RNA structures and uses of the same

Abstract

Disclosed are oligonucleotide molecules comprising a four-way junction core structure. The molecules can deliver up to four different siRNAs or ASO to a targeted tissue.

Description

RNA STRUCTURES AND USES OF THE SAMESEQUENCE LISTING

[0001] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on January 7, 2026, is named 30600_WO.xml and is 616,603 bytes in size.BACKGROUND

[0002] The use of therapeutic oligonucleotides, including but not limited to small interfering RNA (siRNA) and antisense oligonucleotides (ASO), holds great promise for treating diseases and conditions that may otherwise go unaddressed. However, while the potential of oligonucleotide-based therapies is significant, numerous obstacles stand in the way of translating the technology into the clinic.

[0003] Effective delivery of therapeutic oligonucleotides has proven difficult. Providing the active therapeutic specifically to the targeted tissue and avoiding off-target delivery is a challenge. The central nervous system (CNS) presents unique challenges for siRNA delivery due to its complex structure and the presence of the blood brain barrier. This makes the efficient delivery and distribution of siRNA within the CNS particularly difficult. The treatment of neurological disorders such as Alzheimer's, Parkinson's, and Huntington's diseases through gene silencing has garnered significant interest, necessitating advancements in siRNA delivery mechanisms.

[0004] Various modifications, including the incorporation of 5'-vinylphosphonate (5'VP), replacing phosphodiester (PO) with phosphorothioate (PS) on the RNA backbone, and altering the 2'-hydroxyl (2'OH) with modifications such as 2'-fluoro (2'F) and 2'-O-methyl (2'OMe) enhance siRNA stability and potency. However, despite these advancements, clinical applications of siRNA, and in the treatment of CNS diseases in particular, are limited by delivery challenges.Traditional siRNA-lipid conjugates have demonstrated improved cellular uptake but come with the drawback of potential lipid-related toxicity. Divalent siRNA may in some instances aid in improving distribution and potency in the CNS (see, e.g., Alterman JF et al., A divalent siRNA chemical scaffold for potent and sustained modulation of gene expression throughout the central nervous system, Nat Biotechnol, 37: 884-864 (2019); Belgrad J et al., A programmable dualtargeting siRNA scaffold supports potential two-gene modulation in the central nervous system, Nucleic Acids Res, 52: 6099-6113 (2024)).

[0005] One solution that has been proposed to deliver oligonucleotide therapeutics is by use of an RNA scaffold or nanoparticle (referred to herein as RNA structures), which does not itself have a therapeutic effect, but is capable of delivering active therapeutic ingredients. RNA structures are disclosed in, for example, U.S. Patent No. 11,976,092.

[0006] As noted above, the difficulties faced with therapeutic oligonucleotide delivery are amplified in the context of those oligonucleotides destined for the central nervous system (CNS). The blood-brain barrier (BBB) is a selective, semipermeable boundary which greatly limits the ability of molecules, particularly those of larger size, to access the CNS.

[0007] Accordingly, there is a need for improved RNA-based delivery mechanisms for therapeutic oligonucleotides, and particularly those destined for the CNS for the treatment of various diseases and conditions. The present disclosure addresses this need.SUMMARY OF THE INVENTION

[0008] The present disclosure provides, in some embodiments, a RNA structure comprising, a first oligonucleotide comprising a first core nucleotide sequence comprising SEQ ID NO: 1; a second oligonucleotide comprising a second core nucleotide sequence comprising SEQ ID NO: 2; a third oligonucleotide comprising a third core nucleotide sequence comprising SEQ ID NO: 3;and a fourth oligonucleotide comprising a fourth core nucleotide sequence comprising SEQ ID NO: 4, optionally one or more nucleotides of the first, second, third, and fourth oligonucleotides are independently modified nucleotides, and optionally one or more intemucleotide linkages of the first, second, third, and fourth oligonucleotides are modified internucleotide linkages.

[0009] In some embodiments, the first oligonucleotide forms a duplex with both the second oligonucleotide and the fourth oligonucleotide, the second oligonucleotide forms a duplex with both the first oligonucleotide and the third oligonucleotide, the third oligonucleotide forms a duplex with both the second oligonucleotide and the fourth oligonucleotide, and the fourth oligonucleotide forms a duplex with both the first oligonucleotide and the third oligonucleotide.

[0010] In some embodiments, one or more nucleotides in at least one of the first, second, third, and fourth oligonucleotides are modified nucleotides. In some embodiments, each nucleotide in at least one of the first, second, third, and fourth oligonucleotide is a modified nucleotide. In some embodiments, each nucleotide in the first, second, third, and fourth oligonucleotide is a modified nucleotide. In some embodiments, the modified nucleotide is a 2'-O-methyl modified nucleotide, 2'-F modified nucleotide, 2'-O-C16 alkyl modified nucleotide, or 2'-O-MOE modified nucleotide. In some embodiments, each of the first, second, third, and fourth oligonucleotides are between 20 to 70 nucleotides in length.

[0011] In some embodiments, the first core nucleotide sequence comprises SEQ ID NO: 5; the second core nucleotide sequence comprises SEQ ID NO: 6; the third core nucleotide sequence comprises SEQ ID NO: 7; and the fourth core nucleotide sequence comprises SEQ ID NO: 8. In some embodiments, the first core nucleotide sequence comprises SEQ ID NO: 9; the second core nucleotide sequence comprises SEQ ID NO: 6; the third core nucleotide sequence comprises SEQ ID NO: 10; and the fourth core nucleotide sequence comprises SEQ ID NO: 8.

[0012] In some embodiments, one or more of the first, second, third, and fourth oligonucleotides comprise a second nucleotide sequence, optionally one or more nucleotides of the second nucleotide sequence are independently modified nucleotides, and optionally one or more intemucleotide linkages of the second nucleotide sequence are modified internucleotide linkages.

[0013] In some embodiments, one or more nucleotides in the second nucleotide sequence are modified nucleotides. In some embodiments, each nucleotide in the second nucleotide sequence is a modified nucleotide. In some embodiments, the modified nucleotide is a 2'-O-methyl modified nucleotide, 2'-F modified nucleotide, 2'-O-C16 alkyl modified nucleotide, or 2'-O-MOE modified nucleotide.

[0014] In some embodiments, the second nucleotide sequence is a sense strand of a double stranded RNA (dsRNA). In some embodiments, the sense strand of the dsRNA does not bind to any one of the first core nucleotide sequence, the second core nucleotide sequence, the third core nucleotide sequence, or the fourth core nucleotide sequence. In some embodiments, the sense strand of the dsRNA is positioned at the 5' end or 3' end of the first core nucleotide sequence, the second core nucleotide sequence, the third core nucleotide sequence, and / or the fourth core nucleotide sequence. In some embodiments, the sense strand of the dsRNA is positioned at the 5' end of the first core nucleotide sequence, the second core nucleotide sequence, the third core nucleotide sequence, and / or the fourth core nucleotide sequence. In some embodiments the sense strand of the dsRNA is positioned at the 3' end of the first core nucleotide sequence, the second core nucleotide sequence, the third core nucleotide sequence, and / or the fourth core nucleotide sequence. In some embodiments, the first, second, third, and fourth oligonucleotides comprise the same sense strand sequence. In some embodiments, the first, second, third, and fourtholigonucleotides comprise two different sense strand sequences. In some embodiments, the first, second, third, and fourth oligonucleotides comprise three different sense strand sequences. In some embodiments, the first, second, third, and fourth oligonucleotides comprise four different sense strand sequences. In some embodiments, the second nucleotide sequence comprises a sequence that is one of SEQ ID NOs: 11-18.

[0015] In some embodiments, the RNA structure further comprises an antisense oligonucleotide (ASO) attached to the second nucleotide sequence. In some embodiments, the second nucleotide sequence is an antisense oligonucleotide (ASO).

[0016] In some embodiments, the RNA structure further comprises a third nucleic acid sequence that comprises a nucleic acid sequence complementary to the sense strand of the dsRNA, and the third nucleic acid sequence forms a duplex with the sense strand of the dsRNA. In some embodiments, the third nucleic acid sequence comprises a sequence that is one of SEQ ID NOs: 19-26. In some embodiments, the third nucleic acid sequence comprises the antisense strand of an siRNA. In some embodiments, the siRNA targets HPRT, SNCA, MAPT, APP, SARM1, SOD1, ATXN2, ATXN3, APOE, BACE1, FMRI, LRRK2, HTT, SCN10A, SCN9A, CACNA1B, PRNP, ACVR2a, ACVR2b.

[0017] In some embodiments, the RNA structure comprises one or more of SEQ ID NOs: 1-26. In some embodiments, the RNA structure further comprises an antisense oligonucleotide (ASO) that is reversibly attached to the third nucleic acid sequence. In some embodiments, the ASO targets HPRT, SNCA, MAPT, APP, SARM1, SOD1, ATXN2, ATXN3, APOE, BACE1, FMRI, LRRK2, HTT, SCN10A, SCN9A, CACNA1B, PRNP, ACVR2a, ACVR2b.

[0018] The present disclosure provides, in some embodiments, a pharmaceutical composition comprising the RNA structures described herein and one or more pharmaceutically acceptable excipients.

[0019] The present disclosure provides, in some embodiments, a method of treating a disease or disorder in a patient in need thereof, comprising administering the RNA structures or the pharmaceutical composition described herein. In some embodiments, the disease or disorder is a genetic disease or disorder. In some embodiments, the disease or disorder is a central nervous system (CNS) disease or disorder. In some embodiments, the CNS disorder is Alzheimer’s disease, Parkinson’s disease, frontotemporal dementia (FTD), progressive supranuclear palsy (PSP), amyotrophic lateral sclerosis (ALS), Down’s syndrome, multiple sclerosis.

[0020] The present disclosure provides, in some embodiments, a method for delivering a molecule to the central nervous system (CNS) of a subject in need thereof, comprising administering the RNA structure or the pharmaceutical composition described herein. In some embodiments, the subject has a CNS disease or disorder.

[0021] The present disclosure provides, in some embodiments, a method for detecting the formation of an RNA nanoparticle, comprising: a) assaying the RNA nanoparticle of any one of claims 1-32 or the pharmaceutical composition of claim 33 by size-exclusion chromatography (SEC); b) separating RNA nanoparticles from one or more intermediate molecules identified in a); and c) assaying the separated RNA nanoparticles of b) using ion-pairing reverse phase (IPRP) chromatography. In some embodiments, the SEC in a) is performed using a 200A, 1.9 pm, 4.6 mm x 300 mm column. In some embodiments, the SEC column temperature is maintained at 25°C. In some embodiments, the IPRP chromatography of c) further comprises assaying the RNA nanoparticle or composition using a peak-based multiple heart-cutting sampling mode. In someembodiments, the IPRP chromatography is performed using an analytical 300A, 1.7 pm, 2.1 mm x 150 mm column. In some embodiments, the IPRP column temperature is maintained at 75°C.BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1A is a schematic of four-way junction (4WJ) RNA structure with 4 copies of SNCA siRNA. The RNA strand A is an RNA structure scaffold strand of the disclosure, which includes a core nucleotide sequence B (scaffold strand) and a second nucleotide sequence (sense strand) B’ that is attached to core nucleotide sequence B (scaffold strand). The strand C is the third nucleotide sequence (antisense strand) that is not attached to the core nucleotide sequence B. The circles D represent C 16 lipid that is located at the 6th nucleotide position on scaffold strand B.

[0023] Figure IB shows in vivo knock down of SNCA in four tissues: frontal cortex (FCX), brain stem (BS), cerebellum (CB), spinal cord (SC). Two-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001, **** P < 0.0001, all unnoted comparisons are ns, p > 0.05. One BS data point and one SC data point from the same mouse in the 4WJ-4SNCA-2C16 (SF6) group were removed from due to their abnormally high readouts, which are inconsistent with the mice from the same group and all the rest of the groups

[0024] Figure 2A is a schematic of HPRT siRNA-Toc, HPRT siRNA-Toc + SNCA siRNA-Toc, 4WJ-4HPRT-Toc, 4WJ-2HPRT-2SNCA-Toc (four- way junction (4WJ) RNA structure with two HPRT siRNA and two SNCA siRNA). B represents the core nucleotide sequences; E and E’ represent the HPRT siRNA (antisense and sense, respectively); and F and F’ represent SNCA siRNA (antisense and sense, respectively). D represents tocopherol (toe) at 3' end of nanoparticle.

[0025] Figures 2B and 2C show in vivo knock down of HPRT (Fig.2B) and SNCA (Fig.2C) in four CNS tissues; frontal cortex (FCX), brain stem (BS), cerebellum (CB), spinal cord (SC).

[0026] Figure 2D shows in vivo knock down of HPRT in two peripheral tissues: liver and kidney. Two-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001, **** P < 0.0001, all unnoted comparisons are ns, p > 0.05.

[0027] Figures 3A-3C show in vivo knock down of SNCA (Fig. 3A), MAPT (Fig. 3B) and APP (Fig.3C) in four tissues: frontal cortex (FCX), brain stem (BS). cerebellum (CB), spinal cord (SC). Two-way ANOVA, * p < 0.05, ** p < 0.01. *** p < 0.001, **** P < 0.0001, all unnoted comparisons are ns, p > 0.05.

[0028] Figures 4A-4B show the effect of lipid conjugation position in siRNA (Fig. 4A) and RNA structures that comprise an siRNA (Fig.4B) on knockdown of SNCA mRNA in CNS tissue.

[0029] Figure 4C shows the effect of lipid conjugation position on white matter retention in the brain and spinal cord.

[0030] Figure 5A shows a schematic of the 2D-LC / QTOF system configuration used in Example 4.

[0031] Figure 5B shows the results of size-exclusion chromatography (SEC) column screening for separation of fully assembled RNA structures and intermediates for lipid conjugated RNA structures and non-lipid conjugated RNA structures.

[0032] Figure 5C provides both qualitative and quantitative analysis of antisense and sense strands in assembled RNA structures using the IPRP method.

[0033] Figures 5D-5E show SEC x SEC thermodynamic stability analysis for RNA structures. (FIG. 5D). A total of 5 cuts at the main peak region were collected in the first dimension SEC chromatography (FIG. 5E).

[0034] Figure 5F provides an overview of the online SEC x IPRP method for a two-step evaluation for assembled RNA structures. ’D Native SEC analysis provided the size distributionanalysis for RNA structures and2D denatured TPRP analysis revealed single stand composition of RNA structures.

[0035] Figure 5G shows results from the online SEC x IPRP analysis of RNA structures.

[0036] Figure 5H shows the SEC-MALS analysis of the molecular weight distribution and heterogeneity in solution for RNA structures.

[0037] Figure 51 shows native SEC-MS confirmation of RNA structure assembly, with an overlay of raw data (right) and fit data (left).DETAILED DESCRIPTION OF THE INVENTION

[0038] Unless otherwise indicated, this disclosure uses the definitions provided below.

[0039] As used herein, “attached,” “attachment,” and the like refer to direct association via covalent or non-covalent association (e.g., bond) or conjugation of two or more molecules together with no intermediate molecules in-between or to the indirect attachment of two or more molecules together that is mediated via one or more intermediate molecules (e.g., linkers). Non-covalent associations include, but are not limited to, charge interactions, affinity interactions, metal coordination, physical adsorption, host-guest interactions, hydrophobic interactions, TT stacking interactions, hydrogen bonding interactions, van der Waals interactions, magnetic interactions, electrostatic interactions, dipole-dipole interactions, and / or combinations thereof. Covalent associations include, but are not limited to, bonds in which a pair of electrons is shared between one or more atoms in each molecule involved.

[0040] As used herein, the term “cargo compound” refers to any molecule, compound, or composition that can be loaded onto the RNA structures as described herein and can elicit a physiological reaction upon delivery to a subject (e.g., by release from the RNA structure or whileattached or otherwise coupled to the RNA structure). Tn some embodiments, the cargo compound may be a therapeutic nucleic acid (an antisense strand).

[0041] As used herein, “conjugated,” “coupled,” or “coupled to” have the same meaning as “attached.”

[0042] As used herein, “core nucleotide sequence” and “scaffold strand” are used interchangeably and refer to the same nucleotide sequence, such as of a portion of the RNA structure.

[0043] As used herein, “deoxyribonucleic acid (DNA)” and “ribonucleic acid (RNA)” generally refer to any ribonucleotide or deoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. RNA can be in the form of non-coding RNA such as tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), anti-sense RNA, RNAi (RNA interference construct), siRNA (short interfering RNA), microRNA (miRNA), or ribozymes, aptamers, guide RNA (gRNA), or coding mRNA (messenger RNA).

[0044] As used herein, the term “functional group” refers to structures that add a functionality to the molecules described herein.

[0045] As used herein, “identity,” “identical to,” and the like refer to the degree of sequence relatedness between two or more nucleotides or polypeptides as determined by identifying the number of matches between strings of such sequences. “Identity” can be readily calculated by known methods, including, but not limited to. those described in Computational Molecular Biology, Lesk, A. M., Ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., Ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., Eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; andSequence Analysis Primer, Gribskov, M. and Devereux, J., Eds., M Stockton Press, New York, 1991; and Carillo, EL, and Lipman, D., SIAM J. Applied Math. 1988, 48: 1073. The percent (%) identity between two sequences can be determined by using analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, Madison Wis.) that incorporates the Needelman and Wunsch, (J. Mol. Biol., 1970, 48: 443-453,) algorithm (e.g., NBLAST, and XBLAST).

[0046] As used herein, “nucleic acid sequence,” “oligonucleotide,” and “polynucleotide” refer to a string of linked nucleotides or modified nucleotides comprising natural or modified nucleobase-sugar-phosphate combinations and refer to, among others, DNA, RNA, hybrid molecules comprising DNA and RNA. “Nucleic acid sequence,” “oligonucleotide,” and “polynucleotide” also encompasses such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells. For instance, the terms include DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs comprising unusual nucleobases, such as inosine, or modified nucleobases, such as tritylated bases, are polynucleotides as the terms are used herein. “Nucleic acid sequence,” “oligonucleotide,” and “polynucleotide” also include PNAs (peptide nucleic acids), phosphorothioates, and other variants of the phosphate backbone of native nucleic acids. Natural nucleic acids have a phosphate backbone, while artificial nucleic acids may contain other types of backbones but contain the same nucleobases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are “nucleic acid sequences,” “oligonucleotides,” and “polynucleotides” as the terms are used herein. In particular, “nucleic acid sequence,” “oligonucleotide,” and “polynucleotide” also include 2'Fluoro, 2' O-methyl, LNA (locked nucleic acids), and other variants of 2' modifications of the ribose or deoxyribose (sugar) moiety of native nucleic acids.

[0047] As used herein, “self-assembly” refers to the ability of nucleic acids (and, in some instances, preformed nucleic acid nanostructures (e.g., crystals)) to anneal to each other, in a sequence-specific manner, in a predicted manner, and without external control. In some aspects, nucleic acid structure self-assembly methods include combining nucleic acids (e.g., singlestranded nucleic acids, or oligonucleotides) in a single vessel and allowing the nucleic acids to anneal to each other, based on sequence complementarity. In some aspects, this annealing process involves placing the nucleic acids at an elevated temperature and then reducing the temperature gradually in order to favor sequence-specific binding. Various nucleic acid structures or selfassembly methods are known and described herein and in U.S. Patent No. 11,976,092.Core Structure

[0048] The present disclosure provides, in some embodiments, the scaffold of RNA structures comprising a first oligonucleotide comprising a first core nucleotide sequence comprising SEQ ID NO: 1; a second oligonucleotide comprising a second core nucleotide sequence comprising SEQ ID NO: 2; a third oligonucleotide comprising a third core nucleotide sequence comprising SEQ ID NO: 3; and a fourth oligonucleotide comprising a fourth core nucleotide sequence comprising SEQ ID NO: 4 see Table 1 A). In some embodiments, the first oligonucleotide forms a duplex with both the second oligonucleotide and the fourth oligonucleotide, the second oligonucleotide forms a duplex with both the first oligonucleotide and the third oligonucleotide, the third oligonucleotide forms a duplex with both the second oligonucleotide and the fourth oligonucleotide, and the fourth oligonucleotide forms a duplex with both the first oligonucleotide and the third oligonucleotide.In some embodiments, the first, second, third, and fourth oligonucleotides form a nanostructure that has four double stranded arms.

[0049] In some embodiments, the RNA structures described herein comprise a first oligonucleotide comprising a first core nucleotide sequence comprising SEQ ID NO: 1; a second oligonucleotide comprising a second core nucleotide sequence comprising SEQ ID NO: 2; a third oligonucleotide comprising a third core nucleotide sequence comprising SEQ ID NO: 3; and a fourth oligonucleotide comprising a fourth core nucleotide sequence comprising SEQ ID NO: 4, wherein optionally one or more nucleotides of the first, second, third, and fourth core nucleotide sequences are independently modified nucleotides, and wherein optionally one or more intemucleotide linkages of the first, second, third, and fourth core nucleotide sequences are modified intemucleotide linkages.

[0050] In some embodiments, the RNA structures described herein comprise a first core nucleotide sequence comprising a first oligonucleotide comprising a first core nucleotide sequence having at least 90% identity to SEQ ID NO: 1; a second oligonucleotide comprising a second core nucleotide sequence comprising a nucleotide sequence having at least 90% identity to SEQ ID NO: 2; a third oligonucleotide comprising a third core nucleotide sequence comprising a nucleotide sequence having at least 90% identity to SEQ ID NO: 3; and a fourth oligonucleotide comprising a fourth core nucleotide sequence comprising a nucleotide sequence having at least 90% identity to SEQ ID NO: 4, wherein optionally one or more nucleotides of the first, second, third, and fourth core nucleotide sequences are independently modified nucleotides, and wherein optionally one or more intemucleotide linkages of the first, second, third, and fourth core nucleotide sequences are modified intemucleotide linkages.

[0051] In some embodiments, the RNA structures described herein comprise a first core nucleotide sequence comprising a first oligonucleotide comprising a first core nucleotide sequence having at least 95% identity to SEQ ID NO: 1; a second oligonucleotide comprising a second core nucleotide sequence comprising a nucleotide sequence having at least 95% identity to SEQ ID NO: 2; a third oligonucleotide comprising a third core nucleotide sequence comprising a nucleotide sequence having at least 95% identity to SEQ ID NO: 3; and a fourth oligonucleotide comprising a fourth core nucleotide sequence comprising a nucleotide sequence having at least 95% identity to SEQ ID NO: 4, wherein optionally one or more nucleotides of the first, second, third, and fourth core nucleotide sequences are independently modified nucleotides, and wherein optionally one or more intemucleotide linkages of the first, second, third, and fourth core nucleotide sequences are modified intemucleotide linkages.Table 1A. Unmodified Core Scaffold Nucleotide Sequences.

[0052] In some embodiments, one or more nucleotides of the first, second, third, and fourth core nucleotide sequences are modified nucleotides. In some embodiments, each nucleotide of the first, second, third, and fourth core nucleotide sequences is a modified nucleotide. In some embodiments, one or more intemucleotide linkages of the first, second, third, and fourth core nucleotide sequences are modified intemucleotide linkages. In some embodiments, each intemucleotide linkage of the first, second, third, and fourth core nucleotide sequences are modified intemucleotide linkages. In some embodiments, one or more nucleotides of the first,30600_WGsecond, third, and fourth core nucleotide sequences are modified nucleotides and one or more intemucleotide linkages of the first, second, third, and fourth core nucleotide sequences are modified intemucleotide linkages. In some embodiments, each nucleotide of the first, second, third, and fourth core nucleotide sequences is a modified nucleotide and one or more intemucleotide linkages of the first, second, third, and fourth core nucleotide sequences are modified intemucleotide linkages. In some embodiments, one or more nucleotides of the first, second, third, and fourth core nucleotide sequences are modified nucleotides and each intemucleotide linkage of the first, second, third, and fourth core nucleotide sequences is a modified intemucleotide linkage. In some embodiments, each nucleotide of the first, second, third, and fourth core nucleotide sequences is a modified nucleotide and each intemucleotide linkage of the first, second, third, and fourth core nucleotide sequences is a modified intemucleotide linkage.

[0053] In some embodiments, the modified nucleotides comprise 5'-terminal modifications, 3'-terminal modifications, 2'-intemal sugar modifications, and / or nucleobase modifications. Typical 5' terminal modifications include, but are not limited to, amino, carboxy, phosphate, thiol, maleimide, alkyne, cholesterol, aldehyde, carbon spacers, PEG-spacer, doubler, trebler, photocleavable amino, photocleavable spacer, fluorophores (e.g. Cyanine 3, 3.5, 5, 5.5, 7, Fluorescein, etc.), biotin, desthiobiotin, digoxigenin, quenchers (dabcyl, dabsyl, BlackHole, BBQ650. etc.) or other 5' modifications known to an experienced user of the art. Typical 3' terminal modifications include, but are not limited to, amino, carboxy, phosphate, thiol, alkyne, cholesterol, carbon spacers, PEG-spacer, fluorophores (e.g. Cyanine 3, 3.5, 5, 5.5, 7, Fluorescein, etc.), biotin, desthiobiotin, digoxigenin, quenchers (dabcyl, dabsyl, BlackHole, BBQ650, etc.) or other 3' modifications known to an experienced user of the art. Typical internal modifications include amino-dA, amino-dC, amino-dT, carboxy-dT, 2'0-propargyl, 2'amino, 2'fluoro,2'methoxy, 5-ethynyl-dU, C8-alkyne-dC, C8-alkyne-dT, carbon spacers, Peg-spacer, fluorophores (e.g. Cyanine 3, 3.5, 5, 5.5, 7, Fluorescein, etc.), biotin, desthiobiotin, digoxigenin, quenchers (dabcyl, dabsyl, BlackHole, BBQ650, etc.) or other 5' modifications known to an experienced user of the art. In some embodiments, the modification is an alkyne group attached to a nucleotide. In some embodiments, the modification is a functional group attached to a nucleotide. Suitable functional groups are described elsewhere herein. The alkyne group(s) or functional group(s) present in each synthetic RNA oligonucleotide can facilitate conjugation of a cargo compound at the site(s) containing the alkyne group via, for example, click chemistry. In some embodiments, the 2' or other modification is a 2'Fluoro-, 2'0-methyl-, LNA-, or any other backbone, sugar, or base modified ribonucleotide or any combination of native, backbone, sugar, and base modified ribonucleotides. In some embodiments, the one or more modified nucleotides include, but are not limited to, a 2'-O-methyl modified nucleotide, 2'-F modified nucleotide, 2'-O-C16 alkyl modified nucleotide, or 2'-0-M0E modified nucleotide. In some embodiments, the one or more modified nucleotides include a lipid attached to the nucleotide. In some embodiments, the lipids is tocopherol, cholesterol, a C14 lipid, a C16 lipid, a C18 lipid, or a C20 lipid.

[0054] The present disclosure provides, in some embodiments, RNA nanoparticles comprising a first oligonucleotide comprising a first core nucleotide sequence comprising SEQ ID NO: 5; a second oligonucleotide comprising a second core nucleotide sequence comprising SEQ ID NO: 6; a third oligonucleotide comprising a third core nucleotide sequence comprising SEQ ID NO: 7; and a fourth oligonucleotide comprising a fourth core nucleotide sequence comprising SEQ ID NO: 8 (see Table IB). In some embodiments, the RNA structures described herein comprise a first oligonucleotide comprising a first core nucleotide sequence comprising SEQ ID NO: 9; a second oligonucleotide comprising a second core nucleotide sequence comprising SEQ ID NO: 6; a thirdoligonucleotide comprising a third core nucleotide sequence comprising SEQ TD NO: 10; and a fourth oligonucleotide comprising a fourth core nucleotide sequence comprising SEQ ID NO: 8. In some embodiments, the first oligonucleotide forms a duplex with both the second oligonucleotide and the fourth oligonucleotide, the second oligonucleotide forms a duplex with both the first oligonucleotide and the third oligonucleotide, the third oligonucleotide forms a duplex with both the second oligonucleotide and the fourth oligonucleotide, and the fourth oligonucleotide forms a duplex with both the first oligonucleotide and the third oligonucleotide.

[0055] In some embodiments, the RNA structures described herein comprise a first oligonucleotide comprising a first core nucleotide sequence comprising a nucleotide sequence having at least 90% identity to SEQ ID NO: 5; a second oligonucleotide comprising a second core nucleotide sequence comprising a nucleotide sequence having at least 90% identity to SEQ ID NO: 6; a third oligonucleotide comprising a third core nucleotide sequence comprising a nucleotide sequence having at least 90% identity to SEQ ID NO: 7; and a fourth oligonucleotide comprising a fourth core nucleotide sequence comprising a nucleotide sequence having at least 90% identity to SEQ ID NO: 8. In some embodiments, the RNA structures described herein comprise a first oligonucleotide comprising a first core nucleotide sequence comprising a nucleotide sequence having at least 90% identity to SEQ ID NO: 9; a second oligonucleotide comprising a second core nucleotide sequence comprising a nucleotide sequence having at least 90% identity to SEQ ID NO: 6; a third oligonucleotide comprising a third core nucleotide sequence comprising a nucleotide sequence having at least 90% identity to SEQ ID NO: 10; and a fourth oligonucleotide comprising a fourth core nucleotide sequence comprising a nucleotide sequence having at least 90% identity to SEQ ID NO: 8.

[0056] In some embodiments, the RNA structures described herein comprise a first oligonucleotide comprising a first core nucleotide sequence comprising a nucleotide sequence having at least 95% identity to SEQ ID NO: 5; a second oligonucleotide comprising a second core nucleotide sequence comprising a nucleotide sequence having at least 95% identity to SEQ ID NO: 6; a third oligonucleotide comprising a third core nucleotide sequence comprising a nucleotide sequence having at least 95% identity to SEQ ID NO: 7; and a fourth oligonucleotide comprising a fourth core nucleotide sequence comprising a nucleotide sequence having at least 95% identity to SEQ ID NO: 8. In some embodiments, the RNA structures described herein comprise a first oligonucleotide comprising a first core nucleotide sequence comprising a nucleotide sequence having at least 95% identity to SEQ ID NO: 9; a second oligonucleotide comprising a second core nucleotide sequence comprising a nucleotide sequence having at least 95% identity to SEQ ID NO: 6; a third oligonucleotide comprising a third core nucleotide sequence comprising a nucleotide sequence having at least 95% identity to SEQ ID NO: 10; and a fourth oligonucleotide comprising a fourth core nucleotide sequence comprising a nucleotide sequence having at least 95% identity to SEQ ID NO: 8.Table IB. Modified Core Nucleotide Sequences.Abbreviations - “m” indicates 2'-0Me; and “hd” indicates a 2' C16 alkyl group.

[0057] In some embodiments, the first, second, third, and fourth oligonucleotides are each 20 nucleotides to 70 nucleotides in length, 20 nucleotides to 65 nucleotides in length, 20 nucleotides to 60 nucleotides in length, 20 nucleotides to 55 nucleotides in length, 20 nucleotides to 50 nucleotides in length, 20 nucleotides to 45 nucleotides in length, 20 nucleotides to 40 nucleotides in length, 20 nucleotides to 35 nucleotides in length, 20 nucleotides to 30 nucleotides in length, 25 nucleotides to 70 nucleotides in length, 30 nucleotides to 70 nucleotides in length, 35 nucleotides to 70 nucleotides in length, 40 nucleotides to 70 nucleotides in length, 45 nucleotides to 70 nucleotides in length, 50 nucleotides to 70 nucleotides in length, 55 nucleotides to 70 nucleotides in length, 60 nucleotides to 70 nucleotides in length, or 65 nucleotides to 70 nucleotides in length.

[0058] In some embodiments, the first, second, third, and fourth oligonucleotides are each 20 nucleotides in length, 21 nucleotides in length, 22 nucleotides in length, 23 nucleotides in length, 24 nucleotides in length, 25 nucleotides in length, 26 nucleotides in length, 27 nucleotides in length, 28 nucleotides in length, 29 nucleotides in length, 30 nucleotides in length, 31 nucleotides in length, 32 nucleotides in length, 33 nucleotides in length, 34 nucleotides in length, 35 nucleotides in length, 36 nucleotides in length, 37 nucleotides in length, 38 nucleotides in length, 39 nucleotides in length, 40 nucleotides in length, 41 nucleotides in length, 42 nucleotides in length, 43 nucleotides in length, 44 nucleotides in length, 45 nucleotides in length, 46 nucleotides in length, 47 nucleotides in length, 48 nucleotides in length, 49 nucleotides in length, 50 nucleotides in length, 51 nucleotides in length, 52 nucleotides in length, 53 nucleotides in length, 54 nucleotides in length, 55 nucleotides in length, 56 nucleotides in length, 57 nucleotides in length, 58 nucleotides in length, 59 nucleotides in length, 60 nucleotides in length, 61 nucleotidesin length, 62 nucleotides in length, 63 nucleotides in length, 64 nucleotides in length, 65 nucleotides in length, 66 nucleotides in length, 67 nucleotides in length, 68 nucleotides in length, 69 nucleotides in length, or 70 nucleotides in length.

[0059] In some embodiments, no oligonucleotide sequences are the same length. In some embodiments, at least two oligonucleotide sequences are the same length. In some embodiments, at least three oligonucleotide sequences are the same length. In some embodiments, all oligonucleotide sequences are the same length.

[0060] In some embodiments, the first, second, third, and fourth core nucleotide sequence are each 20 nucleotides to 32 nucleotides in length, 20 nucleotides to 31 nucleotides in length, 20 nucleotides to 30 nucleotides in length, 20 nucleotides to 29 nucleotides in length, 20 nucleotides to 28 nucleotides in length, 20 nucleotides to 27 nucleotides in length, 20 nucleotides to 26 nucleotides in length, 20 nucleotides to 25 nucleotides in length, 20 nucleotides to 24 nucleotides in length, 20 nucleotides to 23 nucleotides in length, 20 nucleotides to 22 nucleotides in length, 20 nucleotides to 21 nucleotides in length, 21 nucleotides to 32 nucleotides in length, 22 nucleotides to 32 nucleotides in length, 23 nucleotides to 32 nucleotides in length, 24 nucleotides to 32 nucleotides in length, 25 nucleotides to 32 nucleotides in length, 26 nucleotides to 32 nucleotides in length, 27 nucleotides to 32 nucleotides in length, 28 nucleotides to 32 nucleotides in length, 29 nucleotides to 32 nucleotides in length, 30 nucleotides to 32 nucleotides in length, or 31 nucleotides to 32 nucleotides in length.

[0061] In some embodiments, the first, second, third, and fourth core nucleotide sequences are each 20 nucleotides in length, 21 nucleotides in length, 22 nucleotides in length, 23 nucleotides in length, 24 nucleotides in length, 25 nucleotides in length, 26 nucleotides in length, 27 nucleotidesin length, 28 nucleotides in length, 29 nucleotides in length, 30 nucleotides in length, 31 nucleotides in length, or 32 nucleotides in length.

[0062] In some embodiments, no core nucleotides sequences are the same length. In some embodiments, at least two core nucleotides sequences are the same length. In some embodiments, at least three core nucleotide sequences are the same length. In some embodiments, all core nucleotide sequences are the same length.Second Sequence

[0063] In some embodiments, one or more of the first, second, third, and fourth oligonucleotides further comprises a second nucleotide sequence. In some embodiments, two or more of the first, second, third, and fourth oligonucleotides further comprises a second nucleotide sequence. In some embodiments, three or more of the first, second, third, and fourth oligonucleotides further comprises a second nucleotide sequence. In some embodiments, each of the first, second, third, and fourth oligonucleotides further comprises a second nucleotide sequence. In some embodiments, each of the first, second, third, and fourth oligonucleotides comprises a core nucleotide sequence and a second nucleotide sequence. In some embodiments, the second nucleotide sequence is not complementary to any of the first, second, third, or fourth core nucleotide sequences, such that the none of the core nucleotide sequences will bind to or duplex with the second nucleotide sequences. In some embodiments, the second nucleotide sequence is the sense strand of a dsRNA. In some embodiments, the second nucleotide sequence is the sense strand of a siRNA. In some embodiments, the second nucleotide sequence is an antisense oligonucleotide (ASO).

[0064] In some embodiments, the second nucleotide sequence is 18 nucleotides to 30 nucleotides in length, 18 nucleotides to 29 nucleotides in length, 18 nucleotides to 28 nucleotides in length, 18nucleotides to 27 nucleotides in length, 18 nucleotides to 26 nucleotides in length, 18 nucleotides to 25 nucleotides in length, 18 nucleotides to 24 nucleotides in length, 18 nucleotides to 23 nucleotides in length, 18 nucleotides to 22 nucleotides in length, 18 nucleotides to 21 nucleotides in length, 18 nucleotides to 20 nucleotides in length, 18 nucleotides to 19 nucleotides in length, 19 nucleotides to 30 nucleotides in length, 20 nucleotides to 30 nucleotides in length, 21 nucleotides to 30 nucleotides in length, 22 nucleotides to 30 nucleotides in length, 23 nucleotides to 30 nucleotides in length, 24 nucleotides to 30 nucleotides in length, 25 nucleotides to 30 nucleotides in length, 26 nucleotides to 30 nucleotides in length, 27 nucleotides to 30 nucleotides in length, 28 nucleotides to 30 nucleotides in length, or 29 nucleotides to 30 nucleotides in length.

[0065] In some embodiments, the second nucleotide sequence is 18 nucleotides in length, 19 nucleotides in length, 20 nucleotides in length, 21 nucleotides in length, 22 nucleotides in length, 23 nucleotides in length, 24 nucleotides in length, 25 nucleotides in length, 26 nucleotides in length, 27 nucleotides in length, 28 nucleotides in length, 29 nucleotides in length, or 30 nucleotides in length.

[0066] In some embodiments, one of the first, second, third, and fourth oligonucleotides comprise a second nucleotide sequence. In some embodiments, two of the first, second, third, and fourth oligonucleotides comprise a second nucleotide sequence. In some embodiments, three of the first, second, third, and fourth oligonucleotides comprise a second nucleotide sequence. In some embodiments, each of the first, second, third, and fourth oligonucleotides comprise a second nucleotide sequence.

[0067] In some embodiments, the RNA structures described herein comprise one unique second nucleotide sequence. In some embodiments, the RNA structures described herein comprise two unique second nucleotide sequences. In some embodiments, the RNA structures described hereincomprise three unique second nucleotide sequences. Tn some embodiments, the RNA structures described herein comprise four unique second nucleotide sequences.

[0068] In some embodiments, the second nucleotide sequence is positioned at the 3' end of the core nucleotide sequence, hi some embodiments, the second nucleotide sequence is positioned at the 5' end of the core nucleotide sequence.

[0069] In some embodiments, the second nucleotide sequence comprises a portion of a nucleic acid sequence of interest. It is appreciated that the second nucleotide sequence(s) may comprise a portion of any mRNA that has been identified for RNA silencing or another nucleic acid-based therapeutic. In some embodiments, the second nucleotide sequence includes, but is not limited to, a portion of an alpha synuclein (SNCA), microtubule-associated protein tau (MAPT), amyloidbeta precursor protein (APP), sterile alpha and TIR motif containing 1 (SARM1), superoxide dismutase 1 (SOD1), ataxin 2 (ATXN2), ataxin 3 (ATXN3), apolipoprotein E (APOE), beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), fragile X messenger ribonucleoprotein 1 (FMRI), leucine-rich repeat kinase 2 (LRRK2), huntingtin (HTT), sodium voltage-gated channel alpha subunit 10 (SCN10A), sodium voltage-gated channel alpha subunit 9 (SCN9A), calcium voltage-gated channel subunit alpha 1 B (CACNA1B), prion protein (PRNP), phosphoinositide kinase, activin A receptor type 2 A (ACVR2a), activin A receptor type 2 A (ACVR2b), or hypoxanthine phosphoribosyltransferase l(HPRT) transcript.

[0070] In some embodiments, the second nucleotide sequence comprises a sequence that is one of SEQ ID NOs: 11-14 see Table 2A). In some embodiments, the second nucleotide sequence comprises a sequence having at least 90% identity to one of SEQ ID NOs: 11-14. In some embodiments, the second nucleotide sequence comprises a sequence having at least 95% identity to one of SEQ ID NOs: 11-14.Table 2A. Unmodified Second Nucleotide Sequences.

[0071] In some embodiments, one or more nucleotides of the second nucleotide sequence are modified nucleotides. In some embodiments, each nucleotide of the second nucleotide sequence is a modified nucleotide. In some embodiments, one or more internucleotide linkages of the second nucleotide sequence are modified intemucleotide linkages. In some embodiments, each internucleotide linkage of the second nucleotide sequence is a modified internucleotide linkage. In some embodiments, one or more nucleotides of the second nucleotide sequence are modified nucleotides and one or more intemucleotide linkages of the second nucleotide sequence are modified intemucleotide linkages. In some embodiments, each nucleotide of the second nucleotide sequence is a modified nucleotide and one or more intemucleotide linkages of the second nucleotide sequence are modified intemucleotide linkages. In some embodiments, one or more nucleotides of the second nucleotide sequence are modified nucleotides and each intemucleotide linkage of the second nucleotide sequence is a modified intemucleotide linkage. In some embodiments, each nucleotide of the second nucleotide sequence is a modified nucleotide and each intemucleotide linkage of the second nucleotide sequence is a modified intemucleotide linkage. In some embodiments, modified nucleotides and / or intemucleotide linkages of the second nucleotide sequence include those modifications described herein.

[0072] In some embodiments, the second nucleotide sequence comprises a sequence that is one of SEQ ID NOs: 15-18 (see Table 2B). In some embodiments, the second nucleotide sequence comprises a sequence having at least 90% identity to one of SEQ ID NOs: 15-18. In someembodiments, the second nucleotide sequence comprises a sequence having at least 95% identity to one of SEQ ID NOs: 15-18.Table 2B. Modified Second Nucleotide Sequences.Abbreviations - “m” indicates 2'-0Me; “f” indicates 2’-fluoro; indicates phosphorothioate linkage.siRNA

[0073] In some embodiments, the RNA structures described herein further comprise a third nucleotide sequence. In some embodiments, the third nucleotide sequence is complementary to the second nucleotide sequence. In some embodiments, the third nucleotide sequence is referred to as the “antisense strand” of siRNA. In some embodiments, the third nucleotide sequence forms a duplex with the second nucleotide sequence. In some embodiments, the third nucleotide sequence is the antisense strand of an siRNA. In some embodiments, the third nucleotide sequence is the antisense strand of an siRNA, and the second nucleotide sequence is the sense strand of an siRNA, and these two strands represent a siRNA when duplexed, even in cases when the second nucleotide sequence resides on the same oligonucleotide as a core nucleotide sequence. In some embodiments, the third nucleotide sequence is not complementary to any of the first, second, third, or fourth core nucleotide sequences, such that the third nucleotide sequence will not bind to any of the first, second, third, or fourth core nucleotide sequences.

[0074] In some embodiments, the duplex of the second nucleotide sequence and the third nucleotide sequence comprises a 3' overhang and / or a 5' overhang. In some embodiments, the duplex of the second nucleotide sequence and the third nucleotide sequence comprises a 3' overhang. In some embodiments, the duplex of the second nucleotide sequence and the third nucleotide sequence comprises a 5' overhang. In some embodiments, the duplex of the second nucleotide sequence and the third nucleotide sequence comprises both a 3' overhang and a 5' overhang.

[0075] In some embodiments, the third nucleotide sequence is 18 nucleotides to 30 nucleotides in length, 18 nucleotides to 29 nucleotides in length, 18 nucleotides to 28 nucleotides in length, 18 nucleotides to 27 nucleotides in length, 18 nucleotides to 26 nucleotides in length, 18 nucleotides to 25 nucleotides in length, 18 nucleotides to 24 nucleotides in length, 18 nucleotides to 23 nucleotides in length, 18 nucleotides to 22 nucleotides in length, 18 nucleotides to 21 nucleotides in length, 18 nucleotides to 20 nucleotides in length, 18 nucleotides to 19 nucleotides in length, 19 nucleotides to 30 nucleotides in length, 20 nucleotides to 30 nucleotides in length, 21 nucleotides to 30 nucleotides in length, 22 nucleotides to 30 nucleotides in length, 23 nucleotides to 30 nucleotides in length, 24 nucleotides to 30 nucleotides in length, 25 nucleotides to 30 nucleotides in length, 26 nucleotides to 30 nucleotides in length, 27 nucleotides to 30 nucleotides in length, 28 nucleotides to 30 nucleotides in length, or 29 nucleotides to 30 nucleotides in length.

[0076] In some embodiments, the third nucleotide sequence is 18 nucleotides in length, 19 nucleotides in length, 20 nucleotides in length, 21 nucleotides in length, 22 nucleotides in length, 23 nucleotides in length, 24 nucleotides in length, 25 nucleotides in length, 26 nucleotides in length, 27 nucleotides in length, 28 nucleotides in length, 29 nucleotides in length, or 30 nucleotides in length.

[0077] In some embodiments the third nucleotide strand (antisense strand) is the same length as the second nucleotide sequence (sense strand). In some embodiments the third nucleotide strand (antisense strand) is not the same length as the second nucleotide sequence (sense strand).

[0078] In some embodiments, one of the first, second, third, and fourth oligonucleotides comprise a second nucleotide sequence and a third nucleotide sequence that is duplexed with the second nucleotide sequence. In some embodiments, two of the first, second, third, and fourth oligonucleotides comprise a second nucleotide sequence and a third nucleotide sequence that is duplexed with the second nucleotide sequence. In some embodiments, three of the first, second, third, and fourth oligonucleotides comprise a second nucleotide sequence and a third nucleotide sequence that is duplexed with the second nucleotide sequence. In some embodiments, each of the first, second, third, and fourth oligonucleotides comprise a second nucleotide sequence and a third nucleotide sequence that is duplexed with the second nucleotide sequence.

[0079] In some embodiments, the RNA structures described herein comprise one unique third nucleotide sequence, such that the molecule comprises one unique siRNA. In some embodiments, the RNA structures described herein comprise two unique third nucleotide sequences, such that the molecule comprises two unique siRNAs. In some embodiments, the RNA structures described herein comprise three unique third nucleotide sequences, such that the molecule comprises three unique siRNAs. In some embodiments, the RNA structures described herein comprise four unique third nucleotide sequences, such that the molecule comprises four unique siRNAs.

[0080] In some embodiments, the siRNA targets a gene transcript. In some embodiments, the gene transcript is one or more of a SNCA, MAPT, APP, SARM1, SOD1, ATXN2, ATXN3, APOE, BACE1, FMRI, LRRK2, HTT, SCN10A, SCN9A, CACNA1B, PRNP, ACVR2a,T1ACVR2b, or HPRT transcript. In some embodiments, the gene transcript is one or more of a HPRT, SNCA, MAPT, or APP transcript.

[0081] In some embodiments, the third nucleotide sequence comprises a sequence that is one of SEQ ID NOs: 19-22 (see Table 3A). In some embodiments, the third nucleotide sequence comprises a sequence having at least 90% identity to one of SEQ ID NOs: 19-22. In some embodiments, the third nucleotide sequence comprises a sequence having at least 95% identity to one of SEQ ID NOs: 19-22.Table 3A. Unmodified Third Nucleotide Sequences.

[0082] In some embodiments, one or more nucleotides of the third nucleotide sequence(s) are modified nucleotides. In some embodiments, each nucleotide of the third nucleotide sequence(s) is a modified nucleotide. In some embodiments, one or more internucleotide linkages of the third nucleotide sequence are modified internucleotide linkages. In some embodiments, each intemucleotide linkage of the third nucleotide sequence is a modified internucleotide linkage. In some embodiments, one or more nucleotides of the third nucleotide sequence are modified nucleotides and one or more intemucleotide linkages of the third nucleotide sequence are modified intemucleotide linkages. In some embodiments, each nucleotide of the third nucleotide sequence is a modified nucleotide and one or more intemucleotide linkages of the third nucleotide sequence are modified intemucleotide linkages. In some embodiments, one or more nucleotides of the third nucleotide sequence are modified nucleotides and each intemucleotide linkage of the thirdnucleotide sequence is a modified internucleotide linkage. In some embodiments, each nucleotide of the third nucleotide sequence is a modified nucleotide and each internucleotide linkage of the third nucleotide sequence is a modified internucleotide linkage. In some embodiments, modified nucleotides and / or internucleotide linkages of the third nucleotide sequence include those modifications described herein.

[0083] In some embodiments, the third nucleotide sequence comprises a sequence that is one of SEQ ID NOs: 23-26 (see Table 3A). In some embodiments, the third nucleotide sequence comprises a sequence having at least 90% identity to one of SEQ ID NOs: 23-26. In some embodiments, the third nucleotide sequence comprises a sequence having at least 95% identity to one of SEQ ID NOs: 23-26.Table 3B. Modified Third Nucleotide Sequences.Abbreviations - “AS” indicates antisense; “m” indicates 2'-OMe; “f” indicates 2'-fluoro; indicates phosphorothioate linkage; unless otherwise noted, the 5' position of the nucleotide sequence can include 5'-phosphate or 5 '-vinylpho sphonate (VP).Antisense Oligonucleotide

[0084] In some embodiments, one or more of the first, second, third, and fourth oligonucleotides further comprises an antisense oligonucleotide (ASO). In some embodiments, two or more of the first, second, third, and fourth oligonucleotides further comprise an ASO. In some embodiments,three or more of the first, second, third, and fourth oligonucleotides further comprise an ASO. In some embodiments, each of the first, second, third, and fourth oligonucleotides further comprises an ASO. In some embodiments, each of the first, second, third, and fourth oligonucleotides comprises a core nucleotide sequence and an ASO. In some embodiments, the ASO is not complementary to any of the first, second, third, or fourth core nucleotide sequences, such that none of the core nucleotide sequences will bind to or duplex with the ASO.

[0085] In some embodiments, the ASO is positioned at the 3' end of the core nucleotide sequence. In some embodiments, the ASO is positioned at the 5' end of the core nucleotide sequence. In some embodiments, the ASO is linear. In some embodiments, the ASO is circular.

[0086] In some embodiments, the RNA structures described herein comprise one unique ASO. In some embodiments, the RNA structures described herein comprise two unique ASOs. In some embodiments, the RNA structures described herein comprise three unique ASOs. In some embodiments, the RNA structures described herein comprise four unique ASOs.

[0087] In some embodiments, a linker connects the ASO to the core nucleotide sequence. In some embodiments, the linker is a thermodynamic linker, a pH responsive linker, a light-sensitive (photocleavable) linker, or an enzyme-cleavable linker. Other suitable linkers will be appreciated by those of ordinary skill in the art. It is appreciated that any linker suitable for connecting two different polynucleotides without adversely affecting the function of the polynucleotides can be used in the molecule described herein.

[0088] In some embodiments, the RNA structures described herein comprise first, second, third, and fourth oligonucleotides comprising a core nucleotide sequence, a second nucleotide sequence, and a third nucleotide sequence (e.g., an siRNA), and an ASO, such that the molecule comprises both an ASO and a siRNA. In some embodiments, the ASO is reversibly attached to the thirdnucleotide sequence. In some embodiments, the ASO and siRNA have different target sequences. In some embodiments, the ASO and siRNA have the same target sequence.

[0089] In some embodiments, the RNA structures described herein comprise first, second, third, and fourth oligonucleotides comprising a core nucleotide sequence and a second nucleotide sequence, and further comprises an ASO. In some embodiments, the ASO forms a duplex with the second nucleotide sequence. In some embodiments, the ASO is not complementary to any of the first, second, third, or fourth core nucleotide sequences, such that the ASO will not bind to any of the first, second, third, or fourth core nucleotide sequences. In some embodiments, the ASO is reversibly attached to the second nucleotide sequence but does not form a duplex with the second nucleotide sequence.

[0090] In some embodiments, the ASO is 10 nucleotides to 30 nucleotides in length, 10 nucleotides to 29 nucleotides in length, 10 nucleotides to 28 nucleotides in length, 10 nucleotides to 27 nucleotides in length, 10 nucleotides to 26 nucleotides in length, 10 nucleotides to 25 nucleotides in length, 10 nucleotides to 24 nucleotides in length, 10 nucleotides to 23 nucleotides in length, 10 nucleotides to 22 nucleotides in length, 10 nucleotides to 21 nucleotides in length, 10 nucleotides to 20 nucleotides in length, 10 nucleotides to 19 nucleotides in length, 10 nucleotides to 18 nucleotides in length, 10 nucleotides to 17 nucleotides in length, 10 nucleotides to 16 nucleotides in length, 10 nucleotides to 15 nucleotides in length, 10 nucleotides to 14 nucleotides in length, 10 nucleotides to 13 nucleotides in length, 10 nucleotides to 12 nucleotides in length, 10 nucleotides to 11 nucleotides in length, 11 nucleotides to 30 nucleotides in length, 12 nucleotides to 30 nucleotides in length, 13 nucleotides to 30 nucleotides in length, 14 nucleotides to 30 nucleotides in length, 15 nucleotides to 30 nucleotides in length, 16 nucleotides to 30 nucleotides in length, 17 nucleotides to 30 nucleotides in length, 18 nucleotides to 30 nucleotides in length, 19nucleotides to 30 nucleotides in length, 20 nucleotides to 30 nucleotides in length, 21 nucleotides to 30 nucleotides in length, 22 nucleotides to 30 nucleotides in length, 23 nucleotides to 30 nucleotides in length, 24 nucleotides to 30 nucleotides in length, 25 nucleotides to 30 nucleotides in length, 26 nucleotides to 30 nucleotides in length, 27 nucleotides to 30 nucleotides in length, 28 nucleotides to 30 nucleotides in length, or 29 nucleotides to 30 nucleotides in length.

[0091] In some embodiments, the ASO is 10 nucleotides in length, 11 nucleotides in length, 12 nucleotides in length, 13 nucleotides in length, 14 nucleotides in length, 15 nucleotides in length, 16 nucleotides in length, 17 nucleotides in length, 18 nucleotides in length, 19 nucleotides in length, 20 nucleotides in length, 21 nucleotides in length, 22 nucleotides in length, 23 nucleotides in length, 24 nucleotides in length, 25 nucleotides in length, 26 nucleotides in length, 27 nucleotides in length, 28 nucleotides in length, 29 nucleotides in length, or 30 nucleotides in length.

[0092] In some embodiments, the ASO targets a gene transcript. In some embodiments, the gene transcript is one or more of a SNCA, MAPT, APP, SARM1, SOD1, ATXN2, ATXN3, APOE, BACE1, FMRI, LRRK2, HTT, SCN10A, SCN9A, CACNA1B, PRNP, ACVR2a, ACVR2b, or HPRT transcript. In some embodiments, the gene transcript is one or more of a HPRT, SNCA, MAPT, or APP transcript.Other API

[0093] In some embodiments, the RNA structures described herein further comprise one or more cargo compounds and / or one or more functional groups. It is appreciated that the cargo compound(s) or functional group(s) can be any biological molecule, chemical molecule, synthetic molecule, or any other molecule that can be attached to the RNA structures described herein.

[0094] In some embodiments, the cargo compound and / or functional group is an active agent. In some embodiments, the cargo compound and / or functional group includes, but is not limited to,DNA, RNA, modified ribonucleotides, amino acids, peptides, polypeptides, antibodies, aptamers, aptazymes, riboswitches, ribozymes, guide sequences for ribozymes that inhibit translation or transcription of essential tumor proteins and genes, hormones, immunomodulators, antipyretics, anxiolytics, antipsychotics, analgesics, antispasmodics, anti-inflammatories, anti-histamines, anti-infectives, and chemotherapeutic s (anti-cancer drugs). Other suitable cargo compounds include sensitizers, (e.g. radiosensitizers) that can make a cell or subject more responsive (or sensitive) to a treatment or prevention and imaging or other diagnostic agents. In some embodiments, the RNA structures described herein are used as a monotherapy. In some embodiments, the RNA structures described herein are used in combination with other active agents for treatment or prevention of a disease or disorder. In some embodiments, the cargo compound and / or functional group comprises a targeting moiety that facilitates the targeting of a specific cell, tissue, or organ.

[0095] Methods of production and annealing conditions for the RNA structures are disclosed herein are described in, for example, in U.S. Patent No. 11,976,092.Pharmaceutical Composition

[0096] In some embodiments, the present disclosure also provides pharmaceutical compositions comprising the RNA structures described herein and a pharmaceutical carrier appropriate for administration to an individual in need thereof. The pharmaceutical compositions described herein can be administered via any suitable administration route. In some embodiments, the pharmaceutical compositions described herein are administered to the subject in need thereof orally, intravenously, ocularly, intraocularly, intramuscularly, intravaginally, intraperitoneally, rectally, parenterally, topically, intranasally, subcutaneously, or by any other suitable administration route.

[0097] Various excipients and other components for the pharmaceutical compositions described herein are described, for example, in U.S. Patent No. 11,976,092.Methods of Assaying

[0098] Also described herein are methods for assaying the purity of an RNA structure and compositions comprising the RNA structures described herein. In some embodiments, the methods comprise assaying an RNA structure or composition comprising RNA structures in two dimensions. In some embodiments, the two dimensions comprise analysis in a first dimension followed by analysis in a second dimension. In some embodiments, the first dimension comprises size-exclusion chromatography (SEC). In some embodiments, the SEC analysis is performed using oan AdvanceBio SEC column (200A, 1.9 pm, 4.6 mm x 300 mm). In some embodiments, the column temperature is maintained at 25°C during the SEC analysis. In some embodiments, a flow rate of 0.2 mL / min is used for the SEC analysis. In some embodiments, the SEC analysis (first dimension) separates the fully assembled RNA structures from other intermediate molecules.

[0099] In some embodiments, the second dimension comprises ion-pairing reverse phase (IPRP) chromatography. In some embodiments, the IPRP chromatography further comprises assaying the RNA structure or composition using a peak-based multiple heart-cutting sampling mode. In some embodiments, the IPRP chromatography is performed using an analytical ACQUITY Premier Oligonucleotide BEHC18 Column (300A, 1.7 pm, 2.1 mm x 150 mm). In some embodiments, the column temperature is maintained at 75 °C during the IPRP chromatography. In some embodiments, a flow rate of 0.2 mL / min is used for the IPRP chromatography. In some embodiments, the IPRP chromatography (second dimension) analyzes the component strands of the fully assembled RNA structures that were separated from the intermediate molecules.Methods of Treatment

[0100] In some embodiments, the present disclosure also relates to methods of treating a subject, comprising administering the RNA structures and / or pharmaceutical compositions described herein to a subject in need thereof. The individual in need thereof can have or can be expected to have a cancer, a genetic disease or disorder, a viral, bacterial, fungal, and / or parasitic infection, or other disease or disorder in need of treatment or prevention. In some embodiments, the RNA structures and / or pharmaceutical compositions described herein are administered in an amount effective to treat the cancer, a genetic disease or disorder, viral, bacterial, fungal, and / or parasitic infection, or other disease or disorder.

[0101] In some embodiments, administration of the RNA structures and / or pharmaceutical compositions described herein can be systemic or localized. In some embodiments, the RNA structures and / or pharmaceutical compositions described herein are administered to the subject in need thereof one or more times per day. In some embodiments, the molecules and / or pharmaceutical compositions are administered once daily. In some embodiments, the RNA structures and / or pharmaceutical compositions are administered twice daily. In some embodiments, an effective amount of the RNA structures and / or pharmaceutical compositions are administered to the subject in need thereof. In some embodiments, the RNA structures and / or pharmaceutical compositions are administered one or more times per week.

[0102] In some embodiments, the RNA structures and / or pharmaceutical compositions are administered in a dosage form. In some embodiments, the amount or effective amount of the RNA structures and / or pharmaceutical compositions is divided into multiple dosage forms. For example, the effective amount can be split into two dosage forms and the first dosage form can be administered, for example, in the morning, and the second dosage form can be administered in theevening. Although the effective amount is given over two doses, in one day, the subject receives the effective amount. In some embodiments, the effective amount is 0.1 to 1000 mg per day.EXAMPLESExample 1: Generation of RNA Structures

[0103] Core nucleotide sequences were generated and tested for constructing a desired RNA structure. The following parameters were used to generate the RNA structures: the number of strands, the number of nucleic acids in a strand, the maximum number of base pairs, the minimum ratio of GC pairs, the maximum ratio of GC pairs, and the maximum ratio of unpaired base pairs. With these parameters provided, the RNA sequences were selected and then validated in experiments as described herein.Synthesis of single -stranded oligonucleotides

[0104] Single strands of the oligonucleotides (z.e., core nucleotide sequence and / or second nucleotide sequence) and the API antisense strands (i.e., ASO or third nucleotide sequence) described in Tables 1A-3B were synthesized on solid support via a MerMade™ 12 (LGC Biosearch Technologies). The oligonucleotides were synthesized via phosphoramidite chemistry at either 5, 10, 25, or 50 pmol scales.

[0105] For the sense strands, the types of solid supports were universal CPG: the Universal UnyLinker (Chemgenes, Catalog No. AT273-27) and 3' Teg-Tocopherol (LGC Biosearch Technologies, Catalog No. BG7-1190) were purchased commercially. For all the antisense strands, commercially available standard support mA was utilized. Standard reagents were used in the oligo synthesis {see Table 4 below; as described, for example, in WO 2024 / 123646 A2, published June 13, 2024, the contents of which are incorporated by reference in its entirety), where 0.1M xanthane hydride in pyridine was used as the sulfurization reagent and 20% DEA in ACN wasused as an auxiliary wash post synthesis. All monomers were made at 0. IM in ACN and contained a molecular sieves trap bag.

[0106] The oligonucleotides were cleaved and deprotected (C / D) at 45 °C for 20 hours. The sense strands were C / D from the CPG using ammonia hydroxide (28-30%, cold), whereas 3% DEA in ammonia hydroxide (28-30%, cold) was used for the antisense strands. C / D was determined complete by IP-RP LCMS when the resulting mass data confirmed the identity of sequence. Depending on scale, the CPG was filtered via 0.45 pm PVDF syringeless filter, 0.22 pm PVDF Steriflip® vacuum filtration, or 0.22 pm PVDF Stericup® Quick release. The CPG was back washed / rinsed with either 30% ACN / RNAse free water or 30% EtOH / RNAse free water then filtered through the same filtering device and combined with the first filtrate. This was repeated twice. The material was then divided evenly into 50 mF falcon tubes to remove organics via Genevac™. After concentration, the crude oligonucleotides were diluted back to synthesized scale with RNAse free water and filtered either by 0.45 pm PVDF syringeless filter, 0.22 pm PVDF Steriflip® vacuum filtration or 0.22 pm PVDF Stericup® Quick release.

[0107] The crude oligonucleotides were purified via AKTA™ Pure purification system using either anion-exchange (AEX) or reverse phase (RP) a source 15Q-RP column. For AEX, an ES Industry Source™115Q column maintaining column temperature at 65°C with MPA: 20mM NaH2PO4, 15% ACN, pH 7.4, and MPB: 20 mM NaH2PO4, IM NaBr. 15% ACN, pH 7.4. For RP, a Source™ 15Q-RP column with MPA: 50mM NaOAc with 10% ACN and MPB: 50mM NaOAc with 80% ACN. In all cases, fractions which contained a mass purity greater than 85% without impurities >5% were combined.

[0108] The purified oligonucleotides were desalted using 15 mF 3K MWCO centrifugal spin tubes at 3500 x g for approximately 30 min. The oligonucleotides were rinsed with RNAse freewater until the eluent conductivity reached < 100 p semi / cm. After desalting was complete, 2-3 mL of RNAse free water was added then aspirated ten times, the retainment was transferred to a 50 mL conical tube, and this was repeated until complete transfer of oligo by measuring concentration of the compound on the filter via nanodrop. The final oligonucleotide was then nano filtered two times via 15 mL 100K MWCO centrifugal spin tubes at 3500 x g for 2 min. The final desalted oligonucleotides were analyzed for concentration (nanodrop at A260) and characterized by IP-RP LCMS for mass purity and UPLC for UV-purity.

[0109] For the preparation of duplexes, equimolar amounts of sense and antisense strand were combined and heated at 65 °C for 10 minutes then slowly cooled to ambient temperature over 40 minutes. Integrity of the duplex was confirmed by UPLC analysis and characterized by LCMS using IP-RP. All duplexes were nano filtered and endotoxin levels were measured via Charles River Endosafe® Cartridge Device to give the final compounds of conjugated RNAi. For in vivo analysis, the appropriate amount of duplex was lyophilized and reconstituted in IX PBS for rodent studies and in CSF for non-human primate studies.Table 4. Oligonucleotide Synthesis Reagents._Construction of RNA Structure

[0110] For an RNA structure with four copies of the same siRNA: RNA structures were assembled in a one-pot protocol by mixing five component strands: the four strands containingboth the core nucleotide sequences which yield the four-way junction and the sense strand (second nucleotide sequence); and the antisense oligo (third nucleotide sequence) to be delivered. That is, the sequences represented by 4WJa-SS, 4WJb-SS, 4WJc-SS, 4WJd-SS, and antisense RNAi agent (SNCA, HPRT, APP, or MAPT AS). These were combined at a defined molar ratio of 1:1:1: 1:4, respectively, in IX PBS buffer. The mixed solution was placed into a thermal cycler and annealed as follows: denatured at 85 °C for 5 min, and slowly cooled down to 4°C with -4°C / min rate, to yield a substantially cruciform molecule.

[0111] For an RNA structure with two or more siRNA: RNA structures were assembled as above, but with two, three, or four different antisense oligonucleotides (third nucleotide sequence). These were combined in appropriate molar ratios. For example, for delivery of two copies of an HPRT siRNA and two copies of a SNCA siRNA, the following strands are combined: 4WJa-HSS, 4WJb-HSS, 4WJC-SSS, 4WJd-SSS, HPRT AS and SNCA AS, in a ratio of 1:1:1:1:2:2, respectively. For the delivery of three siRNAs (e.g., one copy of SNCA, two copies of MAPT and one copy of APP), the following strands are combined: 4WJa-SSS, 4WJb-MSS, 4WJc-MSS, 4WJd-ASS, SNCA AS, MAPT AS, APP AS, in a ratio of 1 : 1 : 1 : 1 : 1 :2: 1, respectively. For the delivery of four siRNAs (e.g., one copy of SNCA, one copy of MAPT, one copy of HPRT, and one copy of APP), the following strands are combined: 4WJa-SSS, 4WJb-MSS, 4WJc-HSS, 4WJd- AS S , SNCA AS , MAPT AS . HRPT AS , APP AS , in a ratio of 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1. respectively. (HSS: HPRT sense strand; SSS: SCNA sense strand; MSS: MAPT sense strand; ASS: APP sense strand; AS: antisense).

[0112] The same protocol was followed to assemble a lipid-bearing RNA structure with tocopherol e.g., positioned at the 3' end of the strand), for example by substituting 4WJa-SSS with 4WJa-SSS-tocopherol, with all other components and steps remaining as described above.

[0113] RNA structure assembly was confirmed by gel electrophoresis. RNA structure samples and ladders were each mixed with 6X Loading dye and loaded in a 12% TBE gel and run at room temperature for 55 min. The gel was stained by SYBR safe for 2 min, and then imaged using an Invitrogen iB right imager.Example 2: In vivo evaluation of RNA structure with four copies of a single RNAi agent

[0114] An RNA structure having a core sequence as described herein, and bearing four copies of SCNA RNAi agent, was tested in C57B1 / 6 mice (n = 6), with PBS as control group {see Table 5). The RNA structure of the present disclosure was tested, either with two C16 lipids conjugated to internal nucleotides, or without (Figure 1A). Dose of test materials were administrated on Day 0 by direct ICV injection. All animals were sacrificed, and tissues were harvested on Day 28.Table 5. Full-length oligonucleotide sequences.Abbreviations - “m” indicates 2’-0Me; “f” indicates 2’-fluoro; indicates phosphorothioate linkage; “hd” indicates a 2' C16 alkyl group; unless otherwise noted, the 5’ position of the AS can include 5'-phosphate or 5'-vinylphosphonate (VP).

[0115] In vivo evaluation of the RNA structures was performed by administering the structure via the intracerebroventricular (ICV) route. ICV administration and tissue harvesting were performed at Biomere Biomedical Research Models, Inc., following their standard protocols. Doses of test materials were administrated on Day 0 by direct ICV dosing at a dose amount of 100 pg (siRNA) / mouse and 10 pl / mouse. For both siRNA and RNA structure group, the dose of siRNA was kept the same, at 100 pg, excluding the weight of any conjugations such as lipid and RNA scaffold. The single infusion was performed unilaterally into the right ventricle using the following coordinates: anterior-posterior (AP) = +0.3 mm; medio-lateral (ML) = +1.0 mm; dorso-ventral (DV) = -0.3 mm (from estimated brain surface). On Day 28, all animals were sacrificed, and the left hemisphere of the brain was harvested, including the frontal cortex, cerebellum, brain stem, and lumbar spinal cord, and used as material for quantitative PCR (qPCR). Briefly, brain tissues and spinal cords were harvested from mice, following institutional ethical guidelines and approved protocols. The left hemisphere of the mouse brain was carefully dissected to separate the frontal cortex, the mid cortex, and hind cortex collectively, the cerebellum, and the brain stem. The spinalcord was sectioned to isolate the lumbar region. Whole livers and kidneys were also collected. These tissues were immediately- snap-frozen in liquid nitrogen to preserve RNA integrity and then stored at 80°C until RNA extraction for qPCR analysis. Tissue samples were loaded onto the 2010 GenoGrinder for homogenization. Total RNA was extracted using the MagMAX mirVana Total RNA Isolation Kit according to the manufacturer’s instructions. The concentration and purity of RNA were determined by spectrophotometry at 260 / 280 nm using Nanodrop8.

[0116] First-strand cDNA was synthesized from 320 ng total RNA using the SuperScript IV VILO Kit in a 20 pL reaction volume following the manufacturer’s instructions. The resulting cDNA was stored at -20°C until use.

[0117] qPCR was conducted on a QuantStudio 5 system using the TaqMan Fast Advanced Master Mix and TaqMan qPCR assay. Each 20 pL reaction mixture contained 10 pL of TaqMan Fast Advanced Master Mix. 0.33 pL of 60X TaqMan qPCR assay for both the target and reference genes, 2 pL of cDNA, and 7.34 pL of nuclease-free water. Probes were acquired from ThermoFisher: mmSNCA_FAM, Mm01188700_ml. Thermal cycling began with a UDG activation step at 50°C for 2 minutes, followed by a polymerase activation step at 95 °C for 1 minute. This was followed by 44 cycles of denaturation at 95 °C for 10 seconds and annealing / extension at 60°C for 30 seconds. A dissociation curve step was added to confirm the specificity of the PCR products, ramping from 65 °C to 95 °C.

[0118] Relative gene expression levels were quantified using the comparative 2A(-AACT) method, normalized to the housekeeping gene ? -Actin. Results were expressed at a percentage change relative to control samples. Reactions were performed in duplicate to ensure the reproducibility and accuracy of the results.

[0119] The RNA structure described herein achieved SNCA knockdown (KD) in brain, with no significant difference in different CNS regions: frontal cortex, brain stem, cerebellum, and spinal cord (Figure IB). Further, no significant difference was noted when comparing RNA structures with and without the C16 modified nucleotides. The RNA structures generally demonstrated effective knockdown of SNCA in all tissues tested.Example 3: In vivo evaluation of RNA Structures with more than one distinct RNAi agent

[0120] The ICV injection protocol as described in Example 2 was used to investigate RNA structures having more than one distinct RNAi agent. Similar to Example 2, brain tissues and spinal cords were harvested from mice, following institutional ethical guidelines and approved protocols. The left hemisphere of the mouse brain was carefully dissected to separate the frontal cortex, the mid cortex, and hind cortex collectively, the cerebellum, and the brain stem. The spinal cord was sectioned to isolate the lumbar region. Whole livers and kidneys were also collected. These tissues were immediately snap-frozen in liquid nitrogen to preserve RNA integrity and then stored at -80°C until RNA extraction for qPCR analysis. Tissue samples were loaded onto the 2010 GenoGrinder for homogenization. Total RNA was extracted using the MagMAX mirVana Total RNA Isolation Kit according to the manufacturer’s instructions. The concentration and purity of RNA were determined by spectrophotometry at 260 / 280 nm using Nanodrop8.

[0121] First-strand cDNA was synthesized from 320 ng total RNA using the SuperScript IV VILO Kit in a 20 pL reaction volume following the manufacturer’s instructions. The resulting cDNA was stored at -20°C until use.

[0122] qPCR was conducted on a QuantStudio 5 system using the TaqMan Fast Advanced Master Mix and TaqMan qPCR assay. Each 20 pL reaction mixture contained 10 pL of TaqMan Fast Advanced Master Mix, 0.33 pL of 60X TaqMan qPCR assay for both the target and referencegenes, 2 pL of cDNA, and 7.34 pL of nuclease-free water. All probes were acquired from ThermoFisher: mmSNCA_FAM, Mm01188700_ml; mmAPP_FAM, Mm00431829_ml; mmMAPT_FAM, Mm00521989_ml; mmHPRT_FAM, Mm03024075; mmACTA_VIC, Mm04394036_gl. Thermal cycling began with a UDG activation step at 50°C for 2 minutes, followed by a polymerase activation step at 95 °C for 1 minute. This was followed by 44 cycles of denaturation at 95°C for 10 seconds and annealing / extension at 60°C for 30 seconds. A dissociation curve step was added to confirm the specificity of the PCR products, ramping from 65°C to 95°C.

[0123] Relative gene expression levels were quantified using the comparative 2A(-AACT) method, normalized to the housekeeping gene 7-Actin. Results were expressed as a percentage change relative to control samples. Reactions were performed in duplicate to ensure the reproducibility and accuracy of the results.Two RNAi agents

[0124] RNA structures with two types of siRNA, made in accordance with the methods described above, were evaluated for KD of both HPRT and SNCA in vivo (Figure 2A). KD was evaluated in four brain regions: frontal cortex (FCX), brain stem (BS), spinal cord (SC), and cerebellum (CB). The KD levels are comparable with HPRT and SNCA siRNA mix control (tocopherol-modified siRNA), especially in brain stem and spinal cord, with no significant difference in knockdown efficiency between tocopherol-modified siRNA and the RNA structures in those tissues (Figures 2B-2C). The RNA structures also showed reduced knockdown in both the liver and kidney (Figure 2D).Three RNAi agents

[0125] RNA structures with three siRNA targeting disease relevant genes (SNCA, MAPT, and APP) were assembled and tested in vivo. The SNCA sense strand (second nucleotide sequence) was attached to 4WJa core nucleotide sequence, the MAPT sense strand (second nucleotide sequence) was attached to 4WJb and 4WJc core nucleotide sequences, and the APP sense strand (second nucleotide sequence) was attached to 4WJd core nucleotide sequence. All sense strands (second nucleotide sequences) were attached at the 3' end of the core nucleotide sequences. SNCA, MAPT, and APP antisense strands (third nucleotide sequence) hybridized with the corresponding oligonucleotides (the sense strand portion), respectively. A total of seven strands (as described above) were mixed in IX PBS buffer and subjected to denaturing at 85°C followed by slowly cooling down to 4°C to promote the self-assembly of the four-way-junction RNA structure with three different types of siRNA attached. Whereas the RNA structure with only a single RNAi agent (directed to SNCA) showed in vivo KD of only SNCA and not MAPT or APP, an RNA structure with the three aforementioned siRNA demonstrated in vivo KD of SNCA, MAPT, and APP (Figures 3A-3C). Thus, the RNA structures described herein can deliver multiple siRNA to facilitate the KD of multiple genes, and do so within one chemical entity. This may be particularly beneficial for delivering siRNA to the tissue in a defined ratio to tailor therapeutic regimens and in some cases provide a synergistic effect.Example 4: In vivo analysis of the effect of lipid conjugation positioning

[0126] The effect of positioning for conjugation of a lipid to the RNA structures disclosed herein was assessed in vivo in rats. Briefly, RNA structures were prepared as described above, with a C16 lipid conjugated at position six in the core nucleotide sequence (SF6), at the 3' end of the second nucleotide sequence, or at position 12 of the second nucleotide sequence (NT13). RNA structures were all synthesized with four copies of the SCNA siRNA antisense strand describedherein. Synthesized RNA structures (300 g) were administered to rats intrathecally, and levels of SNCA knockdown was assessed in rat CNS tissue (SCIO, LDRG (lumbar dorsal root ganglion), BS, FCTX) as described herein by qPCR seven days after administration of the RNA structures.

[0127] Conjugation of a C16 lipid to a SNCA siRNA did not significantly alter knockdown of SNCA in CNS tissue (FIG. 4A). Conjugation of a C16 lipid to position six of the scaffold strand increased knockdown of SNCA in the LDRG and BS relative to the other two conjugation positions, and conjugation of a C16 lipid to the N-terminus of the second strand significantly increased knockdown of SNCA in the FCTX. Internal positioning of the lipid further decreased white matter retention in the brain and spinal cord relative to conjugation of the lipid to the 3' end of the second sequence or position 13 of the second sequence (FIG. 4B). Thus, positioning of the lipid conjugated to the RNA structure affected knockdown of the target gene across different brain regions.Example 5: Characterization of RNA Structures.

[0128] This example describes a chromatography platform designed to address challenges for analyzing RNA-based structures. Size-exclusion chromatography (SEC) was used in the first dimension (1D) to understand RNA structure assembly and separate fully assembled molecules from process impurities (including partially assembled intermediates and free single strands). Ionparing reverse phase (IPRP) chromatography was used in the second dimension (2D) to identify component strands and impurities in the peak of interest using peak-based multiple heart-cutting sampling mode. Additionally, SEC coupled with multi-angle light scattering (MALS) and SEC-HRMS were used as orthogonal methods to confirm the findings from the 2D-LC method for RNA structures.Methods30600_WG

[0129] The instrumentation for the online two-dimensional liquid chromatography / quadrupole time-of-flight mass spectrometry (2D-LC / QTOF) from Agilent Technologies (Waldbronn, Germany) is shown in Figure 5AError! Reference source not found.. All modules were obtained from Agilent Technologies, the 2D-LC was coupled to a 6545 XT Q-TOF mass spectrometry system equipped with a dual nebulizer electrospray ionization (ESI) source (G3251B). The!D Agilent 1290 Infinity II 2D-LC Solution consisted of a 1290 Bio high speed pump (G7132A), a 1290 Bio Multisampler (G7137A), and a Diode Array Detector (DAD) (G7117B) with 1 pL flow cell (G7117 - 60020). The2D LC consisted of a 1290 BIO high speed pump (G7132A) and a Diode Array Detector (DAD) (G7117B) with 1 pL flow cell (G7117 - 60020). Both 'D and2D columns were accommodated in 1290 MCT column compartments (G7116B). The two dimensions were connected by a valve drive (G1170A) equipped with InfinityLab Bio 2D-LC Active Solvent Modulation valve (#5320-0017) connected to two 6-position / 14-port valve heads (#5067-4273) carrying six 40 pL loops each. Raw mass spectrometry data was acquired in the negative mode and stored in profile format with an MS threshold of 300. The drying gas temperature was 320°C and the gas flow was 8 L / min. The acquisition rate was 2 spectra / s. The nebulizer pressure was 45 psi, the fragmentor and skimmer voltage were 175V and 65 V. The acquisition mass range was 600-2500 m / z.

[0130] For analysis of RNA structure assembly purity, the size exclusion chromatography (SEC) method was used to resolve the mixed assembled molecules in the first dimension (XD) and separate them for analysis in the second dimension (2D). Four size-exclusion columns were screened to find the most suitable one for RNA structures. All columns had the same physical dimensions (4.6 mm in inner diameter and 300 mm in length) and different particle surface chemistry and properties. Detailed information for each column can be found below in Table 6.IX PBS or 100 mM ammonium acetate was used as mobile phases. The flow rate was set at 0.2 mL / min and the column temperature was kept at 20°C.Table 6. Columns used for SEC screening.

[0131] Ion-pairing reverse phase (IPRP) method was mainly used to understand the strand composition and critical impurities in the peak of interest.2D IPRP is also a step to quantify the antisense strands in the peak sampled from the first dimension to determine dose for in vitro and in vivo experiments, as well as to determine RNA structure assembly. An analytical ACQUITY Premier Oligonucleotide BEH C18 Column (300A, 1.7 pm, 2.1 mm x 150 mm) was used for denaturing IPRP analysis for RNA structures single strands. The flow rate was set to 0.2 mL / min and the column temperature was kept at 75°C during analysis. Details and gradient information for IPRP method can be found in Table 7.Table 7. Chromatography parameters.

[0132] An SEC x SEC 2D-LC method was used to evaluate the thermodynamic behavior of RNA structures. Time-based high resolution (HighRes) sampling modes were used, 5 cuts were collected at the main peak region, and each cut was set to 4 seconds in collection time (33.3% loop filling). Both dimensions had an analytical AdvanceBio SEC column installed (200A, 1.9 pm, 4.6 mm x 300 mm). The flow rate was maintained at 0.2 mL / min using IX PBS as the mobile phases, and the column temperature was kept at 20°C for both dimensions during analysis.

[0133] The SEC x IPRP 2D-LC method was used to evaluate the assembly of RNA structures. The first dimension (JD) SEC method was used to separate the fully assembled RNA structures with other intermediates, and the second dimension ^D) denatured IPRP was used to analyze the component strands from each peak separated by the first dimension. ’D had an analytical AdvanceBio SEC column installed (200A, 1.9 pm, 4.6 mm x 300 mm). The flow rate was maintained at 0.2 mL / min using 100 mM ammonium acetate as the mobile phase. The columntemperature was kept at 20°C during analysis.2D had an analytical ACQUITY Premier Oligonucleotide BEH C18 Column (300A, 1.7 pm, 2.1 mm x 150 mm) installed for denaturing IPRP analysis for RNA structure single strands. The flow rate was set to 0.2 mL / min and the column temperature was kept at 75°C during analysis. Peak-based multiple heart-cutting (MHC) sampling mode was used, the sampling time was set to 8 seconds (66.7% loop filling), threshold mode (50 mAu), Active- Solvent Modulation (ASM) factor=5.1. Sample loop flush = 3 times, and a high aqueous phase initial hold was programmed at the beginning of the method to account for the time in ASM phase in the method.

[0134] The molecular weight distribution in solution and peak uniformity information was obtained with SEC-MALS in order to understand sample homogeneity. SEC was used for separation of assembled RNA structures and partially assembled intermediates and single strands. The SEC-MALS system was composed of an Agilent 1290 Infinity II LC system coupled with a Wyatt DAWN Multi-Angle Static Light Scattering (MALS) detector. A low-shedding analytical ACQUITY Premier Protein SEC Column (250A, 1.7 pm, 4.6 mm x 300 mm) was installed and IX PBS was used as the mobile phases. Bovine serum albumin (BSA) was used as a control sample to set up the system parameters. Samples and mobile phases were filtered with 0.1 pm filters before analysis. A dn / dc value of 0.1700 mL / g was used for RNA structures. The raw data was acquired and analyzed with ASTRA software (version 8.2.0).

[0135] The 4WJ-4HPRT RNA structures were also analyzed by Q Exactive™ UHMR Hybrid Quadrupole Orbitrap™ coupled with a Vanquish HPLC from Thermo Fisher (Waltham, Massachusetts). The sample was separated on TSKgel SuperSW3000 SEC column (250A, 4 pm, 2.0 mm x 30 cm) at a flow rate of 0.075 mL / min with 175 mM ammonium acetate as mobile phase. The spectra were acquired at a resolution of 12,500 with a m / z range of 1,000 to 10,000. Othermass spectrometer parameters were set as: spray voltage 4kV, capillary temperature, 350°C, S-lens RF level 200, sheath gas 30, aux gas 20, In-source CID 40eV, desolvation voltage -150V, and trapping gas pressure 3. The raw data was analyzed with Unidec with a m / z range of 4,000 to 8,000 Da, mass ranges of 7,000 to 120,000 Da, sampling step of 10 Da, and a peak detection threshold of 0.1.Results

[0136] Among the columns tested during the development of the SEC analytical method, the Agilent AdvanceBio SEC column demonstrated the most effective separation with minimal secondary interactions, likely due to its hydrophilic coating. This performance was superior when compared to the Sepax Unix / Unix-C and ACQUITY Premier SEC columns, as illustrated in Figure 5B. The Sepax Unix-C column, featuring a lay-down monolayer surface, potentially reduced secondary interactions compared to the stand-up monolayer of the Sepax Unix column. Consequently, the Unix-C column demonstrated better chromatographic performance than the Unix column. Notably, the Sepax Unix columns exhibited strong hydrophobic interactions with lipid-conjugated RNA structures, leading to significant retention and preventing their elution within the evaluated time range, in contrast to the other three columns. Additionally, the ACQUITY Premier column, utilizing polyethylene oxide (PEO) phase chemistry, showed secondary interactions with lipid conjugated RNA structures, resulting in peak broadening and diminished separation efficiency. The HMW species eluted first were mainly composed of RNA structures aggregates formed by molecule-molecule interaction. The EMW species eluted later were mainly composed of partially assembled intermediates and single strands. The peak shape and resolution for HMW and LMW species identified by the columns varied significantly,attributed to the interaction between RNA structures and the column's surface chemistry. The AdvanceBio SEC column demonstrated the best separation performance among the four columns.

[0137] A denaturing IPRP method was used to determine the individual strand component and to inform the assembly of RNA structures. Figure 5C illustrates that the method successfully identified all five individual peaks for the HPRT antisense strands and four distinct scaffold sense strands. During denatured IPRP analysis, the assembled RNA structures were denatured and separated into single strands. The component ratio was estimated using the absorbance at 260 nm (A260) for each single strand, normalized to the antisense strands' absorbance and adjusted with the extinction coefficient (e), enabling a novel single strand analysis for RNA structures. Each strand has a unique extinction coefficient, which is determined by the sequence and modifications of the oligonucleotides. The equation used for determining the strand component ratio was:A260scaffold strand * ^antisense strandRelative amount% = — — - : - ; - ;A260antisense strand * ^scaffold strandFigure 5C demonstrates that all sense strands (second nucleotide sequence) had lower molar ratio compared to the antisense strand (third nucleotide sequence). This slightly higher molar ratio of antisense strands aids in forming fully assembled RNA structures, preventing empty delivery vehicles. The equation used for determining the strand component ratio was:A260scaffold strand * ^antisense strandRelative amount% =A260antisense strand * ^scaffold strandFigure 5C shows that all sense strands had lower molar ratio compared to the antisense strand. This slightly higher molar ratio of antisense strands aids in forming fully assembled RNA structures, preventing empty delivery vehicles.

[0138] Online SEC x SEC 2D-LC was employed to evaluate the thermodynamic properties for RNA structures. As shown in Figure 5D, five consecutive cuts (cut 1 to 5) in 'D were sampled from the main peak region and analyzed with the same native SEC method in2D under formulationconditions (IX PBS). Figure 5E indicated that the equilibrium did not favor single strand dissociation or the formation of new intermediates, as no major peaks other than the primary peak were observed in2D. Online SEC x SEC results showed that single strands did not dissociate from assembled RNA structures under formulation conditions.

[0139] Following the first dimension native SEC analysis for RNA structures, an ion-pairing reverse phase (IPRP) method was used to analyze the strand composition to inform assembly homogeneity in the peak of interest. To structurally define the peaks observed from ’D SEC and determine the assembly stage, high-resolution online sampling was used to zoom in on the region of interest to study the single strand composition. Figure 5F shows two representative peaks that were sampled from theSEC and corresponding cuts from these two regions were sent to2D IPRP for strand analysis. One cut was collected from the main peak region (cut 1), which was mostly composed of assembled RNA structures. For this cut. the denatured IPRP method detected 5 major peaks, which were the HPRT antisense strands and all the 4 sense strands. The other cut was collected from the LMW species peak region (cut 2), which was mainly composed of partially assembled RNA structures. Three strands were identified for this cut, which were the antisense strands and two of the sense strands. The average mass for each of the single strands was confirmed with high resolution mass spectrometry and can be found in Table 8. The method also allowed for the monitoring of critical impurities, including for shortmers and phosphodiester (PO) impurities.Table 8. Oligonucleotide theoretical mass (Mw) and measured mass for single strands in RNA structures.AS= antisense strand

[0140] To evaluate the peak uniformity in the primary peak region, three cuts were subsequently collected from the main peak region in 'D SEC. These three cuts were further analyzed in2D denatured IPRP for peak uniformity evaluation through strand component analysis. HPRT antisense and sense strands were shown in Cuts 1, 2, and 3 as demonstrated in Figure 5GError! Reference source not found.. Cuts 1 and 2 had slight differences compared to Cut 3 in single strand component ratios. The consistency in peak uniformity observed across different cuts indicates a high level of precision in the RNA structure assembly process.

[0141] The SEC-MALS analysis provided data on the molecular weight distribution and heterogeneity of RNA structures. For the structure evaluated, three notable peaks were examined: the HWM region, the main peak region, and the LMW region. Figure 5H shows that the average molecular weight measured from SEC-MAES corresponded closely with the reference mass shown in Table 9. confirming the uniformity of RNA structures, particularly in the main peak region. This uniformity is crucial as it indicates a higher degree of precision in the assembly process, ensuring that the structures are consistent in their structure and function.Table 9. SEC-MALS molecular weight profiling of RNA structures.

[0142] Each RNA structure was composed of eight chemically modified oligonucleotides through self-assembly process, which contained a total of 304 nucleotides with a molecular weight (Mw) of 101,698 Da. To address challenges associated with assaying RNA structures, a nativeSEC-MS method was established. Ammonium acetate was utilized as the mobile phase to maintain the assembly and to replace metal ions associated with the RNA nanostructures with ammonium ions, which can be efficiently removed during the ionization process. As a result, clean ions of RNA structure with less than 5% of metal ions were obtained, as shown in Figure 51. Interestingly, these ions were obtained under positive mode rather than negative mode, indicating they are positively charged under native SEC-MS condition. Ions were also observed under negative mode, but the signal was very low. The RNA structure has a mass over charge (m / z) range of 5,000 to 7,500 and a deconvoluted mass of 101,700 Da, which is only a two Da difference from the theoretical mass, 101,698 Da (right side). The accurate mass measurement (less than 25 ppm) confirmed that the RNA structure is correctly assembled. The accurate mass measurement provided by the SEC-HRMS approach ensures that the structural integrity of the RNA structures is maintained, which is essential for their functionality and safety in therapeutic applications. Conclusions

[0143] Without being bound by any particular theory, the findings described in this example enable the analysis of RNA structure assembly and structural integrity, which is vital to the product development and clinical translation of RNA structures. A high degree of assembly uniformity is essential for ensuring the stability and efficacy of RNA structures. The findings will also improve both upstream and downstream process development to produce high quality products for in vitro and in vivo evaluations. These analytical methods are powerful tools that enable process optimization and characterization to understand a wide variety of complex therapeutic RNAs, such as multivalent siRNAs, antibody-siRNA conjugates, circular RNAs, and messenger RNA-based therapeutics.

Claims

CLAIMSWhat is claimed:

1. A RNA structure comprising,a first oligonucleotide comprising a first core nucleotide sequence comprising SEQ ID NO: 1;a second oligonucleotide comprising a second core nucleotide sequence comprising SEQ ID NO: 2;a third oligonucleotide comprising a third core nucleotide sequence comprising SEQ ID NO: 3; anda fourth oligonucleotide comprising a fourth core nucleotide sequence comprising SEQ ID NO: 4,wherein optionally one or more nucleotides of the first, second, third, and fourth oligonucleotides are independently modified nucleotides, andwherein optionally one or more internucleotide linkages of the first, second, third, and fourth oligonucleotides are modified intemucleotide linkages.

2. The RNA structure of claim 1,wherein the first oligonucleotide forms a duplex with both the second oligonucleotide and the fourth oligonucleotide,wherein the second oligonucleotide forms a duplex with both the first oligonucleotide and the third oligonucleotide,wherein the third oligonucleotide forms a duplex with both the second oligonucleotide and the fourth oligonucleotide, andwherein the fourth oligonucleotide forms a duplex with both the first oligonucleotide and the third oligonucleotide.

3. The RNA structure of claim 1 or 2, wherein one or more nucleotides in at least one of the first, second, third, and fourth oligonucleotides are modified nucleotides.

4. The RNA structure of any one of claims 1 -3, wherein each nucleotide in at least one of the first, second, third, and fourth oligonucleotide is a modified nucleotide.

5. The RNA structure of claim 4, wherein each nucleotide in the first, second, third, and fourth oligonucleotide is a modified nucleotide.

6. The RNA structure of any one of claims 1-5, wherein the modified nucleotide is a 2'-O-methyl modified nucleotide, 2'-F modified nucleotide, 2'-O-C16 alkyl modified nucleotide, or 2'-0-M0E modified nucleotide.

7. The RNA structure of any one of claims 1-6, wherein each of the first, second, third, and fourth oligonucleotides are between 20 to 70 nucleotides in length.

8. The RNA structure of any one of claims 1-7,wherein the first core nucleotide sequence comprises SEQ ID NO: 5;wherein the second core nucleotide sequence comprises SEQ ID NO: 6;wherein the third core nucleotide sequence comprises SEQ ID NO: 7: andwherein the fourth core nucleotide sequence comprises SEQ ID NO: 8.

9. The RNA structure of any one of claims 1-7,wherein the first core nucleotide sequence comprises SEQ ID NO: 9;wherein the second core nucleotide sequence comprises SEQ ID NO: 6;wherein the third core nucleotide sequence comprises SEQ ID NO: 10; andwherein the fourth core nucleotide sequence comprises SEQ ID NO: 8.

10. The RNA structure of any one of claims 1-9, wherein one or more of the first, second, third, and fourth oligonucleotides comprise a second nucleotide sequence,wherein optionally one or more nucleotides of the second nucleotide sequence are independently modified nucleotides, andwherein optionally one or more intemucleotide linkages of the second nucleotide sequence are modified intemucleotide linkages.

11. The RNA structure of claim 10, wherein one or more nucleotides in the second nucleotide sequence are modified nucleotides.

12. The RNA structure of claim 11, wherein each nucleotide in the second nucleotide sequence is a modified nucleotide.

13. The RNA structure of any one of claims 10-12, wherein the modified nucleotide is a 2' 0-methyl modified nucleotide, 2'-F modified nucleotide, 2'-O-C16 alkyl modified nucleotide, or 2'-O-MOE modified nucleotide.

14. The RNA structure of any one of claims 10-13, wherein the second nucleotide sequence is a sense strand of a double stranded RNA (dsRNA).

15. The RNA structure of claim 14, wherein the sense strand of the dsRNA does not bind to any one of the first core nucleotide sequence, the second core nucleotide sequence, the third core nucleotide sequence, or the fourth core nucleotide sequence.

16. The RNA structure of claim 14 or 15, wherein the sense strand of the dsRNA is positioned at the 5' end or 3' end of the first core nucleotide sequence, the second core nucleotide sequence, the third core nucleotide sequence, and / or the fourth core nucleotide sequence.

17. The RNA structure of claim 16, wherein the sense strand of the dsRNA is positioned at the 5' end of the first core nucleotide sequence, the second core nucleotide sequence, the third core nucleotide sequence, and / or the fourth core nucleotide sequence.

18. The RNA structure of claim 16, wherein the sense strand of the dsRNA is positioned at the 3' end of the first core nucleotide sequence, the second core nucleotide sequence, the third core nucleotide sequence, and / or the fourth core nucleotide sequence.

19. The RNA structure of any one of claims 14-18, wherein the first, second, third, and fourth oligonucleotides comprise the same sense strand sequence.

20. The RNA structure of any one of claims 14-18, wherein the first, second, third, and fourth oligonucleotides comprise two different sense strand sequences.

21. The RNA structure of any one of claims 14-18, wherein the first, second, third, and fourth oligonucleotides comprise three different sense strand sequences.

22. The RNA structure of any one of claims 14-18, wherein the first, second, third, and fourth oligonucleotides comprise four different sense strand sequences.

23. The RNA structure of any one of claims 14-22, wherein the second nucleotide sequence comprises a sequence that is one of SEQ ID NOs: 11-18.

24. The RNA structure of any one of claims 14-23, wherein the RNA structure further comprises an antisense oligonucleotide (ASO) attached to the second nucleotide sequence.

25. The RNA structure of any one of claims 10-13, wherein the second nucleotide sequence is an antisense oligonucleotide (ASO).

26. The RNA structure of any one of claims 14-23, wherein the RNA structure further comprises a third nucleic acid sequence that comprises a nucleic acid sequence complementary to the sense strand of the dsRNA, and wherein the third nucleic acid sequence forms a duplex with the sense strand of the dsRNA.

27. The RNA structure of claim 26, wherein the third nucleic acid sequence comprises a sequence that is one of SEQ ID NOs: 19-26.

28. The RNA structure of claim 26 or 27 wherein the third nucleic acid sequence comprises the antisense strand of an siRNA.

29. The RNA structure of claim 28, wherein the siRNA targets one or more of HPRT, SNCA, MAPT, APP, SARM1, SOD1, ATXN2, ATXN3, APOE, BACE1, FMRI, LRRK2, HTT, SCN10A, SCN9A, CACNA1B, PRNP, ACVR2a, and ACVR2b.

30. The RNA structure of any one of claims 1-29, wherein the RNA structure comprises one or more of SEQ ID NOs: 1-26.

31. The RNA structure of any one of claim 26. wherein the RNA structure further comprises an antisense oligonucleotide (ASO) that is reversibly attached to the third nucleic acid sequence.

32. The RNA structure of any one of claims 24, 25 and 31, wherein the ASO targets one or more of HPRT, SNCA, MAPT, APP, SARM1, SOD1, ATXN2, ATXN3, APOE, BACE1, FMRI, LRRK2, HTT, SCN10A, SCN9A, CACNA1B, PRNP, ACVR2a, ACVR2b.

33. A pharmaceutical composition comprising the RNA structure of any one of claims 1-32 and one or more pharmaceutically acceptable excipients.

34. A method of treating a disease or disorder in a patient in need thereof, comprising administering the RNA structure of any one of claims 1-32 or the pharmaceutical composition of claim 33.

35. The method of claim 34, wherein the disease or disorder is a genetic disease or disorder.

36. The method of claim 34 or 35, wherein the disease or disorder is a central nervous system (CNS) disease or disorder.

37. The method of claim 36, wherein the CNS disorder is Alzheimer’s disease, Parkinson’s disease, frontotemporal dementia (FTD), progressive supranuclear palsy (PSP), amyotrophic lateral sclerosis (ALS), Down’s syndrome, multiple sclerosis.

38. A method for delivering a molecule to the central nervous system (CNS) of a subject in need thereof, comprising administering the RNA structure of any one of claims 1-32 or the pharmaceutical composition of claim 33.

39. The method of claim 38, wherein the subject has a CNS disease or disorder.

40. A method for detecting the formation of an RNA nanoparticle, comprisinga) assaying the RNA nanoparticle of any one of claims 1-32 or the pharmaceutical composition of claim 33 by size-exclusion chromatography (SEC);b) separating RNA nanoparticles from one or more intermediate molecules identified in a); andc) assaying the separated RNA nanoparticles of b) using ion-pairing reverse phase (IPRP) chromatography.

41. The method of claim 40, wherein the SEC in a) is performed using a 200A, 1.9 pm, 4.6 mm x 300 mm column.

42. The method of claim 40 or 41, wherein the SEC column temperature is maintained at 25°C.

43. The method of any one of claims 40-42, wherein the IPRP chromatography of c) further comprises assaying the RNA nanoparticle or composition using a peak-based multiple heartcutting sampling mode.

44. The method of any one of claims 40-43. wherein the IPRP chromatography is performed using an analytical 300A, 1.7 pm, 2.1 mm x 150 mm column.

45. The method of any one of claims 40-44, wherein the IPRP column temperature is maintained at75°C.

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