may also be Vivo-linked (e.g., Vivo-linked phosphorodiamidate morpholino (VPMO)).

[0076] Preferably an oligonucleotide comprises one or more modifications comprising: a modified sugar moiety, and / or a modified internucleoside linkage, and / or a modified nucleotide and / or combinations thereof. It is not necessary for all positions in a given oligonucleotide to be uniformly modified, and in fact more than one of the modifications described herein may be incorporated in a single oligonucleotide or even at within a single nucleoside within an oligonucleotide.

[0077] In some embodiments, the oligonucleotides are chimeric oligonucleotides that contain two or more chemically distinct regions, each made up of at least one nucleotide. These oligonucleotides typically 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 114925-2218-9179 1701586-000159WOPTAttorney’s Docket No. 701586-000159WOPTaffinity for the target) and a region that is a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Chimeric oligonucleotides of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and / or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures comprise, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065: 5,652,355; 5,652,356; and 5,700,922, each of which is herein incorporated by reference.

[0078] ASO Modifications

[0079] A number of nucleotide and nucleoside modifications have been shown to make the oligonucleotide into which they are incorporated more resistant to nuclease digestion than the native oligodeoxynucleotide: these modified oligos survive intact for a longer time than unmodified oligonucleotides. Specific examples of modified oligonucleotides include those comprising modified backbones, for example, phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. In some embodiments, oligonucleotides may have phosphorothioate backbones; heteroatom backbones, such as methylene(methylimino) or MMI backbones; amide backbones (see De Mesmaeker et al. Ace. Chem. Res. 1995, 28:366-374): morpholino backbones (see Summerton and Weller, U. S. Pat. No. 5,034,506); or peptide nucleic acid (PNA) backbones (wherein the phosphodiester backbone of the oligonucleotide is replaced with a polyamide backbone, the nucleotides being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone, see Nielsen et al., Science 1991, 254, 1497). Phosphorus-containing linkages include, but are 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 nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'; see U. S. Pat. Nos. 3,687,808; 4,469,863; 4.476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5.276,019; 5,278.302; 5,286,717; 5,321,131; 5,399.676; 5,405,939: 5,453,496; 5,455. 233; 5.466.677; 5,476,925: 5.519,126; 5.536.821; 5,541.306: 5,550,111; 5.563. 253; 5.571.799; 5,587,361; and 5,625,050.

[0080] Morpholino-based oligomeric compounds are described in Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510); Genesis, volume 30, issue 3, 2001; Heasman, J., Dev. Biol., 2002, 243, 209-214; Nasevicius et aL, Nat. Genet., 2000, 26, 216-220; Lacerra et al., Proc. Natl.124925-2218-9179 1701586-000159WOPTAttorney’s Docket No. 701586-000159WOPTAcad. Sci., 2000, 97, 9591-9596; and U. S. Pat. No. 5,034,506, issued Jul. 23, 1991. In some embodiments, the morpholino-based oligomeric compound is a phosphorodiamidate morpholino oligomer (PMO) (e.g., as described in Iverson, Curr. Opin. Mol. Ther., 3:235-238, 2001; and Wang et al., J. Gene Med., 12:354-364, 2010; the disclosures of which are incorporated herein by reference in their entireties).

[0081] Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wang et al., J. Am. Chem. Soc., 2000, 122. 8595-8602.

[0082] Modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These comprise those having morpholino linkages (formed in part from the sugar portion of a nucleoside); 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 CH2 component parts; see U. S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264, 562; 5, 264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5.602,240; 5,608.046; 5,610,289; 5,618,704; 5,623.070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which is herein incorporated by reference.

[0083] Modified oligonucleotides are also known that include oligonucleotides that are based on or constructed from arabinonucleotide or modified arabinonucleotide residues. Arabinonucleosides are stereoisomers of ribonucleosides, differing only in the configuration at the 2-position of the sugar ring. In some embodiments, a 2'-arabino modification is 2'-F arabino. In some embodiments, the modified oligonucleotide is 2'-fluoro-D-arabinonucleic acid (FANA) (as described in. for example, Lon et al.. Biochem., 41:3457-3467, 2002 and Min et aL. Bioorg. Med. Chem. Lett., 12:2651-2654, 2002; the disclosures of which are incorporated herein by reference in their entireties). Similar modifications can also be made at other positions on the sugar, particularly the 3' position of the sugar on a 3' terminal nucleoside or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide.

[0084] PCT Publication No. WO 99 / 67378 discloses arabinonucleic acids (ANA) oligomers and their analogues for improved sequence specific inhibition of gene expression via association to complementary messenger RNA.

[0085] Other preferred modifications include ethylene-bridged nucleic acids (ENAs) (e.g., International Patent Publication No. WO 2005 / 042777, Morita et al., Nucleic Acid Res., Suppl 1:241-242, 2001;Surono et al., Hum. Gene Ther., 15:749-757, 2004; Koizumi, Curr. Opin. Mol. Then, 8:144-149, 2006 and Horie et al., Nucleic Acids Symp. Ser (Oxf), 49:171-172, 2005; the disclosures of which are incorporated134925-2218-9179 1701586-000159WOPTAttorney’s Docket No. 701586-000159WOPTherein by reference in their entireties). Preferred ENAs include, but are not limited to, 2'-O,4'-C-ethylene-bridged nucleic acids.

[0086] One or more substituted sugar moieties can also be included, e.g., one of the following at the 2' position: OH, SH, SCH3, F, OCN, OCH3, OCH3 O(CH2)n CH3, O(CH2)n NH2 or O(CH2)n CH3 where n is from 1 to about 10; Cl to CIO lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkar l or aralkyl; Cl; Br; CN; CF3; OCF3; O-, S-. orN-alkyl; O-, S-, orN-alkenyl; SOCH3; SO2 CH3; ONO2; NO2: N3: NH2: heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; 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 preferred modification includes 2'-methoxyethoxy [2'-0 — CH2CH2OCH3. also known as 2'-O-(2-methoxyethyl)] (Martin et al, Helv. Chim. Acta, 1995, 78, 486). Other preferred modifications include 2'-methoxy (2'-0 — CH3), 2'-propoxy (2'-OCH2 CH2CH3) and 2'-fluoro (2'-F). Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group.

[0087] It is not necessary for all positions in a given oligonucleotide to be uniformly modified, and in fact more than one of the modifications described herein may be incorporated in a single oligonucleotide or even at within a single nucleoside within an oligonucleotide.

[0088] In some embodiments, both a sugar and an internucleoside 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, for example, an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U. S. Pat. Nos. 5,539,082; 5,714,331; and 5.719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al, Science, 1991. 254, 1497-1500.

[0089] In some embodiments, an oligonucleotide comprises phosphorothioate internucleotide linkages. In some embodiments, an oligonucleotide comprises phosphorothioate internucleotide linkages between at least two nucleotides. In some embodiments, an oligonucleotide comprises phosphorothioate internucleotide linkages between all nucleotides.144925-2218-9179 1701586-000159WOPTAttorney’s Docket No. 701586-000159WOPT

[0090] It should be appreciated that an oligonucleotide can have any combination of modifications as described herein.

[0091] Synthesis ofASOs

[0092] Methods of synthesizing ASOs that are specific for target sequence and / or that have modified backbones are known in the art. Synthesis of specific ASOs with modified backbones is taught in U. S. Pat. Nos. 5,378,825 and 5.541,307, the relevant disclosures of which are herein incorporated by reference for the purpose and subject matter referenced herein. Synthesis of ASOs having morpholino backbone structures are taught in U. S. Pat. No. 5,034,506, the relevant disclosures of which are herein incorporated by reference for the purpose and subject matter referenced herein.

[0093] There are alternative approaches in the art, such as RNA interference (RNAi) approaches in which oligos base-pair with complementary target mRNA have been used to down-regulate specific genes post-transcriptionally. Moreover, shifting splicing by ASOs for its own sake, or using changes in splicing to alter gene expression by producing isoforms that will have increased or reduced mRNA stability (e.g., via nonsense-mediated mRNA decay (NMD)) has been proposed (herein referred to as “conventional ASO applications”). The novelty of the approach in the present disclosure is the possibility to regulate transcription itself, so that production of transcripts is reduced or increased rather than their stability. With this technology, the co-transcriptional alteration in splicing may not trigger NMD. but it modulates transcription and isoform-specific mRNA synthesis. This distinction could be of particular use in cases where: (i) it is critical to inhibit the mRNA synthesis because the transcripts are not degraded efficiently (e.g., in triplet repeat diseases such as myotonic dystrophy type 1, Huntington's disease, etc.); or (ii) the mRNA naturally has a very short half-life (e.g., the c-myc oncogene), making further reduction in halflife challenging or impossible. The above conventional ASO applications act only as long as the ASO is in the cell.

[0094] Classical RNAi or ASO designs work efficiently for some genes, but other genes only give modest responses or are refractory to the technology. Since splicing is crucial for the expression of 94% of human genes, the methods disclosed herein can be used in cases where classical RNAi or conventional ASO approaches are not effective or do not achieve sufficient repression of expression. In addition, expression of more than 54% of human genes is controlled by alternative promoters whose regulation can be crucial for cell differentiation and development. Mis-regulation of the usage of alternative promoters is linked to various diseases. Disclosed herein is the first technology that, by impacting splicing, controls the usage of alternative promoters to enhance or inhibit specific sites of transcription initiation.Nucleic Acid Vectors154925-2218-9179 1701586-000159WOPTAttorney’s Docket No. 701586-000159WOPT

[0095] Various aspects of the present invention relate to a nucleic acid vector comprising any of the ASOs disclosed herein. As used herein, a “nucleic acid vector” may be any of a number of nucleic acids into which a desired sequence or sequences may be inserted, for example, by restriction digestion and ligation or by recombination for transport between different genetic environments or for expression in a host cell. Vectors are typically composed of DNA. although RNA vectors are also available. Examples of vectors include, but are not limited to plasmids, fosmids, phage lambda, cosmids, single stranded phages, expression vectors, artificial chromosomes, adeno-associated virus (AAV) vectors, and retroviral vectors. These vectors can be cloned with a sequence that codes for the ASO.

[0096] In one embodiment, the nucleic acid vector comprises a nucleotide sequence of SEQ ID NO: 1. In one embodiment, the ASO consists of a nucleotide sequence of SEQ ID NO: 1. In one embodiment, the ASO consists essentially of a nucleotide sequence of SEQ ID NO: 1.

[0097] In one embodiment, the nucleic acid vector comprises a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 1. In one embodiment, the ASO consists of a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 1. In one embodiment, the ASO consists essentially of a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%. 94%. 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 1.

[0098] In one embodiment, the nucleic acid vector comprises a nucleotide sequence of SEQ ID NO: 2. In one embodiment, the ASO consists of a nucleotide sequence of SEQ ID NO: 2. In one embodiment, the ASO consists essentially of a nucleotide sequence of SEQ ID NO: 2.

[0099] In one embodiment, the nucleic acid vector comprises a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 2. In one embodiment, the ASO consists of a nucleotide sequence that is at least 85%. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%. 94%. 95%. 96%. 97%. 98%. 99% or more identical to SEQ ID NO: 2. In one embodiment, the ASO consists essentially of a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 2.

[0100] Cloning, or molecular cloning methods, e.g., for generating a nucleic acid vector comprising a ASO as disclosed herein, is known in the art (see, e.g., Current Protocols in Molecular Biology, Ausubel, F. M., et al., New York: John Wiley & Sons. 2006; Molecular Cloning: A Laboratory Manual, Green, M. R. and Sambrook J., New York: Cold Spring Harbor Laboratory Press, 2012; Gibson, D. G., et aL, Nature Methods 6(5):343-345 (2009), the teachings of which relating to molecular cloning are herein incorporated by reference).Methods of Increasing p53 Activity164925-2218-9179 1701586-000159WOPTAttorney’s Docket No. 701586-000159WOPT

[0101] One aspect disclosed herein provides a method for increasing TP53 gene expression in a cell, the method comprising the steps of contacting a cell with any of the synthetic antisense oligonucleotides disclosed herein, or nucleic acid vector or composition comprising the same.

[0102] In one embodiment, TP53 expression and / or p53 activity is increased by at least 10% as compared to an appropriate control. In one embodiment, TP53 expression and / or p53 activity is increased by 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%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%. at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%. at least 59%, at least 60%, at least 61%, at least 62%, at least 63%. at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 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 more or at least lx, at least 2x, at least 3x, at least 4x, at least 5x, at least 6x, at least 7x, at least 8x, at least 9x, at least lOx, at least 20x, at least 30x, at least 40x, at least 50x, at least 60x, at least 70x, at least 80x, at least 90x, at least lOOx, at least 200x, at least 300x, at least 400x, at least 500x, at least 600x, at least 700x, at least 800x, at least 900x, at least lOOOx, or more as compared to an appropriate control. As used herein, an appropriate control refers to an otherwise identical cell that is not contacted with the at least one ASO or is contacted only with a subset of ASOs, or the level prior to contacting. A skilled one in the art can assess TP53 expression levels and / or p53 activity in a cell using standard techniques known in the art.

[0103] In one embodiment, the cell has reduced p53 protein activity as compared to an appropriate control. In one embodiment, p53 protein activity is reduced by at least 10% as compared to an appropriate control. In one embodiment, p53 protein activity is reduced by 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%, at least 20%, at least 21%, at least 22%, at least 23%. at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%,174925-2218-9179 1701586-000159WOPTAttorney’s Docket No. 701586-000159WOPTat least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 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 more as compared to an appropriate control. As used herein, an appropriate control refers to an otherwise identical cell that is healthy, or obtained from a healthy subject. A skilled one in the art can assess the p53 activity levels in a cell using standard techniques known in the art.

[0104] In one embodiment, the cell is a mammalian cell. In one embodiment, the mammalian cell is a human cell.

[0105] In one embodiment, contacting is in vitro, in vivo, or ex vivo.

[0106] In one embodiment, contacting is performed via liposomal delivery, nanoparticle delivery, or viral delivery system.

[0107] As used herein, the term “contacting” refers to in vitro methods of contacting as well as in vivo methods of contacting. The in vitro methods of contacting cells with an ASO or small molecule involve transfection of the cells with an ASO or small molecules. As used herein, the term “transfection” refers to the artificial delivery’ and introduction of nucleic acids (e.g., SSOs), into a cell (e.g., eukaryotic cell). Methods of transfection are well established in the arts and range from chemical, to biological, and to physical methods. Chemical methods include, but are not limited to, calcium phosphate transfection, cationic polymer transfection, lipofection, and DEAE-Dextran-mediated transfection. Other methods of transfection include, but are not limited to, electroporation, sonoporation, cell squeezing, impalefection, optical transfection, protoplast fusion. Magnetofection, and particle bombardment. Non-limiting examples of cells that can undergo transfection as described herein include NIH 3T3 fibroblast cells, HeLa cells, and CAD cells.

[0108] Another aspect disclosed herein provides a method for increasing TP53 gene expression in a cell, the method comprising the steps of administering to the subject in need thereof any of the synthetic antisense oligonucleotides disclosed herein, or nucleic acid vector or composition comprising the same.

[0109] In one embodiment, the subject in need thereof has reduced p53 protein activity as compared to an appropriate control. In one embodiment, the subject in need thereof has been diagnosed as having, or is at risk of having a disease or disorder caused by reduced p53 protein activity. Exemplary diseases or disorders caused by reduced p53 protein activity is Li-Fraumeni syndrome or a cancer associated with TP53 haploinsufficiency.

[0110] Another aspect disclosed herein provides a method for increasing TP53 gene in a subject in need thereof, the method comprising administering to the subject in need thereof a first synthetic antisense oligonucleotides comprising a nucleotide sequence of SEQ ID NO: 1, or comprising a nucleotide184925-2218-9179 1701586-000159WOPTAttorney’s Docket No. 701586-000159WOPTsequence that is at least 85% identical to the sequence of SEQ ID NO: 1; and a second synthetic antisense oligonucleotides comprising a nucleotide sequence of SEQ ID NO: 2, or comprising a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 2.

[0111] In one embodiment, TP53 expression is increased by at least 10% as compared to an appropriate control. In one embodiment. TP53 expression is increased by 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%. at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%. at least 59%, at least 60%. at least 61%, at least 62%, at least 63%, at least 64%, at least 65%. at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, 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 more or at least lx, at least 2x, at least 3x, at least 4x, at least 5x, at least 6x, at least 7x, at least 8x, at least 9x, at least lOx, at least 20x, at least 30x, at least 40x, at least 50x, at least 60x, at least 70x, at least 80x, at least 90x, at least lOOx, at least 200x, at least 300x, at least 400x, at least 500x, at least 600x, at least 700x, at least 800x, at least 900x, at least lOOOx, or more as compared to an appropriate control. As used herein, an appropriate control refers to an otherwise identical tissue that is not administered the at least one ASO or is administered only with a subset of ASOs, or the level prior to administering. A skilled one in the art can assess TP53 gene levels in a cell using standard techniques known in the art.

[0112] In one embodiment, each of the first and second synthetic antisense oligonucleotide is comprised in different nucleic acid vectors. In one embodiment, each of the first and second synthetic antisense oligonucleotide are administered in a single composition. In one embodiment, each of the first and second synthetic antisense oligonucleotide are administered in different compositions. In one embodiment, the different compositions are administered at substantially the same time, or at different times.

[0113] In one embodiment, the administration is local administration or systemic administration.

[0114] In some embodiments, the term '‘administering” refers to the administration of ASOs to a tissue in a subject. For example, the ASOs can be administered in vivo as an injection, using different deliver routes. The ASOs of the present disclosure can be administered intravenously, intradermally, intraarterially, intralesionally, intratumorally, intracranially, intraarticularly, intraprostaticaly,194925-2218-9179 1701586-000159WOPTAttorney’s Docket No. 701586-000159WOPTintrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, locally, inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in creams, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example. Remington's Pharmaceutical Sciences (1990), incorporated herein by reference).

[0115] In one embodiment, administration is performed via liposomal delivery, nanoparticle delivery or viral delivery system.

[0116] In some embodiments, the ASOs may be administered in the form of a drug. The drug would be a sterile composition comprising tire ASOs in inactive form and a pharmaceutically acceptable carrier. As used herein, the term “drug” requires that a compound or composition be nontoxic and sufficiently pure so that no further manipulation of the compound or composition is needed prior to administration to the subject. The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the cells, nanoparticles and / or agent(s) are combined to facilitate administration. The components of the pharmaceutical compositions are combined in a manner that precludes interaction that would substantially impair their desired pharmaceutical efficiency. Moreover, for animal (e.g.. human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by the Food and Drug Administration (FDA) Office of Biological Standards. The compounds are generally suitable for administration to humans or mammals.Compositions

[0117] One aspect disclosed herein is a composition comprising at least one synthetic antisense oligonucleotide disclosed herein.

[0118] Another aspect disclosed herein is a composition comprising at nucleic acid vector comprising at least one synthetic antisense oligonucleotide disclosed herein.

[0119] In various embodiment, tire composition comprises two distinct synthetic antisense oligonucleotides, or at least two nucleic acid vectors, each of the at least two nucleic acid vectors comprising at least one of the two distinct synthetic antisense oligonucleotides.

[0120] In one embodiment, at least one of the two distinct synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 1, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 1.204925-2218-9179 1701586-000159WOPTAttorney’s Docket No. 701586-000159WOPT

[0121] In one embodiment, at least one of the two distinct synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 2, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 2.

[0122] In various embodiment, the composition comprises a first a synthetic antisense oligonucleotide comprising a nucleotide sequence of SEQ ID NO: 1, or comprising a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 1; and a second synthetic antisense oligonucleotide comprising a nucleotide sequence of SEQ ID NO: 2, or comprising a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 2.

[0123] In various embodiment, the composition comprises a first nucleic acid vector comprising a synthetic antisense oligonucleotide comprising a nucleotide sequence of SEQ ID NO: 1, or comprising a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 1; and a second nucleic acid vector comprising a synthetic antisense oligonucleotide comprising a nucleotide sequence of SEQ ID NO: 2, or comprising a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 2.

[0124] Another aspect disclosed herein is a composition comprising a synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 1, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 1; and a synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 2, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 2: or a nucleic acid vector comprising a synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 1, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 1; and a synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 2, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 2; or at least two nucleic acid vectors, each of the at least two nucleic acid vectors expressing at least one of: a synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 1, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 1; and a synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 2, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 2.

[0125] As used here, the term "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.

[0126] As used here, the term "pharmaceutically-acceptable carrier" means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient,214925-2218-9179 1701586-000159WOPTAttorney’s Docket No. 701586-000159WOPTmanufacturing 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, but are not limited to: (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, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, 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 (PEG): (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 polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (22) C2-C12 alchols. such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, binding agents, fillers, lubricants, coloring agents, disintegrants, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative, water, salt solutions, alcohols, antioxidants, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like can also be present in the formulation. The terms such as "excipient", "carrier", "pharmaceutically acceptable carrier" or the like are used interchangeably herein.Administration

[0127] The ASOs described herein can be administered to a subject having or diagnosed as having a disease or disorder resulting from decreased p53 cellular levels. In some embodiments, the methods described herein comprise administering an effective amount of at least one ASO disclosed herein to a subject in order alleviate at least one symptom associated with the disease or disorder resulting from decreased p53 cellular levels. As used herein, "alleviating at least one symptom " is ameliorating any condition or symptom associated with the disease or disorder (e g., reduced TP53 expression levels). As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique. A variety of means for administering the ASOs described herein to subjects are known to those of skill in the art. In one224925-2218-9179 1701586-000159WOPTAttorney’s Docket No. 701586-000159WOPTembodiment, the ASOs are administered systemically or locally. In one embodiment, the ASOs are administered intravenously. In one embodiment, the ASOs are administered continuously, in intervals, or sporadically. The route of administration of the ASOs will be optimized by a skilled practitioner.

[0128] Tire tenn “effective amount" as used herein refers to the amount of that at least one ASO that can be administered to a subject having or the disease or disorder resulting from decreased p53 cellular levels needed to alleviate at least one or more symptom. The term "therapeutically effective amount" therefore refers to an amount of the at least one ASOs that is sufficient to provide a particular effect when administered to a typical subject. It is not generally practicable to specify an exact “effective amount". However, for any given case, an appropriate “effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.

[0129] In one embodiment, the subject is administered two ASOs in a single composition. In one embodiment, the subject is administered two separate compositions, each comprising a single ASO. When the subject is administered two separate compositions, the two separate compositions can be administered at substantially the same time. Alternatively, the two separate compositions can be administered sequentially. For example, the second separate composition is is administered within at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 96 hours, or more following administration of the first separate composition.

[0130] Effective amounts, toxicity, and therapeutic efficacy can be evaluated by standard pharmaceutical procedures in cell cultures or experimental animals. The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50 / ED50. Compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from cell culture assays. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the ASOs, which achieves a half-maximal inhibition of symptoms) as determined in cell culture, or in an appropriate animal model. Levels in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.

[0131] Dosage234925-2218-9179 1701586-000159WOPTAttorney’s Docket No. 701586-000159WOPT

[0132] " Unit dosage form" as the term is used herein refers to a dosage for suitable one administration. By way of example a unit dosage form can be an amount of therapeutic disposed in a delivery' device, e.g., a syringe or intravenous drip bag. In one embodiment, a unit dosage form is administered in a single administration. In another, embodiment more than one unit dosage form can be administered simultaneously.

[0133] The dosage of the at least one ASOs as described herein can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to administer further cells, discontinue treatment, resume treatment, or make other alterations to the treatment regimen. Tire dosage should not be so large as to cause adverse side effects, such as cytokine release syndrome. Generally, the dosage will vary with the age, condition, and sex of the patient and can be determined by one of skill in the art. The dosage can also be adjusted by the individual physician in the event of any complication.

[0134] Combinational therapy

[0135] In one embodiment, the at least one ASOs described herein is used as a monotherapy. In one embodiment, the at least one ASOs described herein can be used in combination with other known agents and therapies, for example, for a disease or disorder associated with, or caused by reduced p53 levels. Administered "in combination," as used herein, means that two (or more) different treatments are delivered to the subject during the course of the subject's affliction. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as "simultaneous", “at substantially the same time” or "concurrent delivery." In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In some embodiments, delivery' is such that the reduction in a symptom, or other parameter related to the disease or disorder is greater than what would be observed with one treatment delivered in the absence of tire other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered. The at least one ASOs described herein and the at least one additional therapy can be administered simultaneously, in the same or in separate compositions, or sequentially. For sequential administration, the at least one ASOs described herein can be administered first, and the additional agent can be administered second, or the244925-2218-9179 1701586-000159WOPTAttorney’s Docket No. 701586-000159WOPTorder of administration can be reversed. The at least one ASOs can be administered before another treatment, concurrently with the treatment, post-treatment, or during remission of the disorder.

[0136] Parenteral Dosage Forms

[0137] Parenteral dosage forms of the at least one ASOs described herein can be administered to a subject by various routes, including, but not limited to, epidural injection, subcutaneous, intravenous (including bolus injection), intramuscular, and intraarterial. Since administration of parenteral dosage forms typically bypasses the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, controlled-release parenteral dosage forms, and emulsions.

[0138] Suitable vehicles that can be used to provide parenteral dosage forms of the disclosure are well known to those skilled in the art. Examples include, without limitation: sterile water; water for injection USP; saline solution; glucose solution; aqueous vehicles such as but not limited to, sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, com oil, cottonseed oil, peanut oil. sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0139] Controlled and Delayed Release Dosage Forms

[0140] In some embodiments of the aspects described herein, the at least one ASOs is administered to a subject by controlled- or delayed-release means. Ideally, the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time. Advantages of controlled-release formulations include: 1) extended activity of the drug; 2) reduced dosage frequency; 3) increased patient compliance; 4) usage of less total drug; 5) reduction in local or systemic side effects; 6) minimization of drug accumulation; 7) reduction in blood level fluctuations; 8) improvement in efficacy of treatment; 9) reduction of potentiation or loss of drug activity; and 10) improvement in speed of control of diseases or conditions. (Kim, Cherng-ju, Controlled Release Dosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000)). Controlled-release formulations can be used to control a compound of formula (I)'s onset of action, duration of action, plasma levels within the therapeutic window, and peak blood levels. In particular, controlled- or extended-release dosage forms or formulations can be used to ensure that the maximum effectiveness of the at least one ASOs is achieved while minimizing potential adverse effects and safety concerns, which can occur both from under-dosing a drug (i.e., going below the minimum therapeutic levels) as well as exceeding the toxicity level for the drug.254925-2218-9179 1701586-000159WOPTAttorney’s Docket No. 701586-000159WOPT

[0141] A variety of known controlled- or extended -release dosage forms, formulations, and devices can be adapted for use with at least one ASOs described herein. Examples include, but are not limited to, those described in U. S. Pat. Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185, each of which is incorporated herein by reference in their entireties. These dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS® (Alza Corporation, Mountain View, Calif. USA)), multilayer coatings, microparticles, liposomes, or microspheres or a combination thereof to provide the desired release profile in varying proportions. Additionally, ion exchange materials can be used to prepare immobilized, adsorbed salt forms of the disclosed compounds and thus effect controlled delivery of the drug. Examples of specific anion exchangers include, but are not limited to. DUOLITE® A568 and DUOLITE® AP143 (Rohm& Haas. Spring House, Pa. USA).

[0142] Efficacy

[0143] The efficacy of any ASO described herein can be determined by the skilled practitioner. However, a treatment is considered “effective treatment." as the term is used herein, if one or more of the signs or symptoms of the disease or disorder are altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response is induced e.g., by at least 10% following treatment according to the methods described herein. Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and / or the incidence of an injury treated according to the methods described herein or any other measurable parameter appropriate. Efficacy can also be measured by a failure of an individual to worsen as assessed by hospitalization, or need for medical interventions. Methods of measuring these indicators are known to those of skill in the art and / or are described herein.

[0144] Efficacy can be assessed in animal models of a condition described herein, for example, a mouse model, as the case may be. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change in a marker is observed.

[0145] All patents, patent applications, and publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with tire present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.264925-2218-9179 1701586-000159WOPTAttorney’s Docket No. 701586-000159WOPT

[0146] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize tire present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.

[0147] The disclosed invention can further be described in the following numbered paragraphs:1) A synthetic antisense oligonucleotide that binds a region of a TP53 RNA transcript.2) The synthetic antisense oligonucleotide of paragraph 1, wherein the TP53 RNA transcript is a TP53 pre-mRNA transcript.3) The synthetic antisense oligonucleotide of any preceding paragraph, wherein binding modulates splicing of an internal exon of the TP53 transcript.4) The synthetic antisense oligonucleotide of any preceding paragraph, wherein the modulation increases TP53 mRNA levels and p53 protein levels.5) The synthetic antisense oligonucleotide of any preceding paragraph, wherein TP53 mRNA levels and p53 protein levels are increased by at least 10% as compared to an appropriate control.6) Tire synthetic antisense oligonucleotide of any preceding paragraph, wherein the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2. or wherein the antisense oligonucleotide comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.7) A nucleic acid vector comprising a synthetic antisense oligonucleotide of any preceding paragraph.8) The nucleic acid vector of any preceding paragraph, wherein tire synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 1, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 1.9) The nucleic acid vector of any preceding paragraph, wherein the synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 2, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 2.10) A nucleic acid vector comprising a synthetic antisense oligonucleotide comprising a nucleotide sequence of SEQ ID NO: 1, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 1.11) A nucleic acid vector comprising a synthetic antisense oligonucleotides comprising a nucleotide sequence of SEQ ID NO: 2, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 2.12) A composition comprising at least one synthetic antisense oligonucleotide of any preceding paragraph, or any of the nucleic acid vectors of any preceding paragraph.274925-2218-9179 1701586-000159WOPTAttorney’s Docket No. 701586-000159WOPT13) The composition of any preceding paragraph, wherein the composition comprises two distinct synthetic antisense oligonucleotides, or a nucleic acid vector comprising two distinct synthetic antisense oligonucleotides, or at least two nucleic acid vectors, each of the at least two nucleic acid vectors comprising at least one of: a synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 1, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 1; and a synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 2, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 2.14) Tire composition of any preceding paragraph, wherein at least one of the two distinct synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 1, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 1.15) The composition of any preceding paragraph, wherein at least one of the two distinct synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 2, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 2.16) A composition comprising:a synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 1, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 1; and a synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 2, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 2: ora nucleic acid vector comprising a synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 1, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 1; and a synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 2, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 2; orat least two nucleic acid vectors, each of the at least two nucleic acid vectors expressing at least one of: a synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 1, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 1; and a synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 2, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 2.17) A method for increasing TP53 gene expression in a cell, the method comprising: contacting a cell with any of the synthetic antisense oligonucleotides of any preceding paragraph, nucleic acid vectors of any preceding paragraph, or compositions of any preceding paragraph.284925-2218-9179 1701586-000159WOPTAttorney’s Docket No. 701586-000159WOPT18) The method of any preceding paragraph, wherein the cell is a mammalian cell.19) The method of any preceding paragraph, wherein the mammalian cell is a human cell.20) The method of any preceding paragraph, wherein contacting is in vitro, in vivo, or ex vivo.21) The method of any preceding paragraph, wherein contacting is performed via liposomal delivery, nanoparticle delivery, or viral delivery system.22) The method of any preceding paragraph, wherein the cell has reduced p53 protein activity as compared to an appropriate control.23) A method for increasing TP53 gene in a subject in need thereof, the method comprising administering to the subject in need thereof any of the synthetic antisense oligonucleotides of any preceding paragraph, nucleic acid vectors of any preceding paragraph, or compositions of any preceding paragraph.24) The method of any preceding paragraph, wherein administration is local administration or systemic administration.25) The method of any preceding paragraph, wherein the subject in need thereof has reduced p53 protein activity as compared to an appropriate control.26) The method of any preceding paragraph, wherein the subject in need thereof has been diagnosed as having, or is at risk of having a disease or disorder caused by reduced p53 protein activity. 27) The method of any preceding paragraph, wherein the disease or disorder caused by reduced p53 protein activity is Li-Fraumeni syndrome or a cancer associated with TP53 haploinsufficiency.28) The method of any preceding paragraph, wherein administration is performed via liposomal delivery, nanoparticle delivery', or viral delivery system.29) A method for increasing TP53 gene in a subject in need thereof, the method comprising administering to the subject in need thereofa. a first synthetic antisense oligonucleotides comprising a nucleotide sequence of SEQ ID NO: 1, or comprising a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: L andb. a second synthetic antisense oligonucleotides comprising a nucleotide sequence of SEQ ID NO: 2, or comprising a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 230) The method of any preceding paragraph, wherein each of the first and second synthetic antisense oligonucleotide is comprised in different nucleic acid vectors.31) The method of any preceding paragraph, wherein each of the first and second synthetic antisense oligonucleotide are administered in a single composition.294925-2218-9179 1701586-000159WOPTAttorney’s Docket No. 701586-000159WOPT32) The method of any preceding paragraph, wherein each of the first and second synthetic antisense oligonucleotide are administered in different compositions.33) The method of any preceding paragraph, wherein the different compositions are administered at substantially the same time, or at different timesEXAMPLES

[0148] The inventor has previously developed a molecular phenomenon called Exon-Mediated Activation of Transcription Starts (EMATS), in which the splicing of internal exons can control gene expression levels. EMATS refers to exon-mediated activation of transcription activating starts, a mechanistic phenomenon in which the splicing of internal exons in a pre-mRNA enhances transcriptional initiation from nearby promoters. In EMATS-regulated genes, inclusion of promoter-proximal internal exons facilitates recruitment, retention, or activity of the core transcription machinery (including RNA polymerase II) at adjacent transcription start sites (TSSs), thereby increasing nascent transcription and overall gene expression. EMATS is characterized by genomic architecture featuring a promoter-proximal internal exon (typically within about 1-2 kilobases downstream of a TSS) whose splicing status correlates with elevated promoter activity; modulation of that exon’s inclusion (e.g., by antisense oligonucleotides targeting splicing-regulatory elements) results in increased transcript output from the proximal TSS.

[0149] These observations support a reverse ‘'recruitment model” in which specific splicing factors recruit core transcription machinery to the vicinity of transcripts as they are being transcribed, boosting RNAPII occupancy and activity of nearby promoters. This phenomenon opened up novel possibilities for therapeutic manipulation of gene expression using antisense oligonucleotides to enhance or inhibit splicing of promoter-proximal alternative exons. The inventor found that the beneficial effect of ASOs is most pronounced in genes regulated by weak human promoters located near highly included skipped exons. Several tumor suppressor genes, such as TP53, present this genomic architecture.

[0150] Data presented herein demonstrate that this strategy successfully upregulated TP53 expression, resulting in increased levels of both mRNA and full-length protein. Several antisense oligonucleotides (ASOs) designed to block the binding of splicing repressors in various human cell lines. Among these, the combination of two ASOs yielded the most significant enhancement in TP53 mRNA and p53 protein levels (FIGs. 1 and 2). The sequences of these two ASOs are provided herein as SEQ ID NO: 1 and 2.

[0151] The restoration of tumor suppressor p53 protein levels effectively reduced key cancer hallmarks, including proliferation (FIG. 3), migration (FIG. 4), and invasion (FIG. 5). Methods disclosed herein achieved up to a three-fold activation of TP53 expression, leading to more than a three-fold reduction in304925-2218-9179 1701586-000159WOPTAttorney’s Docket No. 701586-000159WOPTthese cancer hallmarks. These findings represent a novel therapeutic strategy for patients with haploinsufficiency of tumor suppressor genes, offering a promising avenue for targeted cancer therapy.314925-2218-9179 1701586-000159WOPT

Claims

Attorney’s Docket No. 701586-000159WOPTCLAIMSWhat is claimed herein is:

1. A synthetic antisense oligonucleotide that binds a region of a TP53 RNA transcript.

2. The synthetic antisense oligonucleotide of claim 1, wherein the TP53 RNA transcript is a TP53 pre-mRNA transcript.

3. The synthetic antisense oligonucleotide of claim 1 and 2, wherein binding modulates splicing of an internal exon of the TP53 transcript.

4. The synthetic antisense oligonucleotide of any of claims 1-3, wherein the modulation increases TP53 mRNA levels and p53 protein levels.

5. The synthetic antisense oligonucleotide of claim 4, wherein TP53 mRNA levels and p53 protein levels are increased by at least 10% as compared to an appropriate control.

6. The synthetic antisense oligonucleotide of any of claims 1-5, wherein the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or wherein the antisense oligonucleotide comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

7. A nucleic acid vector comprising a synthetic antisense oligonucleotide of any of claims 1-6.

8. The nucleic acid vector of claim 7, wherein the synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 1, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 1.

9. The nucleic acid vector of claim 7, wherein the synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 2, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 2.

10. A nucleic acid vector comprising a synthetic antisense oligonucleotide comprising a nucleotide sequence of SEQ ID NO: 1, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 1.324925-2218-9179 1701586-000159WOPTAttorney’s Docket No. 701586-000159WOPT11. A nucleic acid vector comprising a synthetic antisense oligonucleotides comprising a nucleotide sequence of SEQ ID NO: 2, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 2.

12. A composition comprising at least one synthetic antisense oligonucleotide of any of claims 1-6. or any of the nucleic acid vectors of claims 7-11.

13. The composition of claim 12, wherein the composition comprises two distinct synthetic antisense oligonucleotides, or a nucleic acid vector comprising two distinct synthetic antisense oligonucleotides, or at least two nucleic acid vectors, each of the at least two nucleic acid vectors comprising at least one of: a synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 1, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 1; and a synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 2, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 2.

14. The composition of claim 13, wherein at least one of the two distinct synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 1, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 1.

15. The composition of claim 13, wherein at least one of the two distinct synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 2, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 2.

16. A composition comprising:a synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 1, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 1; and a synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO:

2. or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 2; ora nucleic acid vector comprising a synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 1, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 1; and a synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 2, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 2; or334925-2218-9179 1701586-000159WOPTAttorney’s Docket No. 701586-000159WOPTat least two nucleic acid vectors, each of the at least two nucleic acid vectors expressing at least one of: a synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 1, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 1; and a synthetic antisense oligonucleotides comprises a nucleotide sequence of SEQ ID NO: 2, or comprises a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 2.

17. A method for increasing TP53 gene expression in a cell, the method comprising: contacting a cell with any of the synthetic antisense oligonucleotide of any of claims 1-6, nucleic acid vectors of any of claims 7-11, or compositions of any of claims 12-16.

18. The method of claim 17, wherein the cell is a mammalian cell.

19. The method of claim 18, wherein the mammalian cell is a human cell.

20. The method of any of claims 17-19, wherein contacting is in vitro, in vivo, or ex vivo.

21. The method of any of claims 17-20, wherein contacting is performed via liposomal delivery, nanoparticle delivery, or viral delivery system.

22. The method of any of claims 17-21, wherein the cell has reduced p53 protein activity as compared to an appropriate control.

23. A method for increasing TP53 gene in a subject in need thereof, the method comprising administering to the subject in need thereof any of the synthetic antisense oligonucleotide of any of claims 1-6, nucleic acid vectors of any of claims 7-11, or compositions of any of claims 12-16.

24. The method of claim 23 and 24, wherein administration is local administration or systemic administration.

25. The method of claim 23, wherein the subject in need thereof has reduced p53 protein activity as compared to an appropriate control.

26. The method of claim 23, wherein the subject in need thereof has been diagnosed as having, or is at risk of having a disease or disorder caused by reduced p53 protein activity.

27. The method of claim 26, wherein the disease or disorder caused by reduced p53 protein activity is Li-Fraumeni syndrome or a cancer associated with TP53 haploinsufficiency.344925-2218-9179 1701586-000159WOPTAttorney’s Docket No. 701586-000159WOPT28. The method of claim 23, wherein administration is performed via liposomal delivery, nanoparticle delivery, or viral delivery system.

29. A method for increasing TP53 gene in a subject in need thereof, the method comprising administering to the subject in need thereofa first synthetic antisense oligonucleotides comprising a nucleotide sequence of SEQ ID NO: 1, or comprising a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO: 1; anda second synthetic antisense oligonucleotides comprising a nucleotide sequence of SEQ ID NO: 2, or comprising a nucleotide sequence that is at least 85% identical to the sequence of SEQ ID NO:

230. The method of claim 29, wherein each of the first and second synthetic antisense oligonucleotide is comprised in different nucleic acid vectors.

31. The method of claim 29, wherein each of the first and second synthetic antisense oligonucleotide are administered in a single composition.

32. The method of claim 29, wherein each of the first and second synthetic antisense oligonucleotide are administered in different compositions.

33. The method of claim 32, wherein the different compositions are administered at substantially the same time, or at different times.354925-2218-9179 1701586-000159WOPT

Images