Anti sense oligonucleotide with g-aggregate formation and a composition thereof

Abstract

The present invention provides a novel antisense oligonucleotide (ASO) featuring a G-rich sequence capable of forming stable intermolecular G-aggregates under physiological conditions. These G-aggregates enhance the ASO's stability, resistance to enzymatic degradation, and cellular uptake, improving its therapeutic efficacy. The ASO is specifically designed the formula of ASO can target a broad range of RNA species, including portion of mRNA, Antisense RNA, long non-coding RNA, long non-coding uncharacterized mRNA transcripts, long intergenic non-coding RNA, miRNA, opposite strand RNA and divergent strand RNA making it versatile for treating complex diseases. The invention also includes organ-specific delivery systems, using conjugates like GALNAC for liver-targeted delivery, ensuring precise distribution and reduced off-target effects. Furthermore, the G-aggregate structure reduces immune activation and toxicity, allowing for lower dosing. Overall, this invention represents an advanced, safer, and more effective approach for gene modulation therapy.

Description

ANTISENSE OLIGONUCLEOTIDE WITH G-AGGREGATE FORMATION AND A COMPOSITION THEREOFFIELD OF THE INVENTION

[0001] The present disclosure relates to the field of molecular biology, biotechnology, and pharmaceutical sciences. More particularly, it pertains to antisense oligonucleotides comprising guanine-rich nucleic acid sequences capable of forming G-aggregate secondary structures wherein said oligonucleotides displays enhanced stability, cellular uptake, and resistance to enzymatic degradation. It further relates to pharmaceutical compositions comprising such antisense oligonucleotides and methods for modulating gene expression by targeting disease-associated RNA molecules for therapeutic applications.BACKGROUND OF THE INVENTION

[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0003] Antisense oligonucleotides (ASOs) have emerged as a promising therapeutic tool for modulating gene expression, offering potential treatments for a wide range of diseases, including neurodegenerative disorders, metabolic syndromes, and cardiovascular conditions. These single-stranded nucleic acids bind to target RNA molecules, interfering with their function and reducing the expression of specific genes. However, despite significant advancements, ASO therapies still face critical limitations that hinder their clinical efficacy and widespread adoption.

[0004] One of the major challenges in ASO therapeutics is poor cellular uptake and stability. Due to their negatively charged backbone, ASOs struggle to cross the hydrophobic lipid bilayer of cell membranes, leading to inefficient delivery to target tissues. Additionally, once introduced into the bloodstream, ASOs are rapidly degraded by nucleases, significantly reducing their half-life. This necessitates frequent dosing, which complicates treatment regimens and reduces patient compliance. Existing solutions, such as chemically modified backbones and lipid-based carriers, have improved stability to some extent but have not fully resolved the issues.

[0005] Another significant issue is immune system activation and toxicity. Naked ASOs can activate Toll-like receptors (TLRs) that recognize foreign nucleic acids, leading to unintended immune responses such as inflammation. To achieve therapeutic effects, higher doses are often required, which increases the risk of systemic toxicity. This trade-off between efficacy and safety has been a persistent challenge for ASO-based therapies.

[0006] Furthermore, existing ASO delivery systems often suffer from inefficient and non-specific distribution. Traditional delivery methods rely on systemic administration, leading to suboptimal accumulation in target tissues. This non-specific distribution increases the likelihood of off-target effects, reducing the overall therapeutic impact. Current strategies such as lipid nanoparticles and viral vectors present additional concerns related to cytotoxicity, immunogenicity, and inconsistent efficacy across patient populations.

[0007] The lack of versatility in many existing ASOs is another significant limitation. Most ASOs are designed to target a single RNA molecule, limiting their therapeutic applicability to diseases driven by a single pathogenic RNA. Complex diseases such as Parkinson’s disease, Alzheimer’s disease, and metabolic disorders often involve multiple interconnected pathways, making it essential to develop ASOs capable of targeting multiple RNA species simultaneously.

[0008] Given these limitations, there is an urgent need for next-generation ASOs that address the shortcomings of poor stability, inefficient delivery, immune activation, and limited target versatility.OBJECTIVE OF THE INVENTION

[0009] An objective of the present disclosure is to provide antisense oligonucleotides comprising guanine-rich nucleic acid sequences capable of forming G-aggregate secondary structures.

[0010] An objective of the present disclosure is to provide antisense oligonucleotides with improved structural stability under physiological conditions.

[0011] An objective of the present disclosure is to provide antisense oligonucleotides with enhanced resistance to nuclease-mediated degradation.

[0012] An objective of the present disclosure is to provide antisense oligonucleotides exhibiting improved cellular uptake without the need for external delivery vectors.

[0013] An objective of the present disclosure is to provide antisense oligonucleotides capable of efficient intracellular delivery through receptor-mediated mechanisms.

[0014] An objective of the present disclosure is to provide antisense oligonucleotides suitable for targeting disease-associated RNA molecules, including mRNA, IncRNA, and miRNA.

[0015] An objective of the present disclosure is to provide pharmaceutical compositions comprising the disclosed antisense oligonucleotides and pharmaceutically acceptable carriers.

[0016] An objective of the present disclosure is to provide methods for modulating gene expression in a subject by administering the disclosed antisense oligonucleotides.

[0017] An objective of the present disclosure is to provide a therapeutic platform for treating neurodegenerative, genetic, cardiovascular, dermatological, and other RNA-mediated disorders.

[0018] An objective of the present disclosure is to provide antisense oligonucleotides with reduced immune activation and improved safety profdes.FIGURES OF THE INVENTION

[0019] FIG. 1 (a) shows SEM image of control; (b) and (c) shows the location and incorporation of ASO dissolved in PBS suggesting that even without the lipid nanoparticle the G-motif has the ability to enter the cell and show the desirable effect. (Red (dark) arrow Denotes entry of ASO in Cytoplasm, Yellow (light) arrow denotes entry of ASO in nucleus); (d) & E shows the location and incorporation of the ASO drug along with lipid nanoparticles into various places inside the cell. The yellow arrows in fig (d) show the incorporation of the drug into the nucleus of the cell which is already stained with DAPI and stained in blue when observed under microscope whereas the red arrow denotes the incorporation of drug inside the cytoplasm.

[0020] FIGs. 2 shows Real time PCR results of oligonucleotides designed for targeted genes. The results show that the levels of the PTPRZ1, KDM5A, MAPK7, SYNJ1 and SLC33A mRNA in MCF-7 cell lines are significantly decreased 24 h after treatment with one of the embodiments of ASO formulation, of present invention, named as Formulation 1.

[0021] FIG. 3 compares and shows the difference in the downregulation of genes by LNP encapsulated ASO vs control, where control is PBS through RT-PCR. These results show that fold changes in the downregulation of the respected genes by LNP encapsulated ASO are greater than the control.

[0022] FIG. 4 Shows RT-PCR result of different doses of ASO drug compared with the control. Here ASO is dissolved in PBS at different concentration that is responsible fordownregulation of mRNA. From this data it can be concluded that ASO with concentration more than 4000 ng or 80ng / pL is more effective than other doses.

[0023] FIG.5 depicts the results of gel electrophoresis experiments conducted under two conditions: with and without ions. In the absence of ions, the motif sequence exhibits significant mobility, traveling a considerable distance through the gel, approximately equivalent to a 16-nucleotide flow. This indicates that the sequence remains free and does not form complex structures. Conversely, in the presence of ions, the motif forms a G-aggregate, a higher-order structure stabilized by the ionic environment. This structural change is reflected in the gel, where the motifs mobility is notably reduced due to the restricted movement through the gel matrix.

[0024] FIG.6 The image contains two figures showcasing the structural dynamics of SEQ ID NO: 82aunder different conditions.

[0025] FIG. 6 (a): This figure illustrates the 3D structure of SEQ ID NO: 82a (GGGGGTGTAGCTCATA) highlighting its behavior in the presence of Na, K, and Mg ions. The guanine residues within the sequence interact to form a stable G-aggregate, stabilized by the ionic environment. This interaction facilitates the formation of a G-secondary, a cohesive structure composed of four strands folding together. The G-secondary unified structure is vital for binding specific cell-surface receptors and enabling efficient delivery of the associated antisense oligonucleotide (ASO) into the cytoplasm.

[0026] FIG. 6 (b): This figure also depicts the 3D structure of SEQ ID NO: 82a, but in the absence of ions. The sequence does not fold into a single, stable conformation and instead exists in an unfolded or loosely arranged state, lacking the compactness characteristic of ordered motifs. Without ionic stabilization, the strands fail to aggregate or form a cohesive structure, remaining separate and disordered. This reinforces the critical role of ions in folding and stabilizing the sequence into a functional, ordered state.

[0027] Both figures collectively emphasize the transformative effect of ions on the structural integrity and functionality of SEQ ID NO: 82a, particularly in its capacity to form G-aggregates crucial for biological applications.

[0028] FIG. 7 (a) is an agarose gel image of 6G-1 (at 100 ng / ul) in the presence of potassium (K) or phosphate buffered saline (PBS), showing that the G-rich antisense sequence migrates in a distinct double band migration pattern.

[0029] FIG. 7 (b) is an agarose gel image of 5G-1 (lOOng / ul) in the presence of varying concentrations of sodium (Na) and potassium (K), showing that the G-rich antisense sequence migrates in a distinct double band migration pattern.

[0030] FIG. 7 (c) is an agarose gel image of 5G-1 (100 ng / ul) in the presence of varying concentrations of Magnesium (Mg) and Calcium (Ca), showing a distinct single migration band in the presence of these ions.

[0031] FIG. 7 (d) is an agarose gel image of 5G-1 in the presence of varying concentrations of PBS, showing that the G-rich antisense sequence migrates in a distinct double band migration pattern.

[0032] FIG. 8 (a) is an agarose gel image of 5G-2 (100 ng / ul) in varying concentrations of potassium (K) or phosphate buffered saline (PBS), showing that the G-rich antisense sequence migrates in a distinct double band migration pattern.

[0033] FIG. 8 (b) is a native gel image of 5G-3 (100 ng / ul) in the presence of varying concentrations of potassium (K) or phosphate buffered saline (PBS). FIG. 8 (c) is an agarose gel image of 5G-4 (100 ng / ul) in the presence of varying concentrations of potassium (K) or phosphate buffered saline (PBS) showing varying migration patterns indicative of higher order structure.

[0034] FIG. 8 (d) is an agarose gel image of 5G-5 (100 ng / ul) in the presence of varying concentrations of potassium (K) or phosphate buffered saline (PBS) showing varying migration patterns indicative of higher order structure.

[0035] FIG. 9 is an agarose gel image of 4G-1 in the presence of varying concentrations of potassium (K), sodium (Na), magnesium (Mg), calcium (Ca), and phosphate buffered saline (PBS). 4G-1 migrates mostly as a single band.

[0036] FIG. 10 (a-e) are images of agarose gels of 4G-2 (a), 4G-3 (b), 3G-1 (c), 3G-2 (d) and 3G-3 (e) in varying concentrations of potassium and PBS. 4G-2 (a), (b), 3G-1 (c), 3G-2 (d) and 3G-3 (e) exhibited a single migration pattern. 4G-3 (b) exhibited a double banded pattern.

[0037] FIG. 11 (a-d) are image of agarose gels of 3G-4 (a), 3G-5 (b), 3G-6 (c) or NG-1 (d) at 100 ng / ul in the presence of varying concentrations of potassium and phosphate buffered saline (PBS). Gel images show a largely single migration band.

[0038] FIG. 12 (a-b) are agarose gel images of NG-2 (a) and NG-3 (b) at 100 ng / ul in the presence of varying concentrations of potassium (K) and phosphate buffered saline (PBS), indicating a single migration band.

[0039] FIG. 13 (a-e) are graphical representations of UV spectroscopy analyses for 6G-1 (a), 5G-1 with potassium (b), 5G-1 with magnesium (c), 5G-1 with PBS (d), 5G-1 with sodium (e), and 5G-1 with calcium (f).

[0040] FIG. 14 (a-e) are graphical representations of UV spectroscopy analyses for 5G-2 (a), 5G-3 (b), 5G-4 (c), 5G-5(d), 4G-1 in the presence of potassium (e), and 4G-1 in the presence of sodium (f).

[0041] FIG. 15 (a-e) are graphical representations of UV spectroscopy analyses for 4G- 1 in PBS (a), 4G-1 in magnesium (b), 4G-1 in calcium (c), 4G-2 (d), 4G-3 (e), and 3G-1 (f).

[0042] FIG. 16 (a-e) are graphical representations of UV spectroscopy analyses for 3G- 2 (a), 3G-3 (b), 3G-4 (c), 3G-5 (d), 3G-6 (e), andNG-1 (f).

[0043] FIG. 17 (a-e) are graphical representations of UV spectroscopy analyses for NG-1 in potassium (a), NG-1 in PBS (b), NG-1 in magnesium (c), NG-1 in calcium (d), NG-2 (e), and NG-3 (f).

[0044] FIG. 18 (a-f) are graphical representations of Circular Dichroism (CD) spectroscopy analyses of 6G-1 (a), 5G-1 in potassium (b), 5G-1 in magnesium (c), 5G-1 in PBS (d), 5G-1 in sodium (e), and 5G-1 in calcium (f).

[0045] FIG. 19 (a-f) are graphical representations of Circular Dichroism (CD) spectroscopy analyses of 5G-2 (a), 5G-3 (b), 4G-1 in potassium (c), 4G-1 in sodium (d), 4G-1 in PBS (e), and 4G-1 in magnesium (f).

[0046] FIG. 20 (a-e) are graphical representations of Circular Dichroism (CD) spectroscopy analyses of 4G-1 in calcium (a), 4G-3 (b), 3G-1 (c), 3G-4 (d), and 3G-6 (e).

[0047] FIG. 21 (a-e) are graphical representations of Circular Dichroism (CD) spectroscopy analyses of NG-1 in potassium (a), NG-1 in sodium (b), NG-1 in PBS (c), NG-1 in magnesium (d), and NG-1 in calcium (e).

[0048] FIG. 22 (a-b) are graphical representations of Circular Dichroism (CD) spectroscopy analyses of NG-2 (a) and NG-3 (b).

[0049] FIG. 23 (a-g) are graphical representations comparing the Circular Dichroism signal (y axis) as a measure of the increase in temperature (x-axis), exhibiting stability of G-quadruplex like conformations. Assessment of G-quadruplex thermal stability in 5G-1 under varying salt conditions using circular dichroism (CD) spectroscopy at 260 nm. (a) Thermal melt profde in nuclease-free water (FLO) revealed a gradual decrease in CD signal with temperature and a clear melting transition with a Tm of 69.29 °C. (b)-(e) CD signal at 260 nm for 5G-1 treated with 10 mM KC1 (10K), 100 mM KC1 (100K), 4X PBS, and 6X PBS respectively showed minimal change across the temperature range (21-90 °C), indicating enhanced thermal stability of the G-quadruplex. (f)-(g) CD thermal profdes for scrambled control (SC) and SC + 100K revealed progressive loss of CD signal without a defined transition, indicating absence of stable G-quadruplex structure.

[0050] FIG. 24 is an agarose gel image of the results of a DNase I digestion assay to assess the structural stability of 5G-1 under different ionic conditions. 5G-1 was incubated with either water, 10 mM K+, 10 mM Na+, or 1 * PBS, followed by DNase I treatment for 0, 10, 20, and 30 minutes. Samples were resolved on a native agarose gel and visualized.

[0051] FIG. 25 is an agarose gel image of the results of a DNase I digestion assay under additional ionic conditions. 5G-1 was incubated with 4x or 6x PBS, followed by DNase I treatment for 0, 10, 20, and 30 minutes. Samples were resolved on a native agarose gel and visualized.

[0052] FIG. 26 (a-b) is an image of a 2% agarose gel showing scrambled 5G-1 processed under different salt conditions as indicated, followed by DNase I treatment for 0, 10, and 20 minutes. Scrambled 5G-1, being completely randomized, shows a single band with no stability upon DNase I digestion, (a) H20, potassium, and sodium were tested, (b) IX, 4X, and 6X PBS were tested. This suggests that the stability observed in 5G-1 arises from the presence of five consecutive guanines that contribute to G-quadruplex formation, thereby conferring structural stability.

[0053] FIG. 27 is a gel image of 5G-1 pre-folded in 6X PBS and incubated with fetal bovine serum (FBS), blood serum, and tissue homogenate.

[0054] FIG. 28 is a series of gel images (a) aligned with quantitative stability data of a 64 base pair folded form (b) or a 16 base pair single stranded form (c). The 5G-1 demonstrated maximum stability in PBS, lowest stability in FBS, and moderate degradation in tissue homogenate.

[0055] FIG. 29 (a-c) are gel images exhibiting the interaction between 6G-1 (a), 5G-1 (b), and 5G-2 (c), with nucleolin (NCL).

[0056] FIG. 30 (a-c) are gel images exhibiting the interaction between 5G-3 (a), 5G-4 (b), and 5G-5 (c), with nucleolin (NCL).

[0057] FIG. 31 (a-c) are gel images exhibiting the interaction between 4G-1 (a), 4G-2 (b), and 4G-3 (c), with nucleolin (NCL).

[0058] FIG. 32 (a-c) are gel images exhibiting the interaction between 3G-1 (a), 3G-2 (b), and 3G-3 (c), with nucleolin (NCL).

[0059] FIG. 33 (a-c) are gel images exhibiting the interaction between 3G-4 (a), 3G-5 (b), and 3G-6 (c), with nucleolin (NCL).

[0060] FIG. 34 (a-c) are gel images exhibiting the interaction between NG-1 (a), NG-2 (b), and NG-3 (c), with nucleolin (NCL).

[0061] FIG. 35a is a series of fluroescent and grayscale micrographs of cells co-treated with 5G-1, No-5G-1, 5G-1 without LysoTracker, or Scrambled control, alongside untreated Cell Control. Fixed cells were labelled with Calcein Blue (nuclei; blue), LysoTracker (lysosomes; red), and 5G-1-Alexa Fluor-488 (green). Channel-wise and merged images demonstrate that 5G-1 enters cells and forms discrete fluorescent puncta. A subset of 5G-1 signal colocalises with LysoTracker, representing lysosomal sequestration, whereas a distinct population of AF488-positive puncta localises outside lysosomal regions. FIG. 35b is a quantitative analysis using Image J of the Alexa Fluor-488 puncta that do not colocalise with LysoTracker to determine the extent of lysosomal escape.

[0062] FIG. 36a is a series of micrographs examining an endosomal escape assay of NG-1 in SH-SY5Y Cells. The images were quantified using ImageJ software (FIG. 36b).

[0063] FIG. 37a is a series of fluorescent micrographs of cells treated with 5G-1 G-rich antisense oligonucleotide under various conditions. DAPI (blue) labels nuclei, MitoTracker (red) labels mitochondria, and Alexa Fluor 488 (green) detects oligonucleotides. Treatments: Control (untreated), 5G-1, 5G-1 (Mag.) (magnified), 5G-1 Transf. (with transfection agent), and 5G-1 SCR (scrambled control). Merged images show channel overlay. FIG 37b is a bar graph quantifying cytoplasmic (black) versus nuclear (gray) localization. 5G-1 preferentially accumulates in nuclei, enhanced by transfection, while scrambled sequence remains cytoplasmic. The images were quantified using ImageJ software

[0064] FIG. 38a is a series of fluorescent micrographs of cells treated with 4G-1 G-rich antisense oligonucleotide under various conditions. DAPI (blue) labels nuclei, MitoTracker (red) labels mitochondria, and Alexa Fluor 488 (green) detects oligonucleotides. Treatments: Control (untreated), 4G-1, 4G-1 (Mag.) (magnified), 4G-1 Transf. (with transfection agent). Merged images show channel overlay. FIG. 38b is a bar graph quantifying cytoplasmic (black) versus nuclear (gray) localization. 4G-1 preferentially accumulates in nuclei, enhanced by transfection. The images were quantified using ImageJ software.

[0065] FIG. 39a is a series of fluorescent micrographs of cells treated with NG-2 NON-G antisense oligonucleotide under various conditions. DAPI (blue) labels nuclei, MitoTracker (red) labels mitochondria, and Alexa Fluor 488 (green) detects oligonucleotides. Treatments: Control (untreated), NG-2, NG-2 (Mag.) (magnified), NG-2 Transf. (with transfection agent). FIG. 39b is a bar graph quantifying cytoplasmic (black) versus nuclear (gray) localization. The images were quantified using ImageJ software.

[0066] FIG. 40 (a-g) are a series of bar graphs displaying the results of the colorimetric MTT assay, (a) Cytotoxicity Analysis of SH-SY5Y Cells after treatment with 5G-1. (b)Cytotoxicity Analysis of SH-SY5Y Cells after treatment with 4G-1 Analysis, (c) Cytotoxicity Analysis of SH-SY 5Y Cells after treatment with NG- 1. (d) Cytotoxicity Analysis of SH-SY 5Y Cells after treatment with NG-3. (e) Cytotoxicity Analysis of HEK Cells after treatment with 5G-1. (f) Cytotoxicity Analysis of HEK Cells after treatment with NG-2. (g) Cytotoxicity Analysis of HEK Cells after treatment with NG-3.

[0067] FIG 41 (a-c) are a series of bar graphs exhibiting the results of a target mRNA inhibition of LIMK1 (a), FAM168B (b), and GRIK2 (c), in SH-SY5Y Cells after treatment with 5G-1 and scrambled 5G-1.

[0068] FIG 42 (a-b) are a series of bar graphs exhibiting the results of arget mRNA inhibition in HepG2 Cells after treatment with 5G-1 in a dose dependent manner for Plink- 1(a) and Plink-lAS(b). Cells were also treated with scrambled control.

[0069] FIG 43 (a-g) are a series of bar graphs exhibiting the results of a Target mRNA inhibition in SH-SY5Y Cells after treatment with 5G-1 for targets LIMK1 (a), FAM168B (b), PINK1 (c), PINK 1 -AS (d), SYNJ1 (e), DGKQ (f), and GRP63 (g).

[0070] FIG. 44 (a-c) are a series of bar graphs exhibiting the results of a target RNA inhibition experiment. Target mRNA inhibition is shown in SH-SY5Y Cells after treatment with NG-2 and scrambled NG-2 for the target mRNA GPX6 (a), CAV 1 (b), or PRDX6-AS (c).

[0071] FIG. 45 is a bar graph showing Target mRNA inhibition of HTT in SH-SY5Y Cells after treatment with 4G-1 and scrambled 4G-1.

[0072] FIG. 46 is an atomic level interaction Map of the nucleolin P2B1046G-1 G-quadruplex structure.

[0073] FIG. 47 is an atomic level interaction Map of the nucleolin D15G-lG-quadruplex structure.

[0074] FIG. 48 is an atomic level interaction Map of the Nucleolin / P2B335G-2 G quadruplex structure.

[0075] FIG. 49 is an atomic level interaction Map of the Nucleolin P2H45G-3 G quadruplex structure.

[0076] FIG. 50 is an Atomic-Level Interaction Map forp2b415gG quadruplex / Nucleolin structure.

[0077] FIG. 51 is an Atomic-Level Interaction Map forNucleolin / P2B675G5-G Quadruplex structure.

[0078] FIG. 52 is an Atomic-Level Interaction Map for Nucleolin / D224G1G- G Quadruplex structure.

[0079] FIG. 53 is an Atomic-Level Interaction Map for Nucleolin & P2V45-4G2 G-Quadruplex structure.

[0080] FIG. 54 is an Atomic-Level Interaction Map for Nucleolin & PRO2H14G3 G-Quadruplex structure.

[0081] FIG. 55 is an Atomic-Level Interaction Map for Nucleolin & P2B83G1 G-Quadruplex structure.

[0082] FIG. 56 is an Atomic -Level Interaction Map for Nucleolin & P2B153G2G-Quadruplex structure.

[0083] FIG. 57 is an Atomic-Level Interaction Map for Nucleolin & P2B633G3 G-Quadruplex structure.

[0084] FIG. 58 is an Atomic-Level Interaction Map for Nucleolin & P2B793G4 (G-Quadruplex) structure.

[0085] FIG. 59 is an Atomic-Level Interaction Map for Nucleolin & F2B223G5 (G-Quadruplex) structure.SUMMARY OF THE INVENTION

[0086] The present disclosure provided herein relates to antisense oligonucleotides comprising a nucleic acid sequence capable of forming G-rich secondary structures termed G-aggregates. These structures enhance stability, cellular uptake, and therapeutic efficacy for RNA-targeted therapies. The antisense oligonucleotides offer precise targeting of disease-associated RNA molecules and demonstrate improved delivery into cells. Accordingly, the antisense oligonucleotides are useful for the treatment of various diseases or conditions in a subject in need thereof, including, but not limited to, neurodegenerative disorders, cardiovascular and musculoskeletal degenerative diseases, dermatological diseases, rare genetic disorders, Autism Spectrum Disorder, Duchenne Muscular Dystrophy, Huntington’s disease, Friedreich Ataxia, and pustular psoriasis.

[0087] In an aspect, the antisense oligonucleotide comprising a nucleic acid sequence, wherein the antisense oligonucleotide is capable of forming G-aggregates, and wherein the nucleic acid sequence is characterized by Formula I:(I) or a pharmaceutically acceptable salt thereof,wherein the G-rich motif (also referred to herein as “guanine-rich motif’) comprises 2 to 30 naturally or non-naturally occurring nucleotides and at least two adjacent guanines or guanine derivatives; andVariable Segment and Y’ are defined herein.

[0088] In an embodiment, the Gmotif comprises at least 50% guanines or guanine derivatives. In another embodiment, the G-rich motif comprises three, four, five, or six guanines or guanine derivatives. In yet another embodiment, the G-rich motif is two to eight nucleotides in length. In still another embodiment, the G-rich motif is selected from a nucleotide sequence of GGGGG, GGGGA, GGGAT, AGGGG, and AGGGA.

[0089] In an embodiment, the antisense oligonucleotide comprises a nucleic acid sequences with G-rich motifs characterized by the following formula:5 ’ W(n)WGGWZ TGTAG XYRAZNN ... 3 ’motif conserved Variable; wherein:W = A / G (purines, independent occurrence);n = 0, 1 (number of W at the first position);Y = T / U / C (pyrimidines or G independent occurrence);R = C / A (independent occurrence);X = C / T (independent occurrence);Z = T / G / A (independent occurrence); andN = A / T / G / C (any nucleotide, non-repeating sequence).

[0090] In another embodiment, the antisense oligonucleotide is selected from singlestranded DNA, RNA, peptide nucleic acid (PNA), phosphorodiamidate morpholino oligomer (PMO), and combinations thereof. In certain embodiments, the antisense oligonucleotide comprises a modification selected from: locked nucleic acid (LNA), ethylene-bridged nucleic acid (ENA), phosphorothioate backbone, tricyclo-DNA, tricyclo-phosphorothioate, 2’-O-methyl (2’-OMe), 2’-O-methoxyethyl (2'-M0E), 2’-fluoro (2'-F), 2’-O-methyl phosphorothioate, 2’-O- methoxyethyl phosphorothioate, and combinations thereof.

[0091] In yet another embodiment, the nucleic acid sequence is conjugated to a targeting moiety, wherein the targeting moiety is selected from GALNAC, vitamins, flavonoids, and cofactors. In a further embodiment, wherein the targeting moiety is selected from GALNAC, biotin, and tocopherol.

[0092] In an aspect, provided herein the 5 ’-terminus or 3 ’-terminus of the nucleic acid sequence comprises a G-rich motif, and wherein the antisense oligonucleotide is a singlestranded DNA or RNA of Formula IV (IV) or a pharmaceutically acceptable salt thereof, wherein G’, E, R1, R2, R3, and z are defined herein.In yet another aspect, provided herein is an antisense oligonucleotide of 16 to 40 naturally or non-naturally occurring nucleotides in length, wherein the antisense oligonucleotide comprises a nucleic acid sequence, and wherein the nucleic acid sequence is selected from:wherein X is T,U or C.

[0093] In some embodiments, the sequences form intermolecular G-aggregates stabilized by monovalent cations, such as sodium (Na+) or potassium (K+), enhancing structural stability and enabling self-delivery through interactions with cellular receptors like nucleolin, Scavenger receptor and equilibrative nucleic acid transporters (ENATs).

[0094] Provided herein are novel antisense oligonucleotide (ASO) sequences characterized by a G-rich motif capable of forming intermolecular G-aggregates under physiological conditions. These ASOs exhibit enhanced stability, improved cellular uptake, and are specifically designed for the targeted downregulation of RNA species, including mRNA, IncRNA, and miRNA.

[0095] In an aspect, provided herein is a pharmaceutical composition comprising an antisense oligonucleotide and at least one pharmaceutically acceptable carrier. In an embodiment, the pharmaceutical compositions comprising the disclosed ASO sequences andpharmaceutically acceptable carriers, are formulated for the treatment of various diseases, including neurodegenerative disorders, cardiovascular and musculoskeletal degenerative diseases, dermatological diseases, rare genetic disorders, Autism Spectrum Disorder, Duchenne Muscular Dystrophy, Huntington’s disease, Friedreich Ataxia, and pustular psoriasis.

[0096] In still another aspect, provided herein is a method of treating a disease or condition comprising administering to a subject in need thereof an antisense oligonucleotide. In some embodiments, the antisense oligonucleotide described herein can be used to treat neurodegenerative disorders, cardiovascular and musculoskeletal degenerative diseases, dermatological diseases, rare genetic disorders, Autism Spectrum Disorder, Duchenne Muscular Dystrophy, Huntington’s disease, Friedreich Ataxia, and pustular psoriasis.In an aspect, the invention provides methods for downregulating the expression of target genes by administering therapeutically effective doses of the disclosed ASOs.

[0097] In an aspect, the invention provides ASO delivery systems conjugated with targeting moieties such as GALNAC, vitamins, or biotin, enabling enhanced delivery to specific organs, including the liver, central nervous system, or muscles.

[0098] The invention aims to address critical challenges in ASO-based therapies by providing a robust and stable platform for gene modulation. The ASOs provided herein exhibit high specificity, improved therapeutic delivery, and broad applicability, revolutionizing RNA-targeted treatments for complex diseases.

[0099] Various objects, features, aspects, and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments.DETAILED DESCRIPTION OF THE INVENTION

[0100] The following is a full description of the disclosure's embodiments. The embodiments are described in such a way that the disclosure is clearly communicated. The level of detail provided, on the other hand, is not meant to limit the expected variations of embodiments; rather, it is designed to include all modifications, equivalents, and alternatives that come within the spirit and scope of the current disclosure as defined by the attached claims. Unless the context indicates otherwise, the term "comprise" and variants such as "comprises" and "comprising" throughout the specification are to be read in an open, inclusive meaning, that is, as "including, but not limited to."

[0101] When "one embodiment" or "an embodiment" is used in this specification, it signifies that a particular feature, structure, or characteristic described in conjunction with theembodiment is present in at least one embodiment. As a result, the expressions "in one embodiment" and "in an embodiment" that appear throughout this specification do not necessarily refer to the same embodiment. Furthermore, in one or more embodiments, the specific features, structures, or qualities may be combined in any way that is appropriate.

[0102] Unless the content clearly demands otherwise, the singular terms "a," "an," and "the" include plural referents in this specification and the appended claims. Unless the content explicitly mandates differently, the term "or" is normally used in its broad definition, which includes "and / or."

[0103] All processes described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0104] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.

[0105] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0106] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description that follows, and the embodiments described herein, is provided by way of illustration of an example, or examples, of particular embodiments of the principles and aspects of the present disclosure. These examples are provided for the purposes of explanation, and not of limitation, of those principles and of the disclosure.

[0107] It should also be appreciated that the present invention can be implemented in numerous ways, including as a system, a method or a device. In this specification, theseimplementations, or any other form that the invention may take, may be referred to as processes. In general, the order of the steps of the disclosed processes may be altered within the scope of the invention.

[0108] Various terms as used herein are shown below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.Definitions:

[0109] For Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the subject matter of the present disclosure, preferred methods and materials are described. For the purposes of the present disclosure, the following terms are defined below.

[0110] The term “about” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. As used herein when referring to a measurable value such as an amount, a temporal duration, and the like, the term “about” is meant to encompass variations of ±10%, including ±5%, ±1%, and ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.[oni] Unless the context indicates otherwise, the term "comprise" and variants such as "comprises" and "comprising" throughout the specification are to be read in an open, inclusive meaning, that is, as "including, but not limited to."

[0112] When "one embodiment" or "an embodiment" is used in this specification, it signifies that a particular feature, structure, or characteristic described in conjunction with the embodiment is present in at least one embodiment. As a result, the expressions "in one embodiment" and "in an embodiment" that appear throughout this specification do not necessarily refer to the same embodiment. Furthermore, in one or more embodiments, the specific features, structures, or qualities may be combined in any way that is appropriate.

[0113] Unless the content clearly demands otherwise, the singular terms "a," "an," and "the" include plural referents in this specification and the appended claims. Unless the content explicitly mandates differently, the term "or" is normally used in its broad definition, which includes "and / or."

[0114] All processes described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0115] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.

[0116] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0117] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description that follows, and the embodiments described herein, is provided by way of illustration of an example, or examples, of particular embodiments of the principles and aspects of the present disclosure. These examples are provided for the purposes of explanation, and not of limitation, of those principles and of the disclosure.

[0118] Various terms as used herein are shown below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.

[0119] The term “alkyl” refers to saturated, straight- or branched-chain hydrocarbon moieties containing, in certain embodiments, between one and six, or one and eight carbon atoms, respectively. Examples of Ci-6-alkyl moieties include, but are not limited to, methyl, ethyl, propyl, isopropyl, / / -butyl, tert-butyl, neopentyl, n-hexyl moieties; and examples of Ci-8-alkyl moieties include, but are not limited to, methyl, ethyl, propyl, isopropyl, / / -butyl, tertbutyl, neopentyl, n-hexyl, heptyl, and octyl moieties.

[0120] The number of carbon atoms in an alkyl substituent can be indicated by the prefix “Cx-y,” where x is the minimum and y is the maximum number of carbon atoms in the substituent. Likewise, a Cx chain means an alkyl chain containing x carbon atoms.

[0121] The term “nucleobase,” “base pairing moiety,” “nucleobase-pairing moiety,” or “base” refers to the heterocyclic ring portion of a nucleoside, nucleotide, and / or morpholino subunit. Nucleobases may be naturally occurring (e.g., uracil, thymine, adenine, cytosine, and guanine), or may be modified or analogs of these naturally occurring nucleobases, e.g., one or more nitrogen atoms of the nucleobase may be independently at each occurrence replaced by carbon. Exemplary analogs include hypoxanthine (the base component of the nucleoside inosine); 2, 6-diaminopurine; 5-methyl cytosine; C5-propynyl -modified pyrimidines; 10-(9-(aminoethoxy)phenoxazinyl) (G-clamp) and the like.

[0122] Further examples of base pairing moieties include, but are not limited to, uracil, thymine, adenine, cytosine, guanine and hypoxanthine having their respective amino groups protected by acyl protecting groups, 2-fluorouracil, 2-fluorocytosine, 5 -bromouracil, 5-iodouracil, 2, 6-diaminopurine, azacytosine, pyrimidine analogs such as pseudoisocytosine and pseudouracil and other modified nucleobases such as 8-substituted purines, xanthine, or hypoxanthine (the latter two being the natural degradation products). The modified nucleobases disclosed in Chiu and Rana (2003) RNA 9:1034-1048, Limbach et al. (1994) Nucleic Acids Res .22:2183-2196 and Revankar and Rao, Comprehensive Natural Products Chemistry, vol. 7, 313, are also contemplated, the contents of which are incorporated herein by reference.

[0123] Further examples of base pairing moieties include, but are not limited to, expanded-size nucleobases in which one or more benzene rings has been added. Nucleic base replacements described in the Glen Research catalog (www.glenresearch.com); Krueger AT et al. (2007) Acc. Chem. Res. 40:141-150; Kool ET (2002) Acc. Chem. Res. 35:936-943; Benner SA et al. (2005) Nat. Rev. Genet. 6:553-543; Romesberg FE et al. (2003) Curr. Opin. Chem. Biol. 7:723-733; Hirao, I (2006) Curr. Opin. Chem. Biol. 10:622-627, the contents of which are incorporated herein by reference, are contemplated as useful for the synthesis of the oligomers described herein. Examples of expanded-size nucleobases are shown below:

[0124] The terms “antisense oligonucleotide”, also referred to as “ASO,” “antisense oligomer,” “oligonucleotide,” or “oligomer” refer to a compound comprising a plurality of linked nucleosides, nucleotides, or a combination of both nucleosides and nucleotides. The ASO provided herein may be defined as a short, synthetic strand of nucleotides designed to bind to specific RNA sequences to modulate gene expression, typically by inhibiting RNA function or promoting RNA degradation.

[0125] Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. Oligonucleotides containing a modified or substituted base include oligonucleotides in which one or more purine or pyrimidine bases most commonly found in nucleic acids are replaced with less common or non-natural bases. In some embodiments, the nucleobase is covalently linked at the N9 atom of the purine base, or at the N1 atom of the pyrimidine base, to the morpholine ring of a nucleotide or nucleoside.

[0126] Purine bases comprise a pyrimidine ring fused to an imidazole ring, as described by the general formula:Adenine and guanine are the two purine nucleobases most commonly found in nucleic acids. These may be substituted with other naturally-occurring purines, including but not limited to N6-methyladenine, N2-methylguanine, hypoxanthine, and 7-methylguanine.

[0127] Pyrimidine bases comprise a six-membered pyrimidine ring as described by the general formula:Cytosine, uracil, and thymine are the pyrimidine bases most commonly found in nucleic acids. These may be substituted with other naturally-occurring pyrimidines, including but not limited to 5 -methylcytosine, 5 -hydroxymethylcytosine, pseudouracil, and 4-thiouracil. In one embodiment, the oligonucleotides described herein contain thymine bases in place of uracil.

[0128] Other modified or substituted bases include, but are not limited to, 2,6-diaminopurine, orotic acid, agmatidine, lysidine, 2-thiopyrimidine (e.g. 2-thiouracil, 2-thiothymine), G-clamp and its derivatives, 5-substituted pyrimidine (e.g. 5-halouracil, 5-propynyluracil, 5-propynylcytosine, 5 -aminomethyluracil, 5 -hydroxymethyluracil, 5-aminomethylcytosine, 5-hydroxymethylcytosine, Super T), 7-deazaguanine, 7-deazaadenine, 7-aza-2,6-diaminopurine, 8-aza-7-deazaguanine, 8-aza-7-deazaadenine, 8-aza-7-deaza-2,6-diaminopurine, Super G, Super A, and N4-ethylcytosine, or derivatives thereof; N2-cyclopentylguanine (cPent-G), N2-cyclopentyl-2-aminopurine (cPent-AP), and N2-propyl-2-aminopurine (Pr-AP), pseudouracil or derivatives thereof; and degenerate or universal bases, like 2,6-difluorotoluene or absent bases like abasic sites (e.g. 1 -deoxyribose, 1,2-dideoxyribose, l-deoxy-2-O-methylribose; or pyrrolidine derivatives in which the ring oxygen has been replaced with nitrogen (azaribose)). Pseudouracil is a naturally occurring isomerized version of uracil, with a C-glycoside rather than the regular N-glycoside as in uridine.

[0129] Certain modified or substituted nucleobases are particularly useful for increasing the binding affinity of the antisense oligonucleotides of the disclosure. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including2 -aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. In various embodiments, nucleobases may include 5 -methylcytosine substitutions, which have been shown to increase nucleic acid duplex stability by 0.6-1.2°C.

[0130] In some embodiments, modified or substituted nucleobases are useful for facilitating purification of antisense oligonucleotides. For example, in certain embodiments, antisense oligonucleotides may contain three or more (e.g., 3, 4, 5, 6 or more) consecutive guanine bases. In certain antisense oligonucleotides, a string of three or more consecutive guanine bases can result in aggregation of the oligonucleotides, complicating purification. In such antisense oligonucleotides, one or more of the consecutive guanines can be substituted with hypoxanthine. The substitution of hypoxanthine for one or more guanines in a string of three or more consecutive guanine bases can reduce aggregation of the antisense oligonucleotide, thereby facilitating purification.

[0131] As used herein, an “analog” or “nucleic acid analog” refers to a non-naturally occurring nucleic acid molecule. A nucleic acid is a polymer of nucleotide subunits linked together into a linear structure. Each nucleotide consists of a nitrogen-containing aromatic base attached to a pentose (five-carbon) sugar, which is in turn attached to a phosphate group. Successive phosphate groups are linked together through phosphodiester bonds to form the polymer. The two common forms of naturally occurring nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). One end of the chain carries a free phosphate group attached to the 5 ’-carbon atom of a sugar moiety; this is called the 5’ end of the molecule. The other end has a free hydroxyl (-OH) group at the 3 ’-carbon of a sugar moiety and is called the 3’ end of the molecule. A nucleic acid analog can include one or more non-naturally occurring nucleobases, sugars, and / or intemucleotide linkages, for example, a phosphorodiamidate morpholino oligomer (PMO).

[0132] The “backbone” of an oligonucleotide analog (e.g., an uncharged oligonucleotide analogue) refers to the structure supporting the base -pairing moieties; e.g., for a morpholino oligomer, as described herein, the “backbone” includes morpholino ring structures connected by intersubunit linkages (e.g., phosphorus-containing linkages). A “substantially uncharged backbone” refers to the backbone of an oligonucleotide analogue wherein less than 50% of the intersubunit linkages are charged at near-neutral pH. For example, a substantially uncharged backbone may comprise less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5% or even 0% intersubunit linkages which are charged at near neutral pH. In some embodiments, the substantially uncharged backbone comprises at most one charged (at physiological pH) intersubunit linkage for every four uncharged (at physiological pH) linkages,at most one for every eight or at most one for every sixteen uncharged linkages. In some embodiments, the nucleic acid analogs described herein are fully uncharged.

[0133] The term “nucleic acid sequence” or simply “sequence” is the sequence in the nucleic acid analog that is complementary (meaning, in addition, substantially complementary) to a target sequence, e.g., a target sequence in the RNA genome of human. The entire sequence, or only a portion, of the analog compound may be complementary to the target sequence. For example, in an analog having 20 bases, only 12-14 may be targeting sequences. Typically, the targeting sequence is formed of contiguous bases in the analog, but may alternatively be formed of non-contiguous sequences that when placed together, e.g., from opposite ends of the analog, constitute sequence that spans the target sequence. The terms “complementary” and “complementarity” refer to oligonucleotides (i.e., a sequence of nucleotides) related by basepairing rules. For example, the sequence “T-G-A (5 ’-3’)” is complementary to the sequence “T-C-A (5 ’-3’).” Complementarity may be “partial,” in which only some of the nucleic acids’ bases are matched according to base pairing rules. Or, there may be “complete,” “total,” or “perfect” (100%) complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. While perfect complementarity is often desired, some embodiments can include one or more but preferably 6, 5, 4, 3, 2, or 1 mismatches with respect to the target RNA. Such hybridization may occur with “near” or “substantial” complementarity of the antisense oligomer to the target sequence, as well as with exact complementarity. In some embodiments, an oligomer may hybridize to a target sequence at about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% complementarity. Variations at any location within the oligomer are included. In certain embodiments, variations in sequence near the termini of an oligomer are generally preferable to variations in the interior, and if present are typically within about 6, 5, 4, 3, 2, or 1 nucleotides of the 5'-terminus, 3 ’-terminus, or both termini. The terms “complementary” and “complementarity” refer to oligonucleotides (i.e., a sequence of nucleotides) related by basepairing rules. For example, the sequence “T-G-A (5 ’-3’)” is complementary to the sequence “T-C-A (5 ’-3’).” Complementarity may be “partial,” in which only some of the nucleic acids’ bases are matched according to base pairing rules. Or, there may be “complete,” “total,” or “perfect” (100%) complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. While perfect complementarity is often desired, some embodiments can include one or more but preferably 6, 5, 4, 3, 2, or 1 mismatches withrespect to the target RNA. Such hybridization may occur with “near” or “substantial” complementarity of the antisense oligomer to the target sequence, as well as with exact complementarity. In some embodiments, an oligomer may hybridize to a target sequence at about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% complementarity. Variations at any location within the oligomer are included. In certain embodiments, variations in sequence near the termini of an oligomer are generally preferable to variations in the interior, and if present are typically within about 6, 5, 4, 3, 2, or 1 nucleotides of the 5 '-terminus, 3 ’-terminus, or both termini.

[0134] Naturally occurring nucleotide bases include adenine, guanine, cytosine, thymine, and uracil, which have the symbols A, G, C, T, and U, respectively. Nucleotide bases can also encompass analogs of naturally occurring nucleotide bases. Base pairing typically occurs between purine A and pyrimidine T or U, and between purine G and pyrimidine C.

[0135] The terms “G-rich sequence” or “guanine-rich sequence” refer to a nucleic acid sequence that is rich in the nucleotide guanine (G). In some embodiments, the G-rich motif comprises at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 95%, or 90% guanines or guanine derivatives. In some embodiments, the G-rich motif comprises two, three, four, five, or six guanines or guanine derivatives. These G-rich sequences may have a propensity to form higher-order structures, such as G-quadruplexes, under physiological conditions.

[0136] The term “G-aggregate” is a structure formed by the self-association of G-rich sequences, stabilized by monovalent cations (e.g., Na+, K+), resulting in intermolecular aggregates that enhance the stability and cellular uptake of antisense oligonucleotides.

[0137] As used herein, a “cell-penetrating peptide” (CPP) is a relatively short peptide capable of promoting uptake of PMOs by cells, thereby delivering the PMOs to the interior (cytoplasm) of the cells. The CPP typically is about 12 to about 40 amino acids long. The length of the CPP is not particularly limited and varies in different embodiments. In some embodiments, the CPP comprises from 4 to 40 amino acid subunits. In other embodiments, the CPP comprises from 6 to 30, from 6 to 20, from 8 to 25 or from 10 to 20 amino acid subunits.

[0138] In certain embodiments, the linking moiety is attached to an antisense oligonucleotide-peptide conjugate from the oligonucleotide conjugate.

[0139] An “amino acid subunit” is generally an > -amino acid residue (-CO-CHR-NH-); but may also be a □- or other amino acid residue (e.g., -CO-CH2CHR-NH-), where R is an amino acid side chain.

[0140] The term “amino acid” refers to both amino acids present in proteins found in nature (“natural amino acids”) and amino acids not present in proteins found in nature (“non-natural amino acids” or “unnatural amino acid”). Examples of natural amino acids include Alanine (A), Cysteine (C), Aspartic acid (D), Glutamic acid I, Phenyalanine (F), Glycine (G), Histidine (H), Isoleucine (I), Lysine (K), Leucine (L). Methionine (M), Asparagine (N), Proline (P), Glutamine (Q), Arginine I, Serine (S), Threonine (T), Valine (V), Tryptophan (W), and Tyrosine (Y).

[0141] Examples of non-natural amino acids include, but are not limited to, beta-alanine (P-Ala) and 6-aminohexanoic acid (Ahx), y-aminobutyric acid (Abu), homoleucine (Hie), norleucine (Nle), naphthylalanine (Nap), diphenylalanine (Dpa), diaminobutyric acid (Dab), aminopiperidine-carboxylic acid (Pip), aminomethylphenylalanine (Amf), and 2-amino-4-guanidinobutanoic acid (Gba).

[0142] The term “downregulation” is a process of decreasing or inhibiting the expression of a gene, often through mechanisms like RNA interference or antisense oligonucleotide binding, which can reduce the activity of a gene and its associated protein.

[0143] The term “mRNA” or “messenger RNA” refers to a type of RNA that serves as a template for protein synthesis. It carries genetic information from the DNA to the ribosome, where proteins are synthesized.

[0144] The terms “IncRNA” or “long non-coding RNA” refer to a type of RNA molecule longer than 200 nucleotides that does not code for proteins but plays critical roles in regulating gene expression, chromatin structure, and cellular processes.

[0145] The terms “miRNA” or “microRNA” refer to small, non-coding RNA molecule that regulates gene expression by binding to mRNA and either blocking its translation or promoting its degradation.

[0146] The term “gene expression” is a process by which information from a gene is used to synthesize a functional gene product, typically a protein or RNA, impacting cellular functions and traits.

[0147] The term “monovalent cations” refer to ions with a single positive charge, such as sodium (Na+) and potassium (K+), that help stabilize the structure of G-aggregates and facilitate their formation in physiological conditions.

[0148] The term “G-tetrad” refers to a four-stranded DNA or RNA structure formed by guanine-rich sequences through hoogsteen hydrogen bonding interactions between guanine bases, creating a stable secondary structure.

[0149] The term ““receptors” are a class of receptors on cell surfaces that facilitate the uptake of molecules such as oligonucleotides by recognizing and binding to specific structures, aiding in their internalization into cells.

[0150] The term “nucleolin” is a protein that acts as a receptor for nucleic acid nanoparticles, including G-aggregates, facilitating their internalization into cells and promoting delivery to the nucleus.

[0151] The term “targeted delivery” refers to a method of directing therapeutic agents, such as antisense oligonucleotides, to specific tissues or cells, typically using conjugated targeting ligands or delivery vehicles to improve efficacy and minimize off-target effects.

[0152] The term “GALNAC” refers to a chemical moiety derived from N-acetylgalactosamine. GALNAC specifically targets liver cells in drug delivery systems, and may be conjugated to therapeutic oligonucleotides for liver-specific delivery.

[0153] ‘Vitamins” and “cofactors” are small organic molecules that may be conjugated with therapeutic oligonucleotides to enhance their stability, solubility, and bioavailability, particularly in organ-specific delivery systems.

[0154] The term “immune activation” refers to a process by which foreign molecules, such as synthetic oligonucleotides, trigger the immune system to recognize and respond to the presence of these molecules, potentially leading to inflammation or adverse effects.

[0155] The terms “toll-like receptors” or “TLRs” refer to a family of receptors in the immune system that recognize pathogen-associated molecular patterns (PAMPs) and initiate immune responses. Activation of TLRs by foreign nucleic acids can lead to inflammation and immune-related side effects.

[0156] As used herein, an “effective amount” refers to any amount of a substance that is sufficient to achieve a desired biological result. A “therapeutically effective amount” refers to any amount of a substance that is sufficient to achieve a desired therapeutic result.

[0157] As used herein, a “subject” or “patient” is a mammal, which can include a mouse, rat, hamster, guinea pig, rabbit, goat, sheep, cat, dog, pig, cow, horse, monkey, non-human primate, or human. In certain embodiments, a subject is a human.

[0158] ‘Treatment” of an individual (e.g., a mammal, such as a human) or a cell is any type of intervention used to alter the natural course of the individual or cell . Treatment includes, but is not limited to, administration of a pharmaceutical composition, and may be performed either prophylactically or subsequent to the initiation of a pathologic event or contact with an etiologic agent.

[0159] In a general embodiment, the present disclosure provides antisense oligonucleotides comprising a nucleic acid sequence that includes a G-rich motif.The antisense oligonucleotides provided herein improve delivery, therapeutic specificity, and resistance to enzymatic degradation. Also provided herein are methods of treating a disease or condition ina subject in need thereof, comprising administering to the subject an antisense oligonucleotide as described herein.

[0160] Without being bound by a particular theory, the presence of the G-rich motif may influence the structural or functional properties of the oligonucleotide, thereby enhancing its interaction with target genes or gene segments. For example, the antisense oligomers, described herein, may be capable of forming stable G-aggregates to enhance therapeutic efficacy, stability, and targeted gene regulation.

[0161] The stability of the ASO’s provided herein may be improved in physiological environments relative to sequences lacking such motif by the presence of G-rich sequence that forms intermolecular G-aggregates, stabilized by monovalent cations such as sodium (Na+) and potassium (K+). Additionally, the ASO’s provided herein may overcome the traditional challenge of poor cellular uptake by forming G-aggregates with a high charge density. The ASO’s provided herein may also maximize therapeutic efficacy and minimize off-target effects by offering an organ-specific delivery system, enhance the therapeutic flexibility of the ASO by making it compatible with various routes of administration, and minimize the immune response typically associated with ASO therapies.Antisense Oligonucleotides

[0162] In an aspect, provided herein are antisense oligonucleotides comprising a nucleic acid sequence that includes a G-rich motif. In some embodiments, the antisense oligomer is selected from one or more of the chemistries described herein.

[0163] In an aspect, provided herein is an antisense oligomer comprising a nucleic acid sequence, wherein the antisense oligomer is capable of forming G-aggregates, and wherein the nucleic acid sequence is characterized by Formula I: (I) or a pharmaceutically acceptable salt thereof,wherein:the G-rich motif comprises 2 to 30 naturally or non-naturally occurring nucleotides and at least two adjacent guanines or guanine derivatives; Y’ is thymine or uracil, or a derivative thereof;Y’ is thymine or uracil, or a derivative thereof;Variable Segment comprises 2 to 30 naturally or non-naturally occurring nucleotides; andthe nucleic acid sequence is optionally conjugated to a targeting moiety.

[0164] In an embodiment, the nucleic acid sequence is characterized by Formula II.

[0165] In another embodiment, the G-rich motif comprises at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 95%, or 90% guanines or guanine derivatives.

[0166] In yet another embodiment, the G-rich motif comprises two, three, four, five, or six guanines or guanine derivatives.

[0167] In still another embodiment, the G-rich motif is two to eight nucleotides in length. In an embodiment, the G-rich motif is characterized by the Formula Illa:WnWGGWZwherein:W is, independently for each occurrence, adenine or guanine, or a derivative thereof;Z is selected from thymine, uracil, guanine, and adenine, or a derivative thereof; andn is 0 or 1.

[0168] In an embodiment, the G-rich motif is selected from a nucleotide sequence of GGGGG, GGGGA, GGGAX, AGGGG, and AGGGA, wherein X is T,U or C. In a further embodiment, X is T. In some embodiments, the Variable Segment is between two and fourteen nucleotides in length, including ranges such as two to thirteen, two to twelve, two to eleven, two to ten, and two to nine nucleotides. In other embodiments, the Variable Segment is between three and fourteen nucleotides, including three to thirteen, three to twelve, three to eleven, three to ten, and three to nine nucleotides. In yet other embodiments, the Variable Segment ranges from four to fourteen nucleotides, with narrower ranges such as four to thirteen, four to twelve, four to eleven, four to ten, and four to nine nucleotides. In still other embodiments, the Variable Segment is between five and fourteen nucleotides, including five to thirteen, five to twelve, five to eleven, five to ten, and five to nine nucleotides.

[0169] In an embodiment, the Variable Segment is characterized by the Formula Illb:XYRAZNNwherein:N is individually, for each occurrence, selected from adenine, thymine, uracil, guanine, and cytosine, or a derivative thereof;R is cytosine or adenine, or a derivative thereof;X is cytosine or thymine, or a derivative thereof;Y is selected from thymine, uracil, guanine, and cytosine, or a derivative thereof; and Z is selected from thymine, uracil, guanine, and adenine, or a derivative thereof.

[0170] In another embodiment, the nucleic acid sequence is characterized by Formula IIIc:wherein:N is individually, for each occurrence, selected from adenine, thymine, uracil, guanine, and cytosine, or a derivative thereof;R is cytosine or adenine, or a derivative thereof;W is independently, for each occurrence, adenine or guanine, or a derivative thereof; X is cytosine or thymine, or a derivative thereof;Y is selected from thymine, guanine, cytosine, or a derivative thereof;Z is independently, for each occurrence, selected from thymine, guanine, and adenine, or a derivative thereof; andn is 0 or 1.

[0171] In another embodiment, the antisense oligonucleotide is selected from singlestranded DNA, RNA, peptide nucleic acid (PNA), phosphorodiamidate morpholino oligomer (PMO), and combinations thereof. In a further embodiment, the antisense oligonucleotide is a single-stranded DNA or RNA. In an embodiment, the antisense oligonucleotide is a singlestranded DNA.

[0172] In an embodiment, the antisense oligonucleotide comprises a modification selected from locked nucleic acid (LNA), ethylene-bridged nucleic acid (ENA), phosphorothioate backbone, tricyclo-DNA, tricyclo-phosphorothioate, 2’-O-methyl (2’-0Me), 2’-O-methoxyethyl (2'-M0E), 2’-fluoro (2'-F), 2’-O-methyl phosphorothioate, and 2’-O-methoxyethyl phosphorothioate, and combinations thereof.

[0173] In another embodiment, the nucleic acid sequence is conjugated to L, wherein L is an optional linking moiety.

[0174] In another embodiment, the linking moiety is a bond, a polyethylene glycol-based ligand, or an optionally functionalized alkyl-based ligand.

[0175] In another embodiment, the targeting moiety is selected from lipid nanoparticles, small molecule ligands that bind to specific receptors or transporters, sugar-based ligands, lipid-based moieties, peptide-based ligands, protein-based ligands, and polymer-based moieties.

[0176] In another embodiment, the targeting moiety is selected from GALNAC, tocopherol, cholesterol, docosahexaenoic acid (DHA), mannose, transferrin, RGD peptide, cell-penetrating peptides, vitamin B derivatives, phospholipids, monoclonal antibodies, PEG (polyethylene glycol), and progesterone. In a further embodiment, the vitamin B derivatives are biotin, folic acid, and riboflavin.

[0177] In another aspect, provided herein is an antisense oligonucleotide comprising a nucleic acid sequence, wherein the antisense oligonucleotide is capable of forming G-aggregates, wherein the 5 ’-terminus or 3 ’-terminus of the nucleic acid sequence comprises a G-rich motif, and wherein the antisense oligonucleotide is a single-stranded DNA or RNA of Formula IV or a pharmaceutically acceptable salt thereof,wherein:G' is selected from -OH,, , and the G-rich motif of formula:wherein A’ is selected from -OH, and ,y is 2-20;each R1is independently, for each occurrence, selected from OH and -NRaRb, wherein each Raand Rbis independently, for each occurrence, H or -Ci-6 alkyl;each R2is independently, for each occurrence, selected from a naturally or non- naturally occurring nucleobase and the sequence formed by the combination of each R2from 5’ to 3’ is a segment of a nucleic acid sequence;each R3is independently, for each occurrence, selected from H, halogen, -C(O)Ci-6 alkyl, -O-Ci-6 alkyl, -O-Ci-6 alkyl-O-Ci-6 alkyl, and -O-Ci-6 alkyl-C(O)NRaRb;z is 8-40;E is selected from H, -Ci-6 alkyl, -C(O)Ci-6 alkyl, and the G-rich motif of the formula; wherein:R11is OH or -NRaRb;L is an optional linking moiety;E’ is selected from H, -Ci-6 alkyl, -C(O)Ci-6 alkyl; andR4is independently, for each occurrence, selected from a naturally or non-naturally occurring nucleobase and the sequence formed by the combination of each R4from 5 ’ to 3’ is a segment of a nucleic acid sequence, wherein the segment comprises at least two adjacent guanines or guanine derivatives;provided that Formula IV comprises a G-rich motif at G’ or E and the G-rich motif comprises 2 to 30 naturally or non-naturally occurring nucleotides and at least two adjacent guanines or guanine derivatives.

[0178] In an embodiment of Formula IV, G’ is the G-rich motif of formula: IV

[0179] In another embodiment of Formula IV, E is the G-rich motif of formula: IV.

[0180] In yet another embodiment of Formula IV, G’ is the G-rich motif of formula: IV, whereinE is selected from H, -C(O)CH3.

[0181] In still another embodiment of Formula IV, G' is selected from -OH, and ; E is the G-rich motif of the formula: IV; and wherein E’ is H or -C(O)CH3.

[0182] In an embodiment of Formula IV, each R1is OH.

[0183] In another embodiment of Formula IV, each R2is a nucleobase, independently at each occurrence, selected from adenine, guanine, cytosine, 5-methyl-cytosine, and thymine, and uracil.

[0184] In yet another embodiment of Formula IV, each R3is H.

[0185] In still another embodiment of Formula IV, the G-rich motif comprises at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 95%, or 90% guanines or guanine derivatives.

[0186] In an embodiment of Formula IV, the G-rich motif comprises three, four, five, or six guanines or guanine derivatives.

[0187] In another embodiment of Formula IV, the G-rich motif is selected from a nucleotide sequence of GGGGG, GGGGA, GGGAX, AGGGG, and AGGGA, wherien X is T , U or C. In a further embodiment, X is T.

[0188] In yet another embodiment of Formula IV, the antisense oligonucleotide comprises an internal nucleotide sequence of XGXAG, wherein X is T , U or C. In a further embodiment, X is T.

[0189] In still another embodiment of Formula IV, the linking moiety is a bond, a polyethylene glycol-based ligand, or an optionally functionalized alkyl-based ligand.

[0190] In an embodiment of Formula IV, the targeting moiety is selected from lipid nanoparticles, small molecule ligands that bind to specific receptors or transporters, sugar-based ligands, lipid-based moieties, peptide-based ligands, protein-based ligands, and polymer-based moieties.

[0191] In another embodiment of Formula IV, the targeting moiety is selected from GALNAC, tocopherol, cholesterol, docosahexaenoic acid (DHA), mannose, transferrin, RGD peptide, cell-penetrating peptides, vitamin B derivatives, phospholipids, monoclonal antibodies, PEG (polyethylene glycol), and progesterone.In yet another embodiment of Formula IV, the vitamin B derivatives are biotin, folic acid, and riboflavin. In yet another aspect, provided herein is an antisense oligonucleotide of 16 to 40 naturally or non-naturally occurring nucleotides in length, wherein the antisense oligonucleotide comprises a nucleic acid sequence, and wherein the nucleic acid sequence is selected fromwherein X is T, U or C; and the nucleic acid sequence is optionally conjugated, wherein L is an optional linking moiety.

[0192] In an embodiment, X is T.

[0193] In another embodiment of the above antisense oligonucleotide, the linking moiety is a bond, a polyethylene glycol-based ligand, or an optionally functionalized alkyl-based ligand.

[0194] In yet another embodiment of the above the antisense oligonucleotide, the targeting moiety is selected from lipid nanoparticles, small molecule ligands that bind to specific receptors or transporters, sugar-based ligands, lipid-based moieties, peptide-based ligands, protein-based ligands, and polymer-based moieties. In still another embodiment of the above the antisense oligonucleotide, the targeting moiety is selected from GALNAC, tocopherol, cholesterol, docosahexaenoic acid (DHA), mannose, transferrin, RGD peptide,cell-penetrating peptides, vitamin B derivatives, phospholipids, monoclonal antibodies, PEG (polyethylene glycol), and progesterone. In a further embodiment, the vitamin B derivatives are biotin, folic acid, and riboflavin.

[0195] In an embodiment of all of the above aspects, the antisense oligonucleotide is capable of being delivered into the nucleus of a cell.

[0196] In an embodiment of all of the above aspects, the antisense oligonucleotide binds to nucleolin.

[0197] In yet another aspect, provided herein is a pharmaceutical composition comprising the antisense oligonucleotides, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier.

[0198] In still another aspect, provided herein is a method of treating a disease or condition in a subject in need thereof, wherein the method comprises administering a therapeutically effective amount of the antisense oligonucleotide or the composition to the subject.

[0199] In an embodiment, the disease or condition is selected from psoriasis, infertility, and cancer. In another embodiment, the disease or condition is selected from skin psoriasis, sperm dysfunction, lung cancer.

[0200] In another aspect, provided herein is a method of modulating the expression of a target gene in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the antisense oligonucleotide.

[0201] In an aspect, provided herein are antisense oligonucleotide sequences (ASO) featuring a G-rich sequence that can form intermolecular G-aggregates under physiological conditions. This structure enhances stability, boosts cellular uptake, and enables targeted downregulation of RNA molecules. G-aggregates are characterized by the following:1. Formation of Secondary Structures: The G-rich sequence forms intermolecular G-aggregates stabilized by monovalent cations (e.g., Na+, K+).2. Flanking Regions may or may not Form Hairpin Loops: These loops confer specific structural properties, enhancing functionality.3. Enhanced Stability and Delivery: The aggregates exhibit superior resistance to enzymatic degradation and improved cellular uptake due to their high charge density and secondary structure.4. Target Versatility: ASOs complementary to RNA targets such as portion of mRNA, Antisense RNA, long non-coding RNA, long non-coding uncharacterized mRNAtranscripts, long intergenic non-coding RNA, miRNA, opposite strand RNA and divergent strand RNA.5. Conjugate Embodiments: The ASO may optionally include conjugates to improve delivery and therapeutic activity, such as:♦ GALNAC for liver-targeted delivery♦ Vitamins (e.g., Vitamin E, Bl, B2, B3, B9)♦ Cofactors (e.g., FAD, FMN, NAD)♦ Flavonoids, biotin, tocopherol, and others.6. Diseases targeted include psoriasis, epilepsy, Alzheimer's, autism, rheumatoid arthritis, and Parkinson's.

[0202] In preferred embodiments, the present disclosure provides antisense oligonucleotides (ASOs) comprising G-rich sequences as set forth in SEQ ID NO: 87a; SEQ ID NO: 85a; SEQ ID NO: 88a; SEQ ID NO: 84a; SEQ ID NO: 89a; SEQ ID NO: 83a; SEQ ID NO: 82a, capable of forming intermolecular G-aggregates stabilized by monovalent cations, including Na+and K+, wherein said antisense oligonucleotide is designed for targeted downregulation of RNA species, including mRNA, long non-coding RNAs (IncRNAs), and microRNAs (miRNAs).

[0203] In an embodiment, the G-aggregates are stabilized in physiological buffers such as phosphate-buffered saline (PBS) or sodium chloride (NaCl) or Potassium Chloride (KC1) solutions.Enhanced Stability and Delivery: The aggregates exhibit superior resistance to enzymatic degradation and improved cellular uptake due to their high charge density and secondary structure.

[0204] In an embodiment, the present disclosure provides a pharmaceutical composition comprising the antisense oligonucleotide as disclosed herein and a pharmaceutically acceptable carrier, wherein the composition is formulated for the treatment of neurodegenerative disorders, metabolic syndromes, or cardiovascular diseases.

[0205] In an embodiment, the pharmaceutically acceptable carrier enhances solubility, stability, and mitigates immune activation to improve therapeutic efficacy.

[0206] In an embodiment, the present disclosure provides a method for downregulating target gene expression by administering a therapeutically effective dose of the antisense oligonucleotide as disclosed herein to a subject in need thereof.

[0207] In an embodiment, the antisense oligonucleotide is administered via intravenous, intrathecal, subcutaneous, intranasal, sublingual, intramuscular, or localized injection routes.

[0208] In the embodiments, the present disclosure provides a delivery system comprising the antisense oligonucleotides of SEQ ID NO: 87a; SEQ ID NO: 85a; SEQ ID NO: 88a; SEQ ID NO: 84a; SEQ ID NO: 89a; SEQ ID NO: 83a; SEQ ID NO: 82a may or may not be conjugated with a targeting moiety, selected from GALNAC, vitamins, flavonoids, cofactors, or biotin, for enhanced delivery to specific organs including the liver, central nervous system, or muscles.

[0209] In an embodiment, the antisense oligonucleotide is conjugated with GALNAC for liver-targeted delivery.

[0210] In an embodiment, in addition to its primary target, the ASO is also capable of targeting other RNA species, including mRNA, long non-coding RNAs (IncRNAs), and miRNA, allowing for the modulation of multiple genes involved in diverse diseases such as cancer, epilepsy, and autoimmune disorders. This versatility expands the therapeutic potential of the ASO across a wide range of complex, multifactorial diseases.

[0211] Accordingly, the present invention overcomes the challenges of traditional ASO therapies by utilizing G-aggregate formation, where the G-rich sequence forms stable G-tetrads, stabilized by monovalent cations like Na+and K+. This significantly enhances the oligonucleotide’s resistance to enzymatic degradation, prolonging its half-life and maintaining its therapeutic effect over time. The G-aggregate structure also improves cellular uptake by interacting favorably with scavenger receptors such as nucleolin, facilitating more efficient delivery of the ASO into target cells.

[0212] Moreover, the present invention incorporates an organ-specific delivery system, where the ASO can be conjugated with molecules like GALNAC, vitamins, or cofactors to enhance targeted delivery to specific tissues or organs, including the liver, central nervous system, and other vital organs. This targeted approach ensures higher local concentrations of the ASO at the disease site, minimizing systemic exposure and reducing potential side effects.

[0213] In addition to improving the stability, specificity, and targeting of ASO therapies, the present invention aims to reduce the risks of immune activation and toxicity commonly associated with ASO treatments. The use of G-aggregates ensures that the ASO can efficiently cross cell membranes without inducing an immune response, enabling the use of lower doses for effective therapeutic outcomes.

[0214] Conclusively, the antisense oligonucleotides disclosed herein represents a significant advancement in the field of gene modulation therapies, providing a versatile, stable,and highly targeted approach to treat a variety of diseases, offering promise for more effective treatments with fewer side effects compared to existing ASO technologies.Oligomer Chemistry Features

[0215] Also provided herein are both unmodified and modified antisense oligomers. In certain embodiments, the antisense oligomer is an unmodified single-stranded DNA or RNA. In other embodiments, the antisense oligomer is a modified antisense oligomer, comprising single -stranded DNA or RNA containing chemical modifications to the backbone, sugar moieties, nucleobases, or combinations thereof.

[0216] Representative examples of backbone modifications include, without limitation, phosphorothioate-modified oligomers, tricyclo-DNA analogs, tricyclo-phosphorothioate derivatives, and phosphorodiamidate morpholino oligomers (PMOs).

[0217] Representative examples of sugar modifications include, without limitation, 2’-O-methyl (2’-0me), 2’-O-methoxyethyl (2’-0-M0E), 2’-fluoro (2’-F), 2’-O-methyl phosphorothioate oligomers, and 2'-O-methyl phosphorothioate oligomers.

[0218] Representative examples of nucleobase or backbone analog modifications include, without limitation, peptide nucleic acids (PNAs), locked nucleic acids (LNAs), ethylene-bridged nucleic acids (ENAs), and unlocked nucleic acids (UNAs).NUCLEIC ACID SEQUENCES

[0219] As described herein, nucleic acid sequences having a G-rich motif have been shown to be effective in modulating the expression of one or more target genes.

[0220] In an aspect, provided herein is an antisense oligonucleotide which can be 10-75 nucleotides long with the following representative sequence:

[0221] 5’ W(n)WGGWZ TGTAG XYRAZNN... 3; motif conserved VariableIn various aspects, provided herein are antisense oligonucleotide comprising a nucleic acid sequence, or a pharmaceutically acceptable salt thereof, wherein the nucleic acid sequence is selected from:wherein X is T , U or C.

[0222] In a further embodiment, X is T.

[0223] Preferably the antisense oligonucleotide comprises a nucleotide sequence having substantial homology to any of the following nucleotide sequences:SEQ ID NO: 87a AGGGATGTAGCGCAGC,SEQ ID NO: 85a GGGATTGTAGTTCAAT,SEQ ID NO: 88a GGGGATGTAGCTCAAT,SEQ ID NO: 84a GGGGATGTAGCTCAGA,SEQ ID NO: 89a GGGGATGTAGCTCAGT,SEQ ID NO: 83a GGGGATGTAGCTCATA, orSEQ ID NO: 82a GGGGGTGTAGCTCATA.

[0224] In some embodiments, the antisense oligonucleotides are characterized by Formula I, Formula II, Formulae Illa-IIIc, and / or Formula IV.

[0225] For antisense applications, the oligomer can be 100% complementary to the nucleic acid target sequence, or may include mismatches to accommodate variants, provided that the resulting heteroduplex is sufficiently stable to resist degradation by cellular nucleases and other in vivo mechanisms. Mismatches, if present, are generally less destabilizing at theend regions of the hybrid duplex than in the central region. The number of mismatches tolerated depends on oligomer length, G:C base pairs in the duplex, and the position of the mismatch(es) in the duplex, in accordance with established principles of duplex stability.

[0226] Although such an antisense oligomer is not necessarily 100% complementary to the nucleic acid target sequence, it can still bind stably and specifically, thereby modulating the biological activity of the nucleic acid target, such as inhibiting expression of encoded protein(s).

[0227] The targeting sequence bases may include standard DNA bases or analogues, such as uracil and inosine, capable of Watson-Crick base pairing to target-sequence RNA bases.

[0228] An antisense oligomer may be designed to block, inhibit, or modulate mRNA translation, alter pre-mRNA splicing, or induce degradation of targeted mRNAs, and may be said to be “directed to” or “targeted against” a target sequence with which it hybridizes.

[0229] An antisense oligomer with sufficient sequence complementarity to a target RNA sequence may modulate splicing by masking protein binding sites and / or altering the targeted RNA’s three-dimensional structure.

[0230] In certain embodiments, the degree of complementarity between the antisense oligomer and the target RNA is sufficient to form a stable duplex and / or quadruplex. This region of complementarity may be as short as 8-11 bases, but may also range from 12-15 bases or more, including ranges such as 10-40 bases, 12-30 bases, 12-25 bases, 15-25 bases, 12-20 bases, or 15-20 bases, including all integers within these ranges. An antisense oligomer of about 14-15 bases is typically sufficient to achieve unique complementary sequence specificity.

[0231] In some embodiments, oligomers up to 40 bases in length may be suitable, provided that at least a minimum number of bases, e.g., 10-12 bases, are complementary to the target sequence. In some embodiments, facilitated or active uptake in cells is optimized at oligomer lengths of less than about 30 bases.

[0232] Included in the disclosure are antisense oligomers (e.g., single -stranded DNA) that consist of about 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, or 40 bases, in which at least about 6, 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, or 40 contiguous or non-contiguous bases are complementary to the desired target sequences.

[0233] In certain embodiments, antisense oligomers may be 100% complementary to the target sequence, or may include mismatches, e.g., to accommodate variants, as long as a heteroduplex formed between the oligomer and target RNA are sufficiently stable to withstandthe action of cellular nucleases and other modes of degradation which may occur in vivo. Hence, certain oligomers may have substantial complementarity, meaning, about or at least about 70% sequence complementarity, e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence complementarity, between the oligomer and the target mRNA.

[0234] Oligomer backbones that are less susceptible to nuclease degradation are discussed herein. Mismatches, if present, are typically less destabilizing toward the end regions of the hybrid duplex than in the middle.

[0235] The stability of the duplex formed between an oligomer and a target RNA sequence is a function of the binding Tmand the susceptibility of the duplex to cellular enzymatic cleavage. The Tm of an oligomer with respect to complementary-sequence RNA may be measured by conventional methods, such as those described by Hames et al., Nucleic Acid Hybridization, IRL Press, 1985, pp. 107-108 or as described in Miyada C. G. and Wallace R. B., 1987, Oligomer Hybridization Techniques, Methods Enzymol. Vol. 154 pp. 94-107.

[0236] In certain embodiments, antisense oligomers may have a binding Tm, with respect to a complementary-sequence RNA, of greater than body temperature and preferably greater than about 45°C or 50°C. Tm values in the range 60-80°C or greater are also included. At the same time, for purposes of optimizing cellular uptake, it may be advantageous to limit the size of the oligomer. For this reason, compounds that show high Tm (45-50°C or greater) at a length of 25 bases or less are generally preferred over those requiring greater than 25 bases for high Tmvalues.TARGETING MOIETIES

[0237] In some embodiments for antisense application, the nucleic acid sequence of the antisense oligomer is conjugated to a targeting moiety. Targeting moieties may be selected from small molecule ligands, sugar-based ligands, lipid-based moieties, peptide-based ligands, protein-based ligands, and polymer-based moieties.

[0238] In certain embodiments, the ASO can be conjugated or encapsulated within delivery vehicles such as lipid nanoparticles (LNPs), polymeric carriers, or other nanocarriers. It can be observed that even though the G-motif containing ASO has the ability to enter the cells but when encapsulated in the lipid nanoparticle its delivery as well as concentration in the cells is enhanced by any fold. Encapsulation within LNPs has been observed to significantly enhance cellular uptake and intracellular concentration of ASOs, including those comprisingguanine-rich motifs. Such formulations facilitate efficient translocation of the ASO into both the cytoplasm and nucleus, thereby improving target engagement and therapeutic potency.

[0239] In some embodiments, the small molecule ligands that bind to specific receptors or transporters may include hormones, vitamins, and cofactors. A non-limiting example of a hormone useful as a targeting moiety is progesterone. Non-limiting examples of vitamins and co-factors may include, but are not limited to, vitamin B derivatives such as biotin, folic acid, riboflavin, and thiamine.

[0240] In some embodiments, the sugar-based ligands may be used to target lectin receptors or liver-specific receptors. Non-limiting examples of sugar-based ligands, may include, but are not limited to, mannose and carbohydrate moieties that bind to asialoglycoprotein receptor (ASGPR), such as GALNAC.

[0241] In some embodiments, lipid-based moieties may enhance membrane interaction or target lipid-associated receptors. Non-limiting examples of lipid-based moieties, may include, but are not limited to, tocopherol, cholesterol, docosahexaenoic acid (DHA), and phospholipids.

[0242] In some embodiments, the peptide-based ligands may be RGD (arginine-glycine-aspartate) peptides or cell-penetrating peptides, which facilitate cellular uptake of ASOs.

[0243] In some embodiments, the protein-based ligands may be transferrin or monoclonal antibodies.

[0244] In some embodiments, the polymer-based moieties may be a polyethylene glycol (PEG) moiety.

[0245] In a further embodiment, the targeting moiety is selected from GALNAC, tocopherol, cholesterol, docosahexaenoic acid (DHA), mannose, transferrin, RGD peptide, cell-penetrating peptides, vitamin B derivatives, phospholipids, monoclonal antibodies, PEG (polyethylene glycol), and progesterone. In another embodiment, the targeting moiety is selected from GALNAC, biotin, and tocopherol.Target Genes

[0246] Provided herein is a method of modulating the expression of a target gene, comprising administering to a patient in need thereof a therapeutically effective amount of the antisense oligomer disclosed herein, or a pharmaceutical composition thereof.

[0247] Non-limiting examples of genes targeted, include but are not limited to DGKQ1, LIMK1, SYNJ1, and ANK1.Table 1: LIST OF GENES TARGETED BY SEQ ID NO: 82aTable 2: LIST OF GENES TARGETED BY SEQ ID NO: 89

[0248] The nucleic acid sequences disclosed herein may be associated with specific target genes and corresponding conditions or diseases.

[0249] In some embodiments, the target gene is PINK 1 -AS 1, the antisense RNA transcript of the PINK1 (PTEN-induced putative kinase 1) gene. PINK1 plays a pivotal role in mitochondrial health by promoting mitochondrial biogenesis, maintaining energy homeostasis, and initiating mitophagy — the process of clearing damaged mitochondria.

[0250] The antisense transcript PINK1-AS1 suppresses PINK1 expression through transcriptional or post-transcriptional interference. This invention leverages ASOs to downregulate PINK1-AS1, thereby upregulating PINK1 expression. Enhanced PINK1expression restores mitochondrial function, which is particularly beneficial in diseases characterized by mitochondrial dysfunction, including:• Neurodegenerative Diseases: Such as Parkinson’s disease and Alzheimer’s disease, where mitochondrial dysfunction contributes to neuronal loss.• Metabolic Disorders: Including diabetes and obesity, which involve impaired mitochondrial biogenesis and energy regulation.• Cardiovascular Diseases: Where mitochondrial health is crucial for maintaining cardiac muscle function.

[0251] By fine-tuning the expression of PINK1 via the modulation of PINK1-AS1, the ASOs described herein provide a targeted approach to addressing the mitochondrial dysfunction underlying these conditions. In addition to targeting PINK1-AS1, the antisense oligonucleotides (ASOs) described herein are designed to modulate the expression of a broad array of genes implicated in various diseases. These genes include, but are not limited to, ETHE1, DGKQ, FAM168B, LIMK1, SH3BP4, SYNJ1, TMTC4, ANK1, ADCY10, TXNRD2, DNAH17, and many others. The diverse targeting capability of these ASOs enables their use in addressing complex, multifactorial diseases by influencing multiple pathways simultaneously. ETHE1 (Ethylmalonic Encephalopathy 1): ETHE1 encodes a mitochondrial sulfur dioxygenase involved in hydrogen sulfide metabolism. Dysregulation of ETHE1 is linked to metabolic disorders such as ethylmalonic encephalopathy. The downregulation of ETHE1 can help modulate toxic hydrogen sulfide accumulation and improve mitochondrial function.

[0252] DGKQ (Diacylglycerol Kinase Theta): DGKQ regulates diacylglycerol (DAG) levels, critical for intracellular signaling pathways, particularly those involving protein kinase C. Aberrant DGKQ activity is associated with metabolic syndromes and certain cancers. Targeting DGKQ offers potential therapeutic benefits in lipid metabolism disorders and malignancies.

[0253] FAM168B (Family with Sequence Similarity 168 Member B): FAM168B is a lesser-studied gene with emerging links to neurodevelopmental disorders and inflammatory pathways. Modulating its expression may provide novel avenues for treating such conditions.

[0254] LIMK1 (LIM Domain Kinase 1): LIMK1 plays a critical role in actin cytoskeleton dynamics and cell migration. Overexpression of LIMK1 is implicated in cancer metastasis and neurological disorders such as Williams syndrome. Downregulating LIMK1 can inhibit tumor invasion and improve neural network stability.

[0255] SH3BP4 (SH3-Domain Binding Protein 4): SH3BP4 is involved in endosomal trafficking and intracellular protein sorting. Its dysregulation contributes to metabolic disorders, including diabetes and obesity. Targeting SH3BP4 provides a means to correct endocytic pathway disruptions.

[0256] SYNJ1 (Synaptojanin 1): SYNJ1 is critical for synaptic vesicle recycling and membrane trafficking.

[0257] Mutations in SYNJ1 are associated with neurodegenerative disorders such as Parkinson’s disease and epilepsy. Downregulating dysregulated SYNJ1 expression offers therapeutic potential for these conditions.

[0258] TMTC4 (Transmembrane and Tetratricopeptide Repeat Containing 4): TMTC4 is involved in calcium homeostasis and endoplasmic reticulum stress responses. Dysregulated TMTC4 expression is linked to cardiac hypertrophy and metabolic disorders. Targeting TMTC4 can restore calcium balance and reduce stress responses in affected tissues.

[0259] ANK1 (Ankyrin 1): ANK1 regulates the structural integrity of the cell membrane, particularly in red blood cells. Mutations or dysregulation of ANK1 are associated with hereditary spherocytosis and other hematological disorders. Modulating ANK1 levels offers therapeutic potential in such diseases.

[0260] ADCY10 (Adenylate Cyclase 10): ADCY10 generates cyclic AMP in response to bicarbonate levels and is involved in sperm motility and mitochondrial function. Dysregulation of ADCY10 contributes to infertility and metabolic dysfunction. Downregulation of ADCY10 can help normalize mitochondrial signaling pathways.

[0261] TXNRD2 (Thioredoxin Reductase 2): TXNRD2 is a mitochondrial enzyme involved in oxidative stress regulation. Overexpression contributes to cancer cell survival, while under-expression is implicated in cardiovascular diseases. Therapeutic modulation of TXNRD2 can balance oxidative stress responses and improve outcomes in these conditions.

[0262] DNAH17 (Dynein Axonemal Heavy Chain 17): DNAH17 is essential for ciliary movement and function. Mutations or dysregulation of DNAH17 are implicated in primary ciliary dyskinesia and related respiratory disorders. Targeting DNAH17 can help restore ciliary function and alleviate associated symptoms.APPLICATIONS AND FUNCTIONAL USES

[0263] Provided herein are antisense oligonucleotides and pharmaceutical compositions thereof that are suitable for modulating the expression of disease-associated genes in biological systems. The disclosed antisense oligonucleotides are capable of targeting multiple genessimultaneously, making them useful for conditions involving overlapping or interconnected molecular pathways. In certain embodiments, the antisense oligonucleotides are configured to downregulate or modulate the expression of two or more genes associated with disease pathogenesis.

[0264] Representative non-limiting applications of the disclosed antisense oligonucleotides include:• Neurodegenerative Disorders: Modulation of PINK 1 -AS 1, SYNJ1, and LIMK1 gene expression to support mitochondrial function, reduce neuroinflammatory responses, and promote synaptic stability.• Metabolic Syndromes: Concurrent regulation of ETHE1, DGKQ, and SH3BP4 gene expression to address metabolic imbalance, insulin resistance, and lipid dysregulation.• Cancer-Related Conditions: Regulation of LIMK1 and TXNRD2 gene expression to inhibit tumor progression and enhance cellular sensitivity to therapeutic interventions. Precision Gene Regulation Through Multi-Gene Modulation

[0265]

[0173] In various embodiments, the disclosed antisense oligonucleotides are applicable for gene regulation in conditions including neurodegenerative disorders, cardiovascular and musculoskeletal degenerative diseases, dermatological diseases, rare genetic disorders, Autism Spectrum Disorder, Duchenne Muscular Dystrophy, Huntington’s disease, Friedreich Ataxia, and pustular psoriasis.

[0266] The disclosed antisense oligonucleotides may be utilized in vitro or in vivo for research, diagnostic, or therapeutic development purposes. In vivo applications may be carried out in mammalian systems, including humans, laboratory animals, and domestic animals, for evaluating gene modulation effects.

[0267] In some embodiments, the antisense oligonucleotides may be used in combination with one or more additional bioactive agents. Such agents may be provided simultaneously or sequentially, through the same or different delivery routes, or in co-formulated compositions or kits for coordinated application.Delivery Platforms and Formulation Approaches

[0268] Efficient delivery of antisense oligonucleotides to target tissues is important for achieving effective gene modulation. Organ-specific delivery systems are therefore contemplated to improve stability, uptake, and localization while minimizing off-target interactions.

[0269] In one embodiment, the antisense oligonucleotides, in combination with pharmaceutically acceptable carriers, are formulated for oral, intravenous, inhalation, topical, subcutaneous, intraperitoneal, or pulmonary administration.

[0270] In another embodiment, the antisense oligonucleotides are formulated for veterinary and livestock-related applications, including supplementation of animal feed compositions to support gene regulation studies in agricultural species.Dosage and Use Parameters

[0271] The quantity, frequency, and duration of antisense oligonucleotide use may be selected based on the intended application, biological system, and desired gene modulation outcome. Such parameters may be optimized through routine experimental evaluation and monitoring to achieve consistent and reproducible biological effects.PHARMACEUTICAL COMPOSITIONS

[0272] The present disclosure also provides for the formulation and delivery of the disclosed antisense oligomers. Accordingly, an aspect of the present disclosure is a pharmaceutical composition comprising antisense oligomers as disclosed herein and a pharmaceutically acceptable carrier.In certain embodiments, the pharmaceutical compositions comprise an ASO as described herein are a pharmaceutically acceptable carriers that facilitates enhanced stability, solubility, and bioavailability of the ASO. The carriers can also mitigate potential toxicity and immune activation, ensuring the safety and efficacy of the therapeutic formulation.

[0273] Effective delivery of the antisense oligomers to the target nucleic acid is an important aspect of treatment. Routes of conjugate delivery include, but are not limited to, systemic routes such as oral and parenteral routes (e.g., intravenous, subcutaneous, intraperitoneal, and intramuscular), as well as inhalation, transdermal and topical delivery. Accordingly, the ASOs may be administered via a variety of routes, including but not limited to:• Intravenous (IV): For systemic delivery, particularly in conditions affecting multiple organ systems or requiring rapid distribution;• Intramuscular (IM): Offering localized delivery and prolonged release for diseases affecting specific muscle groups or regions;• Subcutaneous (SC): A minimally invasive option for chronic conditions requiring regular dosing;• Intrathecal: Direct delivery to the central nervous system for diseases such asneurodegenerative disorders;• Localized Injections: Tailored delivery to specific organs or tissues, such as the liver, kidneys, or lungs, ensuring high local concentrations of the ASO for maximum therapeutic effect.

[0274] With the above observation, it can be concluded that the drug has the capacity to invade the cell and incorporate into both cytoplasm and inside nucleus.

[0275] In certain embodiments, the ASO can be conjugated or encapsulated within delivery vehicles such as lipid nanoparticles (LNPs), polymeric carriers, or other nanocarriers. It can be observed that even though the G-motif containing ASO has the ability to enter the cells but when encapsulated in the lipid nanoparticle its delivery as well as concentration in the cells is enhanced by any fold. Encapsulation within LNPs has been observed to significantly enhance cellular uptake and intracellular concentration of ASOs, including those comprising guanine-rich motifs. Such formulations facilitate efficient translocation of the ASO into both the cytoplasm and nucleus, thereby improving target engagement and therapeutic potency.

[0276] The selection of an appropriate route of administration, formulation strategy, and the carrier system may be determined by one of skill in the art, taking into account the nature of the disease, the condition of the subject under treatment, the target tissue, and the pharmacological properties of the ASO.

[0277] Antisense oligomers may be administered in any convenient vehicle which is physiologically and / or pharmaceutically acceptable. Such a composition can include any of a variety of standard pharmaceutically acceptable carriers employed by those of ordinary skill in the art. Examples include, but are not limited to, saline, phosphate buffered saline (PBS), water (e.g., sterile water for injection), aqueous ethanol, emulsions such as oil / water emulsions or triglyceride emulsions, tablets and capsules. The choice of suitable physiologically acceptable carrier will vary dependent upon the chosen mode of administration.

[0278] The instant compounds (e.g., a single stranded DNA) can generally be utilized as the free acid or free base. Alternatively, the instant compounds may be used in the form of acid or base addition salts. Acid addition salts of the free amino compounds may be prepared by methods well known in the art, and may be formed from organic and inorganic acids. Suitable organic acids include maleic, fumaric, benzoic, ascorbic, succinic, methane sulfonic, acetic, trifluoroacetic, oxalic, propionic, tartaric, salicylic, citric, gluconic, lactic, mandelic, cinnamic, aspartic, stearic, palmitic, glycolic, glutamic, and benzenesulfonic acids. Suitable inorganic acids include hydrochloric, hydrobromic, sulfuric, phosphoric, and nitric acids. Base additionsalts included those salts that form with the carboxylate anion and include salts formed with organic and inorganic cations such as those chosen from the alkali and alkaline earth metals (for example, lithium, sodium, potassium, magnesium, barium and calcium), as well as the ammonium ion and substituted derivatives thereof (for example, dibenzylammonium, benzylammonium, 2-hydroxyethylammonium, and the like). Thus, the term “pharmaceutically acceptable salt” of Formula (I) and Formula (II) are intended to encompass any and all acceptable salt forms.

[0279] In addition, prodrugs are also included within the context of this invention. Prodrugs are any covalently bonded carriers that release a compound of Formula (I) or Formula (II) in vivo when such prodrug is administered to a patient. Prodrugs are generally prepared by modifying functional groups in a way such that the modification is cleaved, either by routine manipulation or in vivo, yielding the parent compound. Prodrugs include, for example, compounds of this invention wherein hydroxy, amine or sulfhydryl groups are bonded to any group that, when administered to a patient, cleaves to form the hydroxy, amine or sulfhydryl groups. Thus, representative examples of prodrugs include (but are not limited to) acetate, formate and benzoate derivatives of alcohol and amine functional groups of the compounds of Formula (I) and Formula (II). Further, in the case of a carboxylic acid (-COOH), esters may be employed, such as methyl esters, ethyl esters, and the like.

[0280] While the foregoing describes various embodiments of the disclosure, other and further embodiments of the disclosure may be devised without departing from the basic scope thereof. The scope of the invention is determined by the claims that follow. The invention is not limited to the described embodiments, versions, or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.EXAMPLES

[0281] Examples have been set forth below for the purpose of illustration and to describe certain specific embodiments of the disclosure. However, the scope of the claims is not to be in any way limited by the examples set forth herein. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art and such changes and modifications including, without limitation, those relating to the chemical structures, substituents, derivatives, formulations or methods of the disclosure may be made without departing from the spirit of the disclosure and the scope of the appended claims. Definitions of the variables in the structures in the schemes herein are commensurate with those of corresponding positions in the formulae presented herein.Example 1 : Solid-Phase Synthesis of a G-Rich Oligonucleotide

[0282] A. Materials and ReagentsAll reagents were obtained from GMP -certified suppliers and verified for traceability and purity.Solid Support: Controlled pore glass (CPG, 500 A pore size) or polystyrene resin, pre-loaded with the 3 '-terminal nucleotide.Phosphoramidite Monomer: 5'-DMT-2'-deoxyguanosine-3'-phosphoramidite (0.1 M in anhydrous acetonitrile).Activator: 0.25 M ethylthiotetrazole (ETT) in anhydrous acetonitrile.Capping Reagents:Solution A: Acetic anhydride (10% v / v in tetrahydrofuran).Solution B: N-methylimidazole (10% v / v in pyridine).Oxidizing Solution: 0.02 M iodine in tetrahydrofuran / pyridine / water (7:2:1 v / v / v).Detritylation Solution: 3% dichloroacetic acid (DCA) in toluene.Deprotection Solution: Aqueous ammonium hydroxide or AMA (1:1 mixture of ammonium hydroxide and methylamine).Solvents: Anhydrous acetonitrile, THF, pyridine, toluene.B. Synthesis Protocol

[0283] 1. Detritylation

[0284] The solid support was treated with 3% DCA in toluene for 60 seconds at room temperature (20-25°C) to remove the 5'-DMT protecting group. The resin was washed with anhydrous acetonitrile (3 x 1 mL) to remove residual acid and trityl cation.

[0285] 2. Coupling

[0286] A solution of guanine phosphoramidite (0.1 M) and ETT (0.25 M) in acetonitrile was delivered to the resin. A 3-5 molar excess of phosphoramidite was used relative to the available 5'-OH groups. The coupling reaction proceeded for 90 seconds at room temperature. The resin was washed with acetonitrile (3 x 1 mL).

[0287] 3. Capping

[0288] Unreacted 5'-OH groups were capped using a 1:1 mixture of Solution A and Solution B. The capping reaction was carried out for 60 seconds at room temperature. The resin was washed with acetonitrile (3 x 1 mL).

[0289] 4. Oxidation

[0290] The phosphite triester was oxidized using 0.02 M iodine solution in THF / pyridine / water (7:2:1 v / v / v) for 60 seconds at room temperature. The resin was washed with acetonitrile (3 x 1 mL).

[0291] 5. Cycle Repetition

[0292] Steps 1-4 were repeated until the desired G-rich oligonucleotide sequence was assembled. Trityl monitoring was performed after each detritylation step by measuring absorbance at 498 nm to confirm coupling efficiency.

[0293] C. Cleavage and Deprotection

[0294] The completed oligonucleotide was cleaved from the solid support and deprotected using aqueous AMA (1:1 ammonium hydroxide: methylamine) at 60°C for 4 hours in a sealed vessel. The reaction mixture was cooled to room temperature and evaporated under reduced pressure. The crude oligonucleotide was reconstituted in nuclease-free water.

[0295] D. Purification

[0296] Purification was performed using ion-pair reverse-phase HPLC (IP-RP-HPLC) with triethylammonium acetate (TEAA, 0.1 M, pH 7.0) as the mobile phase. Gradient elution with acetonitrile was used to separate full-length product from truncated sequences. For G-rich sequences prone to secondary structure formation, 4 M urea was included in the mobile phase to maintain denatured conditions.

[0297] Desalting was achieved via tangential flow filtration (TFF) using a 3 kDa MWCO membrane, followed by ethanol precipitation (2.5 volumes of cold ethanol, -20°C, centrifugation at 12,000 x g for 20 minutes).

[0298] E. Final Processing

[0299] The purified oligonucleotide was formulated into a sodium salt by exchanging the bufferwith lOmM sodium phosphate (pH7.2) viadiafiltration. Sterile filtration was performed using a 0.2 pm PES membrane. The final product was lyophilized and stored at -20°C.

[0300] F. Quality Control

[0301] Identity: Confirmed by LC-MS.

[0302] Purity: >95% by HPLC.

[0303] UV Absorbance: A260 measured for quantification.

[0304] Sequence Fidelity: Verified by enzymatic digestion followed by MS sequencing.

[0305] G-quadruplex Structure: Confirmed by CD spectroscopy (positive peak at -260 nm) and thermal melting analysis (Tm> 60°C).Example 2: GMP-Compliant Ion-Exchange Chromatography for G-Rich ASO Purification

[0306] To achieve high-purity antisense oligonucleotide (ASO) suitable for therapeutic application, ion-exchange chromatography remains a robust and scalable purification strategy. This example outlines a GMP-compliant protocol for purifying G-rich ASOs using strong anion exchange (SAX) resins and a cation replacement strategy to yield ASOs in pharmaceutically acceptable forms such as potassium salts.A. Resin Selection and Conditions for Cation Substitution

[0307] Strong anion exchange (SAX) resins functionalized with quaternary ammonium (Q) ligands are recommended for GMP-compliant purification. These resins provide high binding capacity, excellent pH and chemical stability, and scalability for manufacturing. They support buffer flexibility, including substitution of NaCl with KC1 in the elution buffer, enabling direct isolation of ASO in potassium salt form (ASO-K).• B. Elution Step: Cation Exchange Optimization for G-rich ASOs• Buffer System and pH Control• Primary Buffer: 20 mM Tris-HCl, pH 8.0• Maintains a slightly alkaline environment conducive to ASO stability.• Compatible with SAX resins using quaternary ammonium ligands.• Strategy for Cation Exchange (Na+K+ / Mg2+ / Ca2+)

[0308] 1. Initial Purification (Na+-form):

[0309] Use a NaCl gradient (0- 1.0 M) in 20 mM Tris-HCl (pH 8.0) for optimal resolution and impurity removal.

[0310] Sodium ions minimize G-quadruplex stabilization, aiding purification.

[0311] 2. Post-purification Cation Exchange:

[0312] After desalting or buffer exchange, replace Na+with K+, Mg2+, or Ca2+as needed.

[0313] For K+conversion, use 50-150 mM KC1 in buffer exchange or dialysis.

[0314] 3. Avoid K+During Ion-Exchange Purification:

[0315] Potassium stabilizes G-quadruplexes and may impair resolution. Exclude K+from mobile phases during core purification.

[0316] 4. Mg2+and Ca2+Considerations:

[0317] Use cautiously due to strong binding and precipitation risk.

[0318] Introduce in post-elution buffer exchange at 10-50 mM concentrations.

[0319] Structural Stability Measures

[0320] Temperature Control:

[0321] Conduct purification at 37-45°C to reduce G-quadruplex formation.

[0322] Avoid temperatures >50°C to prevent backbone degradation.

[0323] Mild Denaturation Strategy:

[0324] Add 1-2 M urea or <5% formamide to mobile phase to maintain single -stranded ASO conformation.

[0325] Analytical Monitoring:

[0326] Assess ASO conformation via Circular Dichroism (CD) or non-denaturing PAGE.

[0327] Monitor UV absorbance (260 nm) and conductivity across elution fractions. Example 3: Conjugation of Targeting Moieties via C6 Linkers

[0328] Antisense oligomers (ASOs) can be modified with linking moieties and purified before attachment to a targeting moiety. To enhance cellular uptake, tissue specificity, and pharmacokinetic profiles of antisense oligonucleotides (ASOs), conjugation with targeting moieties such as Tocopherol, GalNAc, and Cholesterol via C6 linkers were pursued. The C6 linker serves as a flexible and biocompatible bridge between the oligo backbone and the ligand, allowing for sterically favorable binding without interfering with hybridization or biological activity.A. Conjugation ChemistriesTable 2 below summarizes applicable conjugation strategies for the targeting moieties.Table 2: Conjugation Strategies* ASO = antisense oligomer, Ce = linkerremoval of excess coupling reagents and side-products via validated HPLC methods.Ligand-Specific Notes:

[0329] Tocopherol: Can be conjugated via amide coupling or thiol -maleimide coupling.

[0330] GalNAc (triantennary ligands): Generally incorporated pre-synthetically via phosphoramidite building blocks. (Kinberger et al., NAR 2017.)

[0331] Cholesterol: Often conjugated at the 3' end via click chemistry or prefunctionalized CPG.

[0332] B. Solid-Phase Supports and Linker Modifications. Table 3 below summarizes details applicable for solid-phase supports.Table 3: Solid-Phase SupportsTable 4 below summarizes applicable synthetic strategies for linker / spacer design.C. GMP-Compliant Purification Strategies

[0333] 1. Initial Purification Strategies: RP-HPLC or Ion-Exchange Chromatography:

[0334] Used for initial purification to separate conjugated from unconjugated ASOs.

[0335] Gradient selection depends on ligand hydrophobicity (e.g., Cholesterol-modified ASOs elute later due to lipophilicity).

[0336] 2. Buffer Exchange: Desalting / Diafiltration removes excess reagents, and concentrates product.

[0337] 3. Identity Confirmation: Mass Spec & UV260 Integration

[0338] Confirms conjugation and quantifies yield

[0339] Essential for batch release under GMP.

[0340] 4. Residual Reagent Clearance:

[0341] Validate clearance of organic reagents (e.g., DMSO, Cu2+) and side-products per ICH Q3 guidelines.

[0342] Example 4: GMP Compliance in G-Rich ASO Manufacturing

[0343] A. Process Modifications for Regulatory Compliance

[0344] 1. Solid-Phase Synthesis & Conjugation

[0345] Standardize phosphoramidite chemistry under GMP-certified conditions, ensuring verified raw materials and reagents traceability from vendor to batch.

[0346] Implement automated synthesis platforms (e.g., iScale GMP systems) featuring built-in process control, barcoding, and final visual inspection to confirm sequence, concentration, and format.

[0347] For conjugations (e.g., antibody or ligand conjugates), introduce controlled reaction time, molar ratios, temperature, and pH, with validation of conjugation efficiency using mass spectrometry or HPLC-UV methods.

[0348] 2. In-Process Sampling & Salt Exchange

[0349] Include intermediate salt-exchange purification steps during synthesis to reduce non-volatile salts.

[0350] Monitor flow, pressure, and pH; adjust process parameters as needed.

[0351] Document salt-exchange efficiency through residual salt assays using capillary electrophoresis or conductivity.

[0352] 3. Assembly & Final Formulation

[0353] Utilize GMP-compliant cleanrooms and equipment with environmental monitoring for particulates and microbial load.

[0354] Implement container closure integrity testing and evaluate extractables & leachables.

[0355] Enforce SOPs for mixing, buffer preparation, and vial filling.

[0356] 4. Traceability & Change Control

[0357] Maintain robust QMS infrastructure: document control, change management, vendor qualification, 4-eyes review, and CAPA.

[0358] Generate Certificates of Analysis (CoA) for each production lot detailing identity, purity, potency, concentration, format, and residual impurities.

[0359] B. Recommended Quality Control (QC) Parameters

[0360] The following QC parameters are recommended:

[0361] Identity & Structure: Confirm sequence, modifications, conjugation

[0362] Methods: HRMS / tandem MS, MS / MS fragmentation, enzymatic digestion + MS / MS

[0363] Assay / Potency: Quantify API content accurately

[0364] Methods: RP-HPLC-UV assay, duplex ratio by AX-HPLC-UV

[0365] Purity / Impurities: Detect truncated oligos, N- 1 impurities, deaminated species

[0366] Methods: IP-RP-LC-UV / MS, AX-HPLC-UV, CGE, SEC-UV

[0367] Conjugation Efficiency: For modified or conjugated oligos

[0368] Methods: HPLC-MS and UV quantification

[0369] Salt Exchange Efficiency: Ensure low residual salt

[0370] Methods: Anion exchange or conductivity assay

[0371] Residual Solvents: Confirm safety and compliance

[0372] Methods: GC with energy-regulated thresholds

[0373] Particulate Matter: Ensure injectable formulations meet standards

[0374] Methods: Light obscuration, microscopic particle count

[0375] Residual Metals: Monitor catalyst / reagent contamination

[0376] Methods: ICP-MS elemental scan

[0377] Sterility / Endotoxin: Required for parenteral products Methods: Sterility testing + LAL assay

[0378] Container Closure Integrity: Verify packaging sterility Methods: Dye ingress, vacuum decay, bubble leak

[0379] Extractables & Leachables: Evaluate packaging / equipment safety Methods: ICH-guided profding

[0380] Stability Testing: Demonstrate shelf-life Methods: ICH-aligned real-time stability with periodic testing

[0381] C. Integration across GMP Lifecycle

[0382] 1. Early Phase (Method Qualification)

[0383] Develop analytical methods for identity, purity, and potency.

[0384] Perform risk-based qualification per regulatory guidance.

[0385] 2. Late Phase / Commercial (Method Validation)

[0386] Validate methods according to ICH Q2(R1): linearity, accuracy, precision, specificity, detection limits, robustness.

[0387] 3. Reference Standards

[0388] Maintain well-characterized in-house reference standards for identity, assay, and impurities.

[0389] Qualification through MS, HPLC, and CGE.

[0390] 4. Documentation & Change Control

[0391] File detailed SOPs, validation protocols, raw data, CoAs, and change logs.

[0392] Perform annual product reviews and periodic supplier audits.Example 5: Scale-Up and Batch Consistency in G-Rich ASO Manufacturing

[0393] A. Cation Exchange Consistency1. Buffer Preparation & Control

[0394] Use GMP-grade salt solutions (e.g., KC1, K-acetate) with certificates of analysis (Co A) and full traceability.

[0395] Prepare buffers using WFI (Water for Injection) in validated, clean vessels.

[0396] Record pH, conductivity, osmolality, and temperature of every buffer batch to ensure process reproducibility.2. Exchange Conditions (TFF or Diafiltration)

[0397] Standardize the number of diafiltration volumes (DV) or TFF cycles required for optimal ion exchange.

[0398] Integrate in-line conductivity probes to monitor ion transition profiles (e.g., drop in Na+and rise in K+).

[0399] Validate membrane molecular weight cut-offs and system hold-up volumes for each TFF skid used at scale.

[0400] 3. Ion Exchange Resin-Based Strategy (if applicable)

[0401] Pre-condition resins with K+and store under GMP-compliant conditions.

[0402] Maintain consistency in column packing: bed height, flow rate, packing density, and pressure limits.

[0403] Implement resin lifecycle management: limit reuse and requalification based on SOP-defined usage cycles and performance metrics.

[0404] B. Conjugation Reactions and Purification Consistency

[0405] 1. Reagent and Reaction Control

[0406] Source GMP-grade ligands, linkers, and solvents from validated suppliers, with identity and purity confirmation.

[0407] Fix the ligand-to-ASO molar ratio, and control reaction parameters like temperature, time, pH, and solvent polarity.

[0408] Employ automated dosing systems to standardize reagent addition and reduce manual variation.

[0409] 2. Purification Strategy

[0410] Use validated HPLC (e.g., RP-HPLC, IEX) or TFF platforms consistently across batches.

[0411] Maintain column integrity by tracking lot numbers, packing parameters, and cleaning records.

[0412] Standardize chromatographic conditions: flow rate, gradient program, column temperature, and detection wavelength.

[0413] 3. Intermediate Quality Controls

[0414] Perform in-process QC using:

[0415] LC-MS or MALDI-TOF to confirm conjugation mass shift.

[0416] UV-Vis absorbance ratios (e.g., A260 for ASO vs. ligand-specific peak).

[0417] Capillary electrophoresis (CE) for identity and purity checks.

[0418] Quantify:

[0419] Conjugation efficiency (%)

[0420] Residual unreacted ligand

[0421] Free ASO impurities

[0422] C. Final Formulation and Lyophilization Consistency

[0423] 1. Solution Preparation

[0424] Prepare formulation buffers in jacketed stainless steel vessels with inbuilt temperature control and GMP-grade stirring systems.

[0425] Filter final formulation through 0.2 pm sterile filters, and perform bioburden and endotoxin testing post-filtration.

[0426] 2. Fill-Finish Operations

[0427] Use automated filling systems to ensure consistent fill volumes and CCI (Container Closure Integrity).

[0428] Calibrate and validate fill volumes using gravimetric or volumetric checks.

[0429] Use pre-qualified vials, stoppers, and seals, and monitor CCI via dye ingress, vacuum decay, or high-voltage leak detection.

[0430] 3. Uyophilization Control

[0431] Develop and validate a freeze-drying protocol with defined:

[0432] Freezing ramp rate and hold time

[0433] Primary drying shelf temperature and chamber pressure

[0434] Secondary drying duration

[0435] Maintain consistency in:

[0436] Cake morphology

[0437] Reconstitution time

[0438] Residual moisture levels (Karl Fischer titration)

[0439] 4. Post-Uyo Storage and Stability

[0440] Store under qualified temperature-controlled conditions (e.g., -20 °C or 2-8 °C).

[0441] Include real-time and accelerated stability testing (per ICH Q1A guidelines).

[0442] Ensure each batch undergoes release testing for appearance, potency, reconstitution, residual solvent, and microbiological safety.Example 6: Rational design of antisense oligonucleotides to investigate guanine enrichment influences structural organization

[0443] Antisense oligonucleotides were rationally designed to investigate how guanine enrichment influences structural organization, intracellular trafficking, and functional antisense activity. The complete panel of sequences, including non-G-rich controls and G-rich variants containing four (4G), five (5G), or six (6G) consecutive guanine residues, is listed in Table 5. This design enabled a systematic evaluation of guanine-dependent properties across all experimental endpoints.Table 5. Nucleotide Sequences of Antisense Oligomers Categorized by Guanine Content

[0444] Initial structural assessment by agarose gel electrophoresis (FIGS. 7-12) revealed that non-G-rich antisense sequences migrated as a single discrete band, indicative of uniform molecular species. In contrast, antisense oligomers containing five or six consecutive guanines exhibited a distinct double-band migration pattern, consistent with the presence of higher-order or aggregated molecular forms. Oligomers containing four guanines predominantly migrated as a single band, suggesting that although 4G sequences may adopt G-rich conformations, these structures are of lower order or stability and are not readily resolved under agarose gel conditions. This observation indicates a threshold effect, whereby increasing guanine content promotes formation of structurally distinct assemblies.

[0445] To confirm that these differences were not attributable to degradation or concentration variability, UV absorbance spectra were recorded for all sequences (FIGS. 13-17). All antisense oligomers displayed characteristic absorbance maxima around 255-260 nm, confirming comparable nucleic acid integrity across G-rich and non-G-rich variants. These data support the conclusion that the observed electrophoretic differences arise from structural organization rather than experimental artifacts.Example 7: Analysis of higher order structural features

[0446] Higher-order structural features were further elucidated by circular dichroism spectroscopy (FIGS. 18-22). Circular dichroism (CD) spectra of antisense oligomers with varying guanine content was recorded over the wavelength range of 220-320 nm. G-rich antisense oligomers exhibited a characteristic positive ellipticity peak near 260 nm and a negative peak between 240-245 nm, consistent with parallel G-quadruplex-like or G-aggregate spectral signatures. This profile was observed across 4G, 5G, and 6G sequences, with progressively stronger and more defined signals in 5G and 6G oligomers. In contrast, non-G-rich antisense sequences lacked these spectral features. These findings indicate that guanine enrichment promotes ordered secondary and higher-order conformations, with increasing guanine content enhancing structural definition and stability.

[0447] The functional relevance of these structures was assessed through thermal and enzymatic stability analyses (FIGS. 23-28). G-rich antisense oligomers demonstrated iondependent stabilization, particularly under potassium- and PBS-containing conditions, consistent with stabilization of G-quadruplex-like conformations. Scrambled non-G-rich controls lacked defined melting transitions and were more susceptible to thermal denaturation (FIG. 23). Assessment of G-quadruplex thermal stability in 5G-1 under varying salt conditions using circular dichroism (CD) spectroscopy at 260 nm. (A) Thermal melt profile in nuclease-free water (FLO) revealed a gradual decrease in CD signal with temperature and a clear melting transition with a Tm of 69.29 °C. (B)-(E) CD signal at 260 nm for 5G-1 treated with 10 mM KC1 (10K), 100 mM KC1 (100K), 4X PBS, and 6X PBS respectively showed minimal change across the temperature range (21-90 °C), indicating enhanced thermal stability of the G-quadruplex. (F)-(G) CD thermal profiles for scrambled control (SC) and SC + 100K revealed progressive loss of CD signal without a defined transition, indicating absence of stable G-quadruplex structure.

[0448] DNase I digestion assays further showed that G-rich antisense oligomers were substantially more resistant to nuclease-mediated degradation across multiple ionic and biologically relevant environments, whereas non-G-rich sequences were rapidly degraded.These results indicate that G-rich structural organization confers a protective advantage that is likely to be beneficial under physiological conditions.

[0449] A DNase I digestion assay to assess the structural stability of 5G-1 under different ionic conditions (FIG. 24) was performed. 5G-1 was incubated with either water, 10 mM K+, 10 mM Na+, or 1 x PBS, followed by DNase I treatment for 0, 10, 20, and 30 minutes. Samples were resolved on a native agarose gel and visualized. A scrambled sequence and untreated controls (without DNase I) were included for comparison. Intact bands indicate protection from DNase I digestion due to stable secondary structure (e.g., G-quadruplex formation), whereas smeared or diminished signal suggests degradation.

[0450] A DNase I digestion assay to assess the structural stability of 5G-1 under different ionic conditions was also performed (Fig. 25). 5G-1 was incubated with 4 x or 6xPBS, followed by DNase I treatment for 0, 10, 20, and 30 minutes. Samples were resolved on a native agarose gel and visualized.

[0451] FIG. 26 shows 2% agarose gel showing (A, B) scrambled 5G-1 processed under different salt conditions as indicated, followed by DNase I treatment for 0, 10, and 20 minutes. Scrambled 5G-1, being completely randomized, shows a single band with no stability upon DNase I digestion. This suggests that the stability observed in 5G-1 arises from the presence of five consecutive guanines that contribute to G-quadruplex formation, thereby conferring structural stability.

[0452] FIG. 27 is a gel image showing 5G-1 pre-folded in 6x PBS showed two distinct bands in PBS, indicating multiple conformations. Upon incubation with FBS, blood serum, and tissue homogenate, the bands were replaced by smears, suggesting mild nuclease-mediated degradation. The smearing was most prominent in FBS and serum, with comparatively less in tissue homogenate.

[0453] FIG. 28 shows quantitative data aligned with gel-based observations. The 5G-1 demonstrated maximum stability in PBS, lowest stability in FBS, and moderate degradation in tissue homogenate. Notably, the single-stranded 16 bp form was more vulnerable to nuclease-mediated degradation than the 64 bp folded form, underscoring the protective role of secondary / higher-order structures in enhancing ASO stability in biologically active environments.

[0454] Thermal and nuclease stability analyses demonstrated that antisense oligomers containing consecutive guanine residues form structurally robust higher-order conformations that confer enhanced resistance to denaturation and enzymatic degradation. Circular dichroism-based thermal melt analysis of a representative 5G antisense oligomer revealed adefined melting transition in nuclease-free water and minimal signal change across a broad temperature range under potassium-, sodium-, and PBS-containing conditions, indicating ion-stabilized G-rich structural organization. In contrast, scrambled non-G-rich control sequences lacked defined melting transitions and exhibited progressive signal loss, consistent with the absence of stable folded structures. DNase I digestion assays further showed that G-rich antisense oligomers retained intact or partially intact bands in multiple ionic environments and biologically relevant matrices, including PBS, serum, and tissue homogenate, whereas scrambled sequences underwent rapid degradation with loss of discrete banding. Collectively, these results establish that incorporation of G-rich motifs imparts sequence-dependent thermal and enzymatic stability, supporting the functional robustness of the antisense oligomers. Example 8: Nucleolin binding studies

[0455] Given the established affinity of nucleolin for G-quadruplex structures, nucleolin binding studies were performed (FIGS. 29-34). These experiments demonstrated selective interaction between G-rich antisense oligomers and nucleolin, with binding correlating with guanine content and structural stability. Non-G-rich antisense sequences showed minimal or no detectable interaction, suggesting that protein engagement is driven by higher-order structure rather than primary sequence alone. This interaction provides a plausible mechanistic link between G-rich structure formation and intracellular trafficking behavior. Binding studies demonstrated that antisense oligomers containing G-rich motifs selectively interact with nucleolin, a known G-quadruplex-binding protein, whereas scrambled non-G-rich control sequences showed minimal or no detectable interaction. The observed nucleolin association correlated with the presence and stability of guanine-rich higher-order structures, with stronger binding evident for oligomers containing higher numbers of consecutive guanines. This selective interaction was not observed for non-G-rich or scrambled sequences, indicating that nucleolin binding is structure-dependent rather than sequence -lengthdependent. These findings suggest that G-rich structural organization enables specific protein engagement, providing a mechanistic basis for enhanced intracellular trafficking and nuclear localization of the antisense oligomers.Example 8: Enhanced Endosomal Escape of G-Rich Antisense Oligonucleotides

[0456] Consistent with this interpretation, endosomal escape assays (FIGS. 35-36) showed that G-rich antisense oligomers exhibited reduced co-localization with endosomal and lysosomal markers and increased cytosolic distribution compared to non-G-rich controls. Enhanced endosomal escape suggests that G-rich structural features facilitate productive intracellular release, an essential step for antisense activity.

[0457] 5G-1 internalisation and lysosomal escape assessed by non-lysosomal Alexa Fluor-488 puncta. SH-SY5Y Cells were treated with 5G-1, No-5G-1, 5G-1 without LysoTracker, or Scrambled control, alongside untreated Cell Control. Fixed cells were labelled with Calcein Blue (nuclei; blue), LysoTracker (lysosomes; red), and 5G-1-Alexa Fluor-488 (green). Channel-wise and merged images demonstrate that 5G-1 enters cells and forms discrete fluorescent puncta. A subset of 5G-1 signal colocalises with LysoTracker, representing lysosomal sequestration, whereas a distinct population of AF488-positive puncta localises outside lysosomal regions.

[0458] To determine the extent of lysosomal escape, only the Alexa Fluor-488 puncta that do not colocalise with LysoTracker were quantified using ImageJ. The 5G-l-treated group showed a pronounced increase in non-lysosomal AF488 signal compared with untreated and scrambled controls, indicating efficient release of 5G-1 from lysosomal compartments into the cytosol. The No-5G-1 and 5G-1 (No-LysoTracker) groups validated probe- and dye-dependent specificity. Data are expressed as mean ± SEM and analysed by one-way ANOVA (FIG 35; ****p < 0.0001, **p < 0.01).

[0459] Cellular trafficking analyses demonstrated that G-rich antisense oligomers exhibit enhanced endosomal escape compared to non-G-rich and scrambled control sequences. Fluorescence imaging using LysoTracker and Alexa Fluor-488-labelled antisense oligomers revealed that G-rich sequences formed distinct cytosolic puncta with reduced co-localization with lysosomal compartments, indicating efficient release from endosomal structures. Quantitative analysis confirmed a significant increase in non-lysosomal Alexa Fluor-488 signal in cells treated with G-rich antisense oligomers relative to untreated, non-G-rich, and scrambled controls. In contrast, non-G-rich and scrambled sequences showed predominant retention within LysoTracker-positive compartments, consistent with limited endosomal escape. These observations indicate that incorporation of G-rich motifs promotes productive intracellular trafficking by facilitating release from endosomal compartments, a prerequisite for downstream nuclear access and functional antisense activity.Example 9: Nuclear Localization of G-rich antisense oligomers

[0460] Subsequent nuclear localization studies (FIGS. 37-39) revealed that G-rich antisense oligomers efficiently accumulated within the nucleus under native uptake conditions, without the need for transfection reagents. 5G-1 preferentially accumulates in nuclei, enhanced by transfection, while scrambled sequence remains cytoplasmic (FIG 37). Similarly, 4G-1 preferentially accumulates in nuclei, enhanced by transfection (FIG. 38) In contrast, non-G-rich antisense sequences showed limited nuclear localization unless forcibly delivered viatransfection FIG. 39). This distinction indicates that G-rich motifs confer an intrinsic capacity for nuclear access, likely arising from the combined effects of structural stability, protein interaction, and improved intracellular trafficking.

[0461] Subcellular imaging analyses demonstrated that antisense oligomers containing G-rich motifs exhibit efficient and preferential nuclear localization under native uptake conditions, without the requirement for transfection reagents. G-rich antisense oligomers showed pronounced accumulation within the nucleus, indicating an intrinsic ability to access the nuclear compartment following cellular internalization. In contrast, non-G-rich antisense sequences displayed limited or diffuse nuclear localization under native conditions but exhibited detectable nuclear accumulation when introduced via transfection. These observations indicate that while non-G-rich antisense oligomers can localize to the nucleus when forcibly delivered, incorporation of G-rich motifs enables spontaneous and efficient nuclear localization in the absence of transfection, highlighting a distinct delivery advantage of the G-rich antisense design.Example 10: Examination of cytotoxicity between G-rich and non-G-rich oligomers

[0462] Importantly, MTT assays (FIG. 40) confirmed that G-rich antisense oligomers did not induce significant cytotoxicity relative to untreated or non-G-rich controls, demonstrating that enhanced uptake and nuclear localization are not associated with adverse cellular effects.

[0463] Cell viability analysis using the MTT assay demonstrated that G-rich antisense oligomers did not induce significant cytotoxicity at the tested concentrations. Cells treated with G-rich antisense sequences showed viability comparable to untreated controls and scrambled non-G-rich sequences, indicating that incorporation of G-rich motifs does not compromise cellular metabolic activity. These findings confirm that the enhanced intracellular trafficking, nuclear localization, and functional activity of G-rich antisense oligomers occur in the absence of overt cytotoxic effects, supporting their suitability for therapeutic applications.Example 11 : Inhibition of target transcript by G-rich antisense oligomers

[0464] Finally, functional validation by quantitative RT-PCR (FIGS. 41-44) demonstrated that the G-rich antisense oligomer 5G-1 mediated significant inhibition of the target RNA in both SH-SY5Y and HepG2 cells, whereas the non-G-rich antisense sequence NG-2 showed largely non-significant effects. The observed inhibition correlated with enhanced cellular uptake and intrinsic nuclear localization of the G-rich antisense oligomer, supporting the conclusion that structural and trafficking advantages conferred by G-rich motifs translate into effective target modulation across distinct cell types.

[0465] Quantitative RT-PCR analysis showed that the G-rich antisense oligomer 5G-1 mediated significant inhibition of the target transcript in both SH-SY5Y and HepG2 cells, whereas the non-G-rich antisense sequence (NG-2) failed to produce meaningful or statistically significant inhibition. This functional outcome correlated with earlier observations of enhanced cellular internalization, efficient endosomal escape, and preferential nuclear accumulation of G-rich antisense oligomers. In contrast, NG-2 displayed limited intracellular trafficking and minimal nuclear localization, consistent with its lack of inhibitory activity. Collectively, these findings indicate that effective target RNA inhibition is associated with increased cellular uptake and nuclear availability of the antisense oligomer, as enabled by incorporation of G-rich motifs.

[0466] Overall, guanine enrichment promotes formation of stable higher-order structures that enhance intracellular trafficking, nuclear localization, and functional antisense activity without inducing cytotoxicity. These findings demonstrate that G-rich motifs provide a structural and mechanistic advantage for effective antisense oligonucleotide design.Example 12: Identification of Interaction between Nucleolin and G-Quadruplex Structures Table 6 provides the nomenclature used for the docking experiments described herein.’Table 6. G-Quadruplex designationsA. Interaction Analysis: Nucleolin & P2B1046G-1 G-Quadruplex

[0467] An in-depth structural analysis of nucleolin docked with the P2B1046G-1 G-quadruplex was undertaken. Based on the Rank 1 pose (Conformer 2206) and detailed PLIP interaction mapping across multiple DNA chains (B, C, D, E), the complex is characterized by an extensive "wrapping" mechanism. The binding is primarily driven by a high-density electrostatic network (Salt Bridges) and extremely short, high-stability hydrogen bonds. The P2B1046G-1 sequence exhibits superior binding energetics, particularly in its electrostatic component.Table 7. P2B1046G-1 / Nucleolin docking performance

[0468] Key Finding: The electrostatic score (-109.116) represents the dominant force, indicating that the protein's positive domains (likely RBDs or RGG) are perfectly aligned with the negative phosphate tracks of the G-quadruplex.Atomic-Level Interaction Mapping is seen in FIG. 46.

[0469] Nucleolin utilizes Lysine and Arginine residues to anchor itself across multiple DNA strands (Table 8).Table 8. P2B1046G-1 / Nucleolin Salt Bridges""

[0470] The H-bond profile reveals several "ultra-short" bonds (under 2.5 A) which signify exceptionally high stability (Table 9).Table 8. P2B1046G-1 / Nucleolin Hydrogen Bond Network (High Stability)Hydrophobic interactions:

[0471] MET 46 (Chain A) - DG 10 (Chain B): A hydrophobic contact at 2.51 A (Carbon Indices

[0472] 342-1560). This interaction provides a localized "anchor" that shields the hydrogen bond network from the solvent, increasing the residence time of the protein on the DNA.Multi-chain Binding Architecture

[0473] The distribution of interactions across chains B, C, D, and E suggests a specific binding mode:

[0474] 1. Terminal Recognition (Chain B & E): MET 46 and ARG 158 target the distal ends of the

[0475] G-quadruplex, likely locking the flanking sequences or terminal tetrads.

[0476] 2. Core Stability (Chain C & D): GLY 1 and LYS 83 / 85 focus on the central loops or the

[0477] groove architecture of the G-quadruplex.

[0478] 3. The "Glycine Anchor": The GLY 1 interaction (2.34 A) is significantly stronger than typical

[0479] protein-DNA H-bonds, suggesting it is a critical "hotspot" for P2B1046G-1 recognition.Conclusion

[0480] P2B1046G-1 shows a more organized and multi -valent binding interface. The high frequency of salt bridges and the presence of "ultra-short" hydrogen bonds suggest that this sequence forms a more rigid and thermodynamically stable complex with Nucleolin. B. Interaction Analysis: Nucleolin & D15G-1 G-Quadruplex

[0481] This details the structural and energetic analysis of Nucleolin docked with the D15G-1

[0482] G-quadruplex sequence. The analysis is based on the top-ranked pose which shows a total binding score of -106.448. The binding interface is characterized by a strong dependence on electrostatic anchors and a dense hydrogen bond network involving both the protein backbone and side chains across DNA chains B, C, D, and E.

[0483] The D15G-1 sequence demonstrates high affinity, primarily driven by electrostatics, with distinct

[0484] VDW and desolvation profiles that stabilize the complex.Table 9. D15G-1 G-Quadruplex / Nucleolin docking performance

[0485] Key Finding: The electrostatic contribution remains the primary driver of the interaction. However, the VDW energy (16.621) suggests a specific packing density at the interface, indicating a complementary fit between the protein domains and the G-quadruplex grooves.

[0486] Atomic-Level Interaction Mapping is seen in FIG. 47.Table 10. D15G-1 G-Quadruplex / Nucleolin Salt Bridges

[0487] D15G-1 features several key H-bonds, including highly specific interactions with loop residues and terminal bases that define the specificity of the binding site.Table 11. D15G-1 G-Quadruplex / Nucleolin Hydrogen Bond Network (High Stability)Hydrophobic Interactions

[0488] MET 46 (Chain A) - DT 6 (Chain E):A hydrophobic contact at 3.47 A(Carbon Indices342-2475). This provides a non-polar patch that helps orient the protein relative to the E-chain of the G-quadruplex.Multi-Chain Binding Architecture

[0489] The interaction data suggests a specific "wrap-around" binding mode for D15G-1:

[0490] 1. Chain C Dominance: Chain C serves as the primary binding hub, hosting the strongest salt bridge (LYS 85, 2.67 A) and a cluster of hydrogen bonds (ASN 100, ARG 158, SER 159). This suggests the protein RNP domains are centered on this strand.

[0491] 2. Flexible Anchoring: ARG 43 interacts with both Chain B and Chain D, acting as a molecular bridge that stabilizes the relative orientation of these two strands.

[0492] 3. Terminal Locking: The interaction of THR 47 and ARG 48 with Chain E suggests a "locking" mechanism at the end of the quadruplex sequence, preventing dissociation.SUMMARY OF FINDINGS

[0493] The D15G-1 complex is characterized by an exceptionally strong salt bridge with LYS 85 (2.67 A) and a diverse array of polar contacts facilitated by residues ASN 100 and SER 159.

[0494] Key Observation: The D15G-1 sequence demonstrates an extensive utilization of its loop residues (specifically DT6 and DT12) for polar recognition. This interaction profile suggests a high degree of structural complementarity that likely influences the binding kinetics and thermodynamic stability of the Nucleolin-DNA complex.C. Interaction Analysis: Nucleolin & P2B335G-2G-Quadruplex

[0495] Provided herein is a comprehensive structural and energetic analysis of Nucleolin docked with

[0496] the P2B335G-2 G-quadruplex sequence. Based on the top-ranked conformation (Conf 2203), the complex exhibits a total binding score of -106.688. The interaction interface is heavily stabilized by an extensive salt-bridge network involving Lysine and Arginine residues and a dense hydrogen bond profile, particularly at the N-terminal Glycine. The P2B335G-2 sequence shows high thermodynamic stability. The electrostatic component is the dominant contributor to the binding energy.Table 12. P2B335G-2 / Nucleolin docking performance

[0497] Key Finding: The top pose achieves a score of -106.688, driven by powerful electrostatic attractions (-109.116). The VDW score (24.276) indicates a specific orientation where electrostatic complementarity outweighs steric repulsion.

[0498] Atomic-Level Interaction Mapping is seen in FIG. 48.

[0499] The stability of the P2B335G-2 complex relies on multiple "hotspot" residues that anchor the protein to the DNA phosphate backbone across multiple chains (Table 10).Table 13. P2B335G-2G-Quadruplex / Nucleolin Salt Bridges

[0500] The hydrogen bonding profile is diverse, featuring exceptionally short bonds and interactions with both the backbone and side chains.Table 14. P2B335G-2G-Quadruplex / Nucleolin Hydrogen Bond Network (High Stability)Hydrophobic Interactions

[0501] MET 46 (Chain A) - DG 10 (Chain B):A very close hydrophobic contact at 2.51 A. This suggests a high degree of methyl-stacking or van der Waals complementarity between the Methionine side chain and the Guanine base.Multi-Chain Binding Architecture

[0502] The interaction data reveals a sophisticated multi-strand binding mode:

[0503] 1. N-Terminal Anchor: GLY 1 forms an extremely short (2.34 A) and linear (168.48°) hydrogen bond with Chain C, suggesting this is the primary initiation point for binding.

[0504] 2. Chain C / D Hub: LYS 83 and LYS 85 act as a central processing unit, forming salt bridges and H-bonds across both Chains C and D, effectively "zipping" the protein to the quadruplex core.

[0505] 3. Backbone Wrapping: The involvement of Chain B (MET 46, LYS 99) and Chain E (ARG 48, THR 47) indicates the protein significantly wraps around the quadruplex, contacting nearly every available DNA strand.SUMMARY OF FINDINGS

[0506] The P2B335G-2 project identifies a high-affinity interaction profile dominated by electrostatics

[0507] ( -109.116). The structural hallmark of this docking pose is the GLY 1 - Chain C hydrogen bond, which, at 2.34 A, represents one of the strongest observed polar contacts. Furthermore, the extensive salt-bridge network involving LYS 83, 85, and 99 suggests a robust and stable complex capable of maintaining high-affinity binding across a wide range of physiological conditions.D. Interaction Analysis: Nucleolin & P2H45G-3G-Quadruplex

[0508] Detailed here is the structural analysis of Nucleolin docked with the P2H45G-3 G-quadruplex. The primary docking conformation (Conf 5596) yielded a total binding score of -101.842. This specific complex is characterized by an exceptionally favorable Van der Waals (VDW) profile (-27.878) and a series of ultra-short hydrogen bonds (down to 2.00 A), suggesting a high degree of shape complementarity and rigid anchoring across multiple DNA chains. The docking results for P2H45G-3 (Table 15) indicate a stable complex where electrostatic attraction is balanced by significant steric optimization.Table 15. P2H45G-3 / Nucleolin docking performance

[0509] Key Finding: The negative VDW score (-27.878) in the top-ranked pose is a distinctive feature, indicating an optimized packing interface where the protein side chains fit deeply into the G-quadruplex grooves.Atomic-Level Interaction Mapping for P2H45G-3 / Nucleolin is seen in FIG 49.Table 16. P2H45G-3 G-Quadruplex / Nucleolin Salt Bridges

[0510] The hydrogen bonding in this project is remarkably strong, featuring several "ultra-short" bonds that suggest high-intensity polar recognition.Table 17. P2H45G-3 G-Quadruplex / Nucleolin Hydrogen Bond NetworkHydrophobic Interactions

[0511] MET 46 (Chain A) - DG 5 (Chain B):Distance of 3.79 A. Stabilizes the interface near the Guanine base of the B-chain.* THR 2 (Chain A) - DG 9 (Chain C): Distance of 3.91 A. Provides non-polar surface contact near the protein N-terminus.Multi-Chain Binding Architecture

[0512] The binding topology for P2H45G-3 suggests a "Wraparound" interaction model:

[0513] 1. C-Terminal Precision: The C-terminal domain (ARG 158, SER 159) is highly active, specifically targeting Chain D with the shortest observed hydrogen bond (2.00 A).

[0514] 2. Central Hub: Chain C acts as the primary recruitment site, involving a cluster of residues (LYS 83, LYS 85, ARG 43, GLY 1) that provide both electrostatic and polar stability.

[0515] 3. Groove Packing: The highly negative VDW score indicates that the protein does not just sit on the DNA but intercalates its side chains into the DNA grooves, particularly near Chain E and Chain B.Summary of Findings

[0516] The P2H45G-3 project demonstrates a binding mode characterized by tight physical packing and ultra-short polar contacts . While the total score is - 101.842, the individual interaction distances (notably the 2.00 A bond at SER 159) and the favorable VDW contribution (-27.878) suggest a highly specific and geometrically optimized complex with restricted conformational mobility.E. Interaction Analysis: Nucleolin & P2B415G-4 G-Quadruplex

[0517] Detailed herein is the structural analysis of Nucleolin docked with the P2B415G-4 G-quadruplex. The primary docking conformation (Conf 2888) yielded a total binding score of -102.699. The binding profile is dominated by strong electrostatic contributions (Ele: -104.010) and a highly refined hydrogen bond network involving extremely short distances (down to 2.19 A). The interaction is characterized by extensive recruitment of Chain E and Chain C, suggesting a specific longitudinal binding mode.Table 18. P2B415G-4 / Nucleolin docking performanceKey Finding: Conf 2888 shows a significant improvement in the electrostatic (Ele) score compared to lower-ranked poses, marking it as the most energetically favorable configuration for ionic interactions.

[0518] Atomic-Level Interaction Mapping for p2b415gG-quadruplex / Nucleolin is seen at FIG. 50.Table 19. P2B415G-4G-Quadruplex / Nucleolin Salt Bridges

[0519] The hydrogen bonding in this project is exceptionally high-quality, featuring a very short 2.19 A bond at the C-terminus and multiple backbone-sidechain interactions.Table 20. P2B415G-4G -Quadruples / Nucleolin Hydrogen Bond NetworkHydrophobic Interactions

[0520] MET 46 (Chain A) - DG 5 (Chain C): Distance of 3.53 A. This represents atight non-polar contact that stabilizes the protein-DNA interface near the center of the complex.

[0521] Multi-Chain Binding Architecture / The binding topology for P2B415G-4 reveals a specific focus on Chain E:

[0522] 1. Chain E Dominance: This chain serves as the primary docking site, hosting the most salt bridges (ARG 158, LYS 85) and the strongest hydrogen bonds (SER 159, ASN 100).

[0523] 2. C-Terminal Activity: The C-terminal segment (residues 158-161) is highly involved in polar and electrostatic recognition, specifically targeting the DG4 and DG5 positions.

[0524] 3. Electrostatic Continuity: The complex is stabilized by ARG 43 and ARG 48, which span across Chains B, C, and D, effectively acting as molecular "clamps" that hold the G-quadruplex structure against the protein surface.Summary of Findings

[0525] The P2B415G-4 project is defined by a high-affinity electrostatic interface. The total score of -102.699 is underpinned by an exceptionally strong Ele component (-104.010). The presence of multiple salt bridges with distances under 4.0 A, combined with a 2.19 A hydrogen bond at SER 159, indicates a rigid and stable binding event primarily localized around Chain E of the G-quadruplex.F. Interaction Analysis: Nucleolin & P2B675G5 G-Quadruplex

[0526] Detailed herein is the structural analysis of Nucleolin docked with the P2B675G5 G-quadruplex. The primary docking conformation (Conf 666) achieved a total binding score of -104.236. The interaction is characterized by an exceptionally strong electrostatic profile (Ele: -107.392) and high-affinity contact points on Chains C, D, and E. Notably, the binding involves ultra-short distance interactions, including a salt bridge at 2.63 A and a hydrogen bond at 1.90 A, indicating a very high degree of surface complementarity.

[0527] The docking results for P2B675G5 show a highly competitive top-tier set of conformations, with the rank 1 pose showing the strongest electrostatic attraction.Table 21. P2B675G5 / Nucleolin docking performance

[0528] Key Finding: The total score is heavily weighted by the electrostatic component, which compensates for a relatively high VDW (repulsion) score, suggesting a binding mode dominated by phosphate backbone recognition rather than deep pocket burial.

[0529] Atomic-Level Interaction Mapping of Nucleolin / P2B675G5-G Quadruplex is seen at FIG. 51. The complex is stabilized by several high-intensity salt bridges, with LYS 85 providing a particularly tight anchor.Table 22. P2B675G5-G-Quadruplex / Nucleolin Salt Bridges

[0530] The hydrogen bonding profile features one of the shortest recorded donoracceptor distances (1.90 A) at MET 46, signaling a highly specific interactionTable 23. P2B675G5-G-Quadruplex / Nucleolin Hydrogen Bond NetworkHydrophobic Interactions

[0531] MET 46 (Chain A) - DC 15 (Chain E): Distance of 2.64 A. This is a remarkably close hydrophobic contact involving the methionine side chain and the cytosine base / sugar, reinforcing the tight binding seen in the hydrogen bond data for the same residue.Binding Topography and Site Specificity

[0532] The interaction data for P2B675G5 highlights a distinct binding strategy:1. Chain E and C Focus: The protein residues 43-48 (N-terminal region) and 158-161 (C-terminal region) converge on Chains E and C, creating a sandwich-like interface.2. LYS 85 Interaction Hub: LYS 85 acts as a bridge between Chain D and Chain C, utilizing its flexible side chain to secure the phosphate backbones of DT9 and DG7 simultaneously.3. High-Density Contact Zone: Residue DC15 in Chain E is a "hotspot," receiving multiple hydrogen bonds from ARG 43, MET 46, and THR 47, as well as a hydrophobic contact from MET 46.Summary of Findings

[0533] The P2B675G5 project demonstrates a highly compressed binding interface. With a total score of -104.236, the complex relies on very short-range contacts, specifically the 2.63 A salt bridge (LYS 85) and the 2.84 A hydrogen bond (MET 46). The extensive involvement of MET 46 in both polar and non-polar capacities suggests it is the primary hinge point for the Nucleolin-P2B675G5 interaction.G. Interaction Analysis: Nucleolin & D224G1 G-Quadruplex

[0534] Provided herein is a detailed structural analysis of the docking simulation between Nucleolin (protein) and the D224G1 G-quadruplex (DNA sequence). Based on pyDockDNA scoring and PLIP interaction profiling, the Rank 1 configuration (Conformer 8891) demonstrates a high-affinity bindinginterface primarily driven by electrostatic interactions (Salt Bridges) and hydrogen bonding, particularly involving Arginine and Lysine residues. The docking simulation produced several high-scoring poses. Conformer 8891 was identified as the top-ranked pose based on the lowest total binding energy.Table 24. Nucleolin & D224G1G-Quadruplex docking performance

[0535] Key Finding: The binding energy is overwhelmingly dominated by Electrostatic components

[0536] (-105.566). This is consistent with the interaction between the positively charged RNA Binding Domains (RBDs) or the RGG domain of Nucleolin and the negatively charged phosphate backbone of the G-quadruplex. Atomic Level interaction mapping can be seen in FIG. 52.Nucleolin / D224G1G-Quadruplex Salt Bridges

[0537] Nucleolin utilizes multiple Lysine and Arginine residues to anchor itself to the DNA phosphate backbone. This suggests a "clamping" mechanism common in G-quadruplex binding proteins.

[0538] LYS 199: High-occupancy interaction with multiple phosphate oxygen atoms (Indices 1533, 1552, etc.).

[0539] LYS 183 & 83: Engage in extensive salt bridges with phosphate groups, stabilizing the

[0540] complex through long-range electrostatics.

[0541] ARG 143 & 148: Provide directional stability to the phosphate groups near the loops or the G-tetrad edges.

[0542] ARG 158: Significant interaction (5.44 A) with the phosphate backbone, likely helping orient the DNA within the binding pocket.Table 25. Si\ic\eo\ nlD224GlG-Qiiadruplex Hydrogen Bond NetworkHydrophobic Interactions

[0543] MET 146: Involved in a hydrophobic contact (2.76 A). While electrostatics dominate, these hydrophobic patches likely assist in the desolvation of the interface, contributing to the specificity of the binding site.

[0544] Structural Implications for D224G1

[0545] The D224G1 sequence forms a G-quadruplex structure. The PLIP data suggests:1. Backbone Wrapping: The abundance of salt bridges with Lysine (199, 183, 85, 83) indicates that Nucleolin effectively "wraps" around the anionic phosphate exterior of the G-quadruplex.2. Loop Recognition: Interactions from GLN 151 and MET 146 likely target the external loops or the terminal G-tetrads, which are the most accessible parts of the G- quadruplex for protein binding.3. Charge Neutralization: The high electrostatic score in pyDockDNA indicates that Nucleolin neutralizes the high negative charge density of the G4 structure, which is a prerequisite for stable nucleoprotein complex formation.Conclusion

[0546] The Rank 1 pose (8891) represents a stable, electrostatically-driven complex. The interaction is characterized by high-density salt bridges involving Lysine / Arginine residues and a supporting H-bond network from residues like Gin and Thr.H. Interaction Analysis: Nucleolin & P2V45-4G2 G-Quadruplex

[0547] Provided herein is an analysis of the binding characteristics of Nucleolin with the P2V45-4G2 G-quadruplex. The docking simulation (Conf 5551) yielded atop rank with a total score of -105.894. The interaction is remarkably distinct due to a highly optimized hydrogen bonding network at the protein's N-terminus (GLY 1) and a significant electrostatic contribution (Ele: -106.620). The binding mode is characterized by high surface complementarity across multiple DNA chains (B, C, D, and E).Table 26. Nucleolin & P2V45-4G2 G-Quadruplex docking performance

[0548] Key Finding: The VDW score is relatively low (7.264) compared to other complexes, suggesting a "cleaner" fit with fewer steric clashes at the interface. Atomic Level interaction mapping can be seen in FIG. 53.Table 27. Nucleolin & P2V45-4G2 G-Quadruplex Hydrogen Bond Network

[0549] The complex is stabilized by a wide-reaching electrostatic network involving five distinct protein residues.Table 28. Nucleolin & P2V45-4G2 G-Quadruplex Salt BridgesHydrophobic Interactions

[0550] MET 46 (Chain A) - DG 10 (Chain D): Distance of 2.66 A. This extremely close contact reinforces the protein's grip on the Chain D groove, complementing the hydrogen bonds formed by the same residue.Binding Topography and Site Specificity

[0551] The interaction profile for P2V45-4G2 reveals a "multi-pronged" binding strategy:1. The N-Terminal Hook: GLY 1 forms a dual-bond interaction with DG6 on Chain E at a distance of only 1.98 A. This suggests the N-terminus of the protein "hooks" into the G-quadruplex loop / groove with very high affinity.2. Central Electrostatic Hub: LYS 83 and LYS 85 act as central coordination points, managing contacts across Chains B, C, and E simultaneously.3. Groove Recognition: The combination of MET 46 (Hydrophobic / H-bond) and ARG 48 (Salt Bridge) on Chain D suggests a specific recognition of the DT11-DG10 sequence.Summary of Findings

[0552] The P2V45-4G2 complex stands out for its exceptional N-terminal affinity. The 1.98 A hydrogen bond at GLY 1 is among the strongest recorded, providing a foundational anchor that allows the rest of the protein (specifically residues 43-48 and 83-85) to distribute electrostatic contacts across four different DNA chains. This results in a highly stable complex with a low steric clash profile (VDW 7.264).I. Interaction Analysis: Nucleolin & PRO2H14G3 Analysis

[0553] Analyzed herein is the interaction between Nucleolin and PRO2H14G3 represents a high-affinity binding event (Conf 282) with a total score of - 108.051. This complex is driven by even stronger electrostatic forces (Ele: -109.540) than P2V45-4G2. The interface is characterized by a dense network of interactions involving Chain B and Chain C, with a particularly significant contribution from the flexible N-terminal region and the LYS 83 / 85 cluster.Table 29. Nucleolin & PRO2H14G3G-Quadruplex docking performance

[0554] Key Finding: While the electrostatic attraction is stronger here, the VDW score (14.894) is higher than in P2V45-4G2, indicating a more "crowded" or tightly packed interface that may involve more significant conformational adjustments. Atomic Level Interaction Mapping is seen in FIG. 54.Table 30. Nucleolin & PRO2H14G3G-Quadruplex Hydrogen Bond NetworkTable 31. Nucleolin & PRO2H14G3G-Quadruplex Salt BridgesHydrophobic Interactions

[0555] MET 46 (Chain A) - DC 10 (Chain B): Distance of 2.47 A. This represents a highlyintimate hydrophobic contact that complements the MET 46 hydrogen bond, providing adual-mode of stabilization for the protein-DNA interface on Chain B.Binding Topography and Site Specificity1. N-Terminal Dominance: Similar to P2V45-4G2, the GLY 1 residue provides a powerful initial anchor, though here it targets Chain C (2.10 A) instead of Chain E.2. LYS Cluster Efficiency: LYS 83 and LYS 85 form a concentrated binding patch that manages three separate DNA chains (C, D, and E), acting as a "molecular staple." 3. Chain B Specialization: MET 46 and THR 47 specifically target the DC10 / DT11 region of Chain B through a combination of hydrophobic and polar interactions. Summary of Findings

[0556] The PRO2H14G3 complex is characterized by superior electrostatic attraction and a highly specific interaction at the MET 46 residue. The combination of an ultra-close hydrophobic contact (2.47 A) and multiple salt bridges below 4.0 A (LYS 83 and LYS 85) creates a robust and highly stable binding architecture.J. Interaction Analysis: Nucleolin & P2B83G1 G-Quadruplex

[0557] Provided herein is a docking simulation between Nucleolin and the P2B83G1 G-quadruplex revealinf a highly stable, electrostatically driven complex. Confirmation 2360 achieved the top rank with a Total Score of-105.315. The binding is characterized by exceptional surface complementarity (VDW: 5.051) and a multi-chain "clamping" mechanism where the protein RRM domains bridge across four DNA chains (B, C, D, and E). A defining feature of this interaction is the ultra-short salt bridge atLYS 85 (3.04A), suggesting a localized area of very high binding affinity.Table 32. Nucleolin & P2B83G1 G-Quadruplex docking performanceEnergetic Breakdown:

[0558] Electrostatic Dominance: With a score of -105.820, electrostatics contribute nearly 100% of the stabilization energy, compensating for the desolvation penalty.

[0559] Precision Packing: The VDW score of 5.051 is remarkably low for a protein-DNA interface of this size, suggesting that the protein fits into the G4 grooves with high geometric precision and minimal steric strain. Atomic Level Interaction Mapping can be seen in FIG. 55. The N-Terminal Anchor ( GLY 1 )

[0560] The N-terminal GLY 1 residue acts as a structural "velcro," utilizing multiple hydrogen bonds to secure the protein to Chain E.Primary H-Bond: donor N3 to DT10 (02) at 2.48 A (Angle: 163.35°).Secondary H-Bond: acceptor N3 from DT10 (03) at 3.70 A.Implication: This dual-mode interaction locks the flexible N-terminus, providing a stable starting point for the RRM domain to wrap around the G4 structure.The ARG 43 / MET46 / ARG 48 Cluster (Chain C & D)

[0561] This cluster forms the core of the binding interface, managing two different DNA chains simultaneously.ARG 43 (Bifunctional): Sidechain H-Bond: To Chain D (DT14) at 3.39 A.Salt Bridge: To the Phosphate group of Chain C (DC 12) at 3.60 A.MET 46 (Hydrophobic / Polar Dualism):Hydrophobic Contact: A very tight 2.70 A contact with the carbon atoms of DT14 (Chain D).Backbone H-Bond: To DT14 (03) at 3.50 A.ARG 48: Provides a secondary electrostatic anchor to Chain D (DC15) at 4.29 A. The LYS 83 / 85 Anchor ( Chain E & B)

[0562] The Lysine residues provide the strongest electrostatic attraction, focusing on the phosphate backbone. LYS 85 (Primary Anchor): Forms an exceptionally strong salt bridge with Chain E (DA8) at 3.04 A. This is the shortest and likely most stable ionic interaction in the complex. LYS 83: Supports Chain E by anchoring to DA9 at 4.42 A. LYS 85 (Distal): Extends its influence to Chain B (DC6) at 5.08 A, helping to close the "clamp" around the quadruplex.Table 33. Chain Specific Contribution Summary of Nucleolin and the P2B83G1 G- quadruplex" "" "Structural Topology & Conclusion

[0563] The binding of Nucleolin to P2B83G1 is non-symmetrical and highly optimized for specific regions of the G-quadruplex.

[0564] Groove Snugness: The low VDW energy indicates that the protein RRM domains are not merely sitting on top of the G4 but are likely threading or snugly fitting into the loops / grooves.

[0565] Electrostatic Steering: The high density of ARG and LYS interactions suggests that Nucleolin is "steered" into position by the negative charge of the DNA backbone before being locked in place by shorter-range H-bonds and hydrophobic contacts.

[0566] Stability: The presence of interactions below 3.0 A (GLY 1 and LYS 85) suggests that once the complex forms, it is highly resistant to thermal fluctuations.

[0567] Final Verdict: P2B83G1 represents a high-precision binding event where Nucleolin utilizes its flexible N-terminus and a specific ARG / LYS cluster to achieve a multivalency "clamping" effect across the G4 structure.K. Interaction Analysis: Nucleolin & P2B153G2 G-Quadruplex

[0568] Detailed herein is the he binding profile of Nucleolin with the P2B153G2 G-quadruplex. The docking simulation identified Conformation 979 as the top-ranked pose with a Total Score of -115.079. This complex exhibits higher binding energy compared to previous models, driven by an extensive hydrogen bonding network (notably involving the C and E chains) and significant electrostatic stabilization (Ele: -116.925).Table 34. Nucleolin & P2B153G2G-Quadruplex docking performance

[0569] Analysis: The high VDW score in Rank 1 (18.460) relative to later ranks suggests a binding mode that prioritizes electrostatic "clamping" over deep groove penetration, yet maintains a very high total affinity.Table 35. Nucleolin & P2B153G2 G-Quadruplex Hydrogen Bond NetworkTable 36. Nucleolin & P2B153G2G-Quadruplex Salt BridgesHydrophobic Interactions

[0570] MET 46 (Chain A) - DA 9 (Chain D): Distance of 3.64 A.

[0571] MET 46 (Chain A) - DC 11 (Chain B): Distance of 3.75 A.

[0572] Significance: MET 46 acts as a hydrophobic bridge between the B and D chains, contributing to the "clamping" stability.Table 37. Chain Specific Contribution Summary of Nucleolin and the P2B153G2G- QuadruplexStructural Topology & Conclusion

[0573] The Nucleolin-P2B153G2 complex is one of the most energetically favorable G4 interactions analyzed.1. The "Super- Anchor": The residues 156-159 (ASP-GLY-ARG-SER) form a continuous high-affinity patch that interacts with Chain E and C.2. N-Terminal Locking: The GLY 1 interaction with Chain E ensures the protein's flexible start is rigidly positioned.3. Electrostatic Dominance: The very high Ele score (-116.925) suggests that P2B153G2 offers a highly optimized charged surface for Nucleolin' s RRM domains. Final Verdict: This configuration suggests a robust and highly specific binding mode, characterized by short-range H-bonds ( 1.95 A) that likely result in a very low dissociation rate. L. Interaction Analysis: Nucleolin & P2B633G3G-Quadruplex

[0574] Detailed herein is the docking of Nucleolin with the P2B633G3 G-quadruplex. The primary binding mode (Rank 1) achieved a total score of -96.745. While the electrostatic energy is slightly lower than the P2B153G2 complex, the P2B633G3 complex shows a veryfavorable Van der Waals (VDW) contribution (-16.485), indicating a tighter physical fit between the protein and the DNA grooves.Table 38. Nucleolin & P2B633G3G -Quadruples docking performance

[0575] Key Insight: The negative VDW score in the top poses is a strong indicator of high shape complementarity, typically associated with deep groove binding or stable stacking interactions. Atomic Level Interaction Mapping can be seen at FIG. 57.Table 39. Nucleolin & P2B633G3G G-Quadruplex Hydrogen Bond NetworkTable 40: Nucleolin & P2B633G3G G-Quadruplex Salt BridgesHydrophobic Interactions• THR 2 (Chain A) - DG 7 (Chain D): Distance of 3.39 A.• Significance: This interaction at the N-terminus helps orient the protein relative to the D-chain loop of the G-quadruplex.• Chain-Specific Binding Profile• Chain B: Acts as the primary anchor for the N-terminus (GLY 1, THR 2) and the C- terminal RRM patch (SER 159, ARG 158).• Chain C: Heavily stabilized by Lysine clusters (LYS 83, LYS 99) and MET 46.• Chain D: Notable for the ARG 43 salt bridge and multiple Lysine interactions (83, 85).• Chain E: Specifically recognized by the C-terminal TYR 163 and GLN 151.ConclusionThe Nucleolin-P2B633G3 complex demonstrates a "multi-pronged" binding strategy: 1. Shape Complementarity: The low VDW scores suggest the protein fits snugly into the quadruplex architecture.2. Lysine / Arginine Clusters: A dense network of basic residues (43, 83, 85, 99, 158) neutralizes the DNA phosphate backbone across three different chains (B, C, and D).3. Terminal Stability: The interaction between the C-terminal TYR 163 and Chain E (2.88 A) provides a strong directional constraint on the protein's orientation.Final Verdict: P2B633G3 shows excellent structural fit. While the total energy is slightly less than P2B153G2, the negative VDW values suggest a more physically "locked" complex. M. Interaction Analysis: Nucleolin & P2B793G4 (G-Quadruplex)

[0576] Detailed herein is the the binding of Nucleolin to the P2B793G4 G-quadruplex. The docking results reveal a highly stable complex (Rank 1 score: -101.038) primarily driven by intense electrostatic attractions (-101.301). The interface is characterized by exceptionally short hydrogen bonds and a dense network of salt bridges, suggesting a high-affinity interaction.Table 41. Nucleolin & P2B793G4 -Quadruplex docking performance

[0577] Key Finding: The binding is overwhelmingly governed by electrostatics. While the Van der Waals (VDW) component is slightly positive in the top pose, the massiveelectrostatic pull compensates, resulting in a total score exceeding -100. Atomic Level interaction Mapping can be seen in FIG. 58.Table 42. Nucleolin & P2B793G4 G-Quadruplex Hydrogen Bond NetworkTable 43: Nucleolin & P2B793G4 G-Quadruplex Salt BridgesHydrophobic & Auxiliary Interactions

[0578] THR 2 - DG 7 (Chain E): Hydrophobic contact at 3.58 A. This interaction assists in the spatial positioning of the N-terminal tail within the loop of Chain E Chain-Specific Binding Contributions• Chain B: Anchored by ARG 43 and LYS 99; contains the MET 46 hydrogen bond.• Chain C: The "Affinity Hotspot." This chain hosts the strongest hydrogen bond (SER 159, 1.99 A) and the tightest salt bridges (LYS 85 and ARG 158).• Chain D: Interacts with the mid-domain (ASN 100) and C-terminus (SER 161, TYR 163, • GLN 151).• Chain E: Recognized by the N-terminal residues (GLY 1, THR 2) and basic clusters (ARG 43, LYS 83 / 85).Structural Conclusion

[0579] The docking of Nucleolin to P2B793G4 presents a highly robust interaction model. The primary driving force is the electrostatic complementarity between the protein's basic surfaces and the DNA's polyanionic backbone. The structural stability is further reinforced by the SER 159 - DG3 hydrogen bond, which, at 1.99 A, likely represents a major specificity determinant for this quadruplexvariant. The widespread interaction across chains B through E suggests that Nucleolin effectively “wraps" or "clamps" around the quadruplex structure rather than binding to a single face.N. Interaction Analysis: Nucleolin & F2B223G5 ( G-Quadruplex)

[0580] Detailed herein is the analysis of the binding of Nucleolin to the F2B223G5 G-quadruplex. The docking results demonstrate a highly stable interaction with a Rank 1 total score of -98.569. The binding is characterized by intense electrostatic complementarity (-100.706) and a remarkably dense network of salt bridges and ultra-short hydrogen bonds, particularly involving the N-terminal residues and lysine clusters.Table 44. Nucleolin & F2B223G5 -Quadruplex docking performance

[0581] Key Finding: Similar to other G-quadruplex complexes, the F2B223G5 interaction is primarily driven by electrostatics. Interestingly, Rank 2 shows much higher VDW complementarity, but Rank 1 is preferred due to superior electrostatic and desolvation energies. Atomic Level Interaction Mapping can be seen in FIG. 59.Table 45. Nucleolin & F2B223G5 G-Quadruplex Hydrogen Bond NetworkTable 46: Nucleolin & F2B223G5 G-Quadruplex Salt BridgesHydrophobic Interactions

[0582] MET 46 - DG 14 (Chain E): A very tight hydrophobic contact at 2.01 A. This is an unusually close interaction that suggests a deep insertion of the Methionine side chain into a hydrophobic pocket or groove of the G-quadruplex.Chain-Specific Binding Contributions• Chain B: Focused on the ARG 43 salt bridge, providing an initial electrostatic anchor.• Chain C: Highly significant for specificity, containing the strongest hydrogen bond (THR47) and the ARG 158 salt bridge.• Chain D: Acts as a major landing site for the N-terminal anchor (GLY 1) and the high- affinity LYS 85 salt bridge (2.78 A).• Chain E: Dominated by the MET 46 interaction and ARG 48 salt bridge, contributing significantly to the VDW and hydrophobic stability.

[0583] Structural Conclusion

[0584] The binding of Nucleolin to F2B223G5 represents a potent molecular recognition event. While electrostatics remain the dominant force, the presence of an ultra-tight hydrophobic contact (MET 46 at 2.01 A) and an exceptionally short hydrogen bond (THR 47 at 2.10 A) distinguishes this complex. The interaction is multi -valent, spanning chains B through E, which effectively stabilizes the G-quadruplex architecture against unfolding. Conclusion:

[0585] Without being bound by any particular theory, based on the comparative docking analysis, the G2 topology exhibits the thermodynamically beneficial binding properties for Nucleolin, with the P2B153G2 complex achieving the highest global binding affinity (-115.079 kcal / mol) among all tested variants. While the interaction across all groups (G1-G5) appears to be universally driven by a dominant electrostatic "clamping" mechanism — where Lysine and Arginine residues neutralize the anionic phosphate backbone — the G2 and G1 series frequently demonstrate superior stability compared to the more variable G3, G4, and G5 groups. The G1series exhibits the most consistent "wrapping" binding mode (averaging —106 kcal / mol), but the G2 variants appear to offer the most accessible geometry for Nucleolin’s RNA-binding domains, facilitating a "super-anchor" effect via the N-terminal Glycine hook and Methionine hinge that maximizes both electrostatic saturation and surface complementarity.ADVANTAGES OF THE PRESENT INVENTION

[0586] The ASOs disclosed herein provide several advantages, particularly in enhanced stability of G-aggregates formed by the G-rich sequence offer superior resistance to enzymatic degradation, significantly increasing the ASO’s half-life and maintaining its therapeutic effect for longer periods.

[0587] The ASOs disclosed herein provide prolonged shelf-life, reducing the need for frequent administration and improving patient compliance.

[0588] The ASOs disclosed herein provide improved cellular uptake wherein high charge density from G-aggregate formation allows for spontaneous cellular uptake, eliminating the need for additional carriers or delivery agents, which can introduce toxicity or immunogenicity concerns.

[0589] The ASOs disclosed herein provide enhanced ability to interact with scavenger receptors (e.g., nucleolin), improving internalization and delivery into target cells.

[0590] The present invention provides a sequence (SEQ ID NO: 82a) that provides targeted Gene Regulation displaying specific downregulation of PINK 1 -AS 1, upregulating PINK1 expression and restoring mitochondrial health, particularly useful in treating neurodegenerative diseases like Parkinson’s and Alzheimer’s.

[0591] The ASOs disclosed herein provide a versatile RNA targeting, enabling modulation of mRNA, IncRNA, and miRNA, allowing the ASO to target multiple diseaserelevant genes simultaneously.

[0592] The ASOs disclosed herein provide organ-specific delivery wherein the ASO can be conjugated with targeting molecules like GALNAC for liver-specific delivery, improving tissue targeting and reducing off-target effects. Further, Selective delivery to specific organs or tissues (e.g., central nervous system, liver) maximizes therapeutic efficacy and minimizes systemic exposure.

[0593] The ASOs disclosed herein provide reduced immune activation wherein the G-aggregate structure minimizes the activation of Toll-like receptors (TLRs), reducing the risk of inflammation and immune-related side effects, which is common with traditional ASO therapies.

[0594] The ASOs disclosed herein reduces the need for immunosuppressive agents, and improves the overall safety profde of the therapy.

[0595] The ASOs disclosed herein provide flexibility in administration as ASO is compatible with multiple routes of administration including IV, SC, IM, intrathecal, and localized injections, offering flexibility based on disease indications and patient needs. Nevertheless, said ASO has potential for prolonged release formulations, allowing for less frequent dosing, improving patient adherence and convenience.

[0596] The ASOs disclosed herein provide improved therapeutic efficacy as it possesses ability to target multiple RNA species simultaneously, allowing for synergistic modulation of several disease pathways, particularly in complex, multifactorial diseases.

[0597] The ASOs disclosed herein provide restoration of mitochondrial function by targeting PINK 1 -AS 1 and enhance PINK1 expression, improve cellular health, and slow disease progression in neurodegenerative conditions.

[0598] The ASOs disclosed herein provide reduced toxicity by lowering the dosing requirements due to improved stability and cellular uptake, reducing the risk of toxicity typically associated with higher doses of conventional ASOs.

[0599] The ASOs disclosed herein eliminate the need for adjuvants or additional carriers, minimizing side effects related to these additives.

Claims

1. We Claim:

1. An antisense oligonucleotide comprising a nucleic acid sequence, wherein the antisense oligonucleotide is capable of forming G-aggregates, and wherein the nucleic acid sequence is characterized by Formula I:(I)or a pharmaceutically acceptable salt thereof,wherein:the G-rich motif comprises 2 to 30 naturally or non-naturally occurring nucleotides and at least two adjacent guanines or guanine derivatives;Y’ is thymine , uracil, cytosineor guanine or a derivative thereof;Variable Segment comprises 2 to 30 naturally or non-naturally occurring nucleotides; and the nucleic acid sequence is optionally conjugated to a targeting moiety.

2. The antisense oligonucleotide as claimed in claim 1, or a pharmaceutically acceptable salt thereof, wherein the nucleic acid sequence is characterized by Formula II.

3. The antisense oligonucleotide as claimed in claim 1, wherein the G-rich motif comprises at least 50% guanines or guanine derivatives.

4. The antisense oligonucleotide as claimed in claim 1, or a pharmaceutically acceptable salt thereof, wherein the G-rich motif comprises three, four, five, or six guanines or guanine derivatives.

5. The antisense oligonucleotide as claimed in claim 1, or a pharmaceutically acceptable salt thereof, wherein the G-rich motif is two to eight nucleotides in length.

6. The antisense oligonucleotide as claimed in claim 1, or a pharmaceutically acceptable salt thereof, wherein the G-rich motif is characterized by the Formula Illa:WnWGGWZ(Illa),wherein W is, independently for each occurrence, adenine or guanine, or a derivative thereof;Z is selected from thymine, uracil, guanine, and adenine, or a derivative thereof; andn is 0 or 1.

7. The antisense oligonucleotide as claimed in claim 1, or a pharmaceutically acceptable salt thereof, wherein the G-rich motif is selected from a nucleotide sequence of GGGGG, GGGGA, GGGAT, AGGGG and AGGGA.

8. The antisense oligonucleotide as claimed in claim 1, or a pharmaceutically acceptable salt thereof, wherein the Variable Segment is two to nine nucleotides in length.

9. The antisense oligonucleotide as claimed in claim 1, or a pharmaceutically acceptable salt thereof, wherein the Variable Segment is characterized by the Formula Illb:XYRAZNN(Illb),wherein:N is individually, for each occurrence, selected from adenine, thymine, uracil, guanine, and cytosine, or a derivative thereof;R is cytosine or adenine, or a derivative thereof;X is Cytosine, Thymine or a derivative thereof;Y is selected from thymine, uracil, guanine, and cytosine, or a derivative thereof; and Z is selected from thymine, uracil, guanine, and adenine, or a derivative thereof.

10. The antisense oligonucleotide of any one of claims 1-6, 8, or 9, or a pharmaceutically acceptable salt thereof, wherein the nucleic acid sequence is characterized by Formula IIIc:(IIIc),wherein N is individually, for each occurrence, selected from adenine, thymine, uracil, guanine, and cytosine, or a derivative thereof;R is cytosine or adenine, or a derivative thereof;W is independently, for each occurrence, adenine or guanine, or a derivative thereof;X is Cytosine, Thymine or a derivative thereof;Y is selected from thymine, guanine, cytosine, or a derivative thereof;Z is independently, for each occurrence, selected from thymine, guanine, and adenine, or a derivative thereof; andn is 0 or 1.

11. The antisense oligonucleotide as claimed in claim 1„ or a pharmaceutically acceptable salt thereof, wherein the antisense oligomer is selected from single-stranded DNA, RNA, peptide nucleic acid (PNA), phosphorodiamidate morpholino oligomer (PMO), and combinations thereof.

12. The antisense oligonucleotide as claimed in claim 1, or a pharmaceutically acceptable salt thereof, wherein the antisense oligonucleotide comprises a modification selected from locked nucleic acid (LNA), ethylene-bridged nucleic acid (ENA), phosphorothioate backbone, tricyclo-DNA, tricyclo-phosphorothioate, 2’-O-methyl (2’-OMe), 2’-O-methoxyethyl (2'-M0E), 2’-fluoro (2'-F), 2’-O-methyl phosphorothioate, and 2’-O-methoxyethyl phosphorothioate, and combinations thereof.

13. The antisense oligonucleotide as claimed in claim 1, or a pharmaceutically acceptable salt thereof, wherein the nucleic acid sequence is conjugated to L, wherein L is an optional linking moiety.

14. The antisense oligonucleotide of claim 13, or a pharmaceutically acceptable salt thereof, wherein the linking moiety is a bond, a polyethylene glycol-based ligand, or an optionally functionalized alkyl-based ligand.

15. The antisense oligonucleotide as claimed in claim 1, or a pharmaceutically acceptable salt thereof, wherein the targeting moiety is selected from lipid nanoparticles, small molecule ligands that bind to specific receptors or transporters, sugar-based ligands, lipid-based moieties, peptide-based ligands, protein-based ligands, and polymer-based moieties.

16. The antisense oligonucleotide as claimed in claim 1, or a pharmaceutically acceptable salt thereof, wherein the targeting moiety is selected from GALNAC, tocopherol, cholesterol, docosahexaenoic acid (DHA), mannose, transferrin, RGD peptide, cell-penetrating peptides, vitamin B derivatives, phospholipids, monoclonal antibodies, PEG (polyethylene glycol), and progesterone..

17. The antisense oligonucleotide of claim 16, or a pharmaceutically acceptable salt thereof, wherein the vitamin B derivatives are biotin, folic acid, and riboflavin.

18. An antisense oligonucleotide comprising a nucleic acid sequence, wherein the antisense oligonucleotide is capable of forming G-aggregates, wherein the 5 ’-terminus or 3’-terminus of the nucleic acid sequence comprises a G-rich motif, and wherein the antisense oligonucleotide is a single-stranded DNA or RNA of Formula IV:or a pharmaceutically acceptable salt thereof,wherein: G' is selected from -OH, and the G-rich motif of formula IVwherein A’ is selected from -OH,y is 2-20;each R1 is independently, for each occurrence, selected from OH and -NRaRb, wherein each Ra and Rb is independently, for each occurrence, H or -Cl -6 alkyl;each R2 is independently, for each occurrence, selected from a naturally or non-naturally occurring nucleobase and the sequence formed by the combination of each R2 from 5’ to 3’ is a segment of a nucleic acid sequence;each R3 is independently, for each occurrence, selected from H, halogen, -C(O)Cl-6 alkyl, -O-Cl-6 alkyl, -O-Cl-6 alkyl-O-Cl-6 alkyl, and -O-Cl-6 alkyl-C(O)NRaRb;z is 8-40;E is selected from H, -Cl-6 alkyl, -C(O)Cl-6 alkyl, and the G-rich motif of the formulawherein:R11is OH or -NRaRb;L is an optional linking moiety;E’ is selected from H, -Ci-6 alkyl, -C(O)Ci-6 alkyl;R4is independently, for each occurrence, selected from a naturally or non-naturally occurring nucleobase and the sequence formed by the combination of each R4from 5’ to 3’ is a segment of a nucleic acid sequence, wherein the segment comprises at least two adjacent guanines or guanine derivatives;provided that Formula IV comprises a G-rich motif at G’ or E.

19. The antisense oligonucleotide as claimed in claim 18, or a pharmaceutically acceptable salt thereof, wherein G’ is the G-rich motif of formula IV.

20. The antisense oligonucleotide as claimed in claim 18, or a pharmaceutically acceptable salt thereof, wherein E is the G-rich motif of formula IV.

21. The antisense oligonucleotide as claimed in claim 18, or a pharmaceutically acceptable salt thereof, whereinG’ is the G-rich motif of formula IVE is selected from H, -C(O)CH3,The antisense oligonucleotide as claimed in claim 18, or a pharmaceutically acceptable salt thereof, whereinG' is selected from -OH,E is the G-rich motif of the formula IV,wherein E’ is H or -C(O)CH3.

23. The antisense oligonucleotide as claimed in claim 18, or pharmaceutically acceptable salt thereof, wherein each R1is OH.

24. The antisense oligonucleotide as claimed in claim 18„ or pharmaceutically acceptable salt thereof, wherein each R2is a nucleobase, independently at each occurrence, selected from adenine, guanine, cytosine, 5-methyl-cytosine, and thymine, and uracil.

25. The antisense oligonucleotide as claimed in claim 18„ or a pharmaceutically acceptable salt thereof, wherein each R3is H.

26. The antisense oligonucleotide as claimed in claim 18, wherein the G-rich motif comprises at least 50% guanines or guanine derivatives.

27. The antisense oligonucleotide as claimed in claim 18, or a pharmaceutically acceptable salt thereof, wherein the G-rich motif comprises three, four, five, or six guanines or guanine derivatives.

28. The antisense oligonucleotide as claimed in claim 18, or a pharmaceutically acceptable salt thereof, wherein the G-rich motif is selected from a nucleotide sequence of GGGGG, GGGGA, GGGAT, AGGGG , and AGGGA.

29. The antisense oligonucleotide as claimed in claim 18, or a pharmaceutically acceptable salt thereof, wherein the antisense oligonucleotide comprises an internal nucleotide sequence of TGTAG.

30. The antisense oligonucleotide as claimed in claim 18, or a pharmaceutically acceptable salt thereof, wherein linking moiety is a bond, a polyethylene glycol-based ligand, or an optionally functionalized alkyl-based ligand.

31. The antisense oligonucleotide as claimed in claim 18, or a pharmaceutically acceptable salt thereof, wherein the targeting moiety is selected from lipid nanoparticles, small molecule ligands that bind to specific receptors or transporters, sugar-based ligands, lipid-based moieties, peptide-based ligands, protein-based ligands, and polymer-based moieties.

32. The antisense oligonucleotide as claimed in claim 18, or a pharmaceutically acceptable salt thereof, wherein the targeting moiety is selected from GALNAC, tocopherol, cholesterol, docosahexaenoic acid (DHA), mannose, transferrin, RGD peptide, cell-penetrating peptides, vitamin B derivatives, phospholipids, monoclonal antibodies, PEG (polyethylene glycol), and progesterone.

33. The antisense oligonucleotide as claimed in claim 18, or a pharmaceutically acceptable salt thereof, wherein the vitamin B derivatives are biotin, folic acid, and riboflavin.

34. An antisense oligonucleotide of 16 to 40 naturally or non-naturally occurring nucleotides in length, wherein the antisense oligonucleotide comprises a nucleic acid sequence, and wherein the nucleic acid sequence is selected from:wherein X is T,U or C; and the nucleic acid sequence is optionally conjugated to L, wherein L is an optional linking moiety; andwherein the nucleic acid sequence is selected from: SEQ ID NO: 87a (AGGGATGTAGCGCAGC);SEQ ID NO: 85a (GGGATTGTAGTTCAAT);SEQ ID NO: 88a (GGGGATGTAGCTCAAT)SEQ ID NO: 82a (GGGGGTGTAGCTCATA);SEQ ID NO: 84a (GGGGATGTAGCTCAGA);SEQ ID NO: 89a (GGGGATGTAGCTCAGT); andSEQ ID NO: 83a (GGGGATGTAGCTCATA).

35. The antisense oligonucleotide of any one of claims 1-33, or a pharmaceutically acceptable salt thereof, wherein the antisense oligonucleotide is capable of being delivered into the nucleus of a cell.

36. The antisense oligonucleotide of claim 34, or a pharmaceutically acceptable salt thereof, wherein the antisense oligonucleotide binds to nucleolin.

37. A pharmaceutical composition comprising the antisense oligonucleotide of any one of claims 1-36 and a pharmaceutically acceptable carrier.

38. A method of treating a disease or condition in a subject in need thereof, the method comprising administering a therapeutically effective amount of the antisense oligonucleotide of any one of claims 1-36 or the composition of claim 37 to the subject.

39. The method of claim 38, wherein the disease or condition is selected from neurodegenerative disorders, cardiovascular and musculoskeletal degenerative diseases, dermatological diseases, rare genetic disorders, Autism Spectrum Disorder, Duchenne Muscular Dystrophy, Huntington’s disease, Friedreich Ataxia, and pustular psoriasis.

40. A method of downregulating the expression of target genes in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the antisense oligonucleotide of any one of claims 1-36.

41. The method of claim 40, wherein the target gene is selected from PINK1-AS, LIMK1, FAM168B, CACNA1D, GRIK2, LIF-AS2, PINK1-AS, GPX6, STEAP4, TRPC5, HTT, TCIRG1, CHRNB2.

42. The method of claim 40 or claim 41, wherein the antisense oligonucleotide downregulates target RNA species.

43. The method of claim 42, wherein the RNA species is selected from mRNA, long non-coding RNAs (IncRNAs), and microRNAs (miRNAs).

44. A method of delivering an antisense oligonucleotide into the nucleus of a cell in a subject, comprising contacting the cell with the antisense oligonucleotide of claim 1, wherein said antisense oligonucleotide comprises a G-rich motif capable of forming G-aggregates, and wherein said antisense oligonucleotide translocates to the nucleus in the absence of a transfection reagent.

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