Fatty acid conjugates of nucleic acids

Abstract

The present disclosure relates to a conjugate comprising a fatty acid moiety and a nucleic acid moiety, wherein the fatty acid moiety is conjugated to a nucleic acid via polyethylene glycol (PEG) and a glutamic acid group.The nucleic acid moiety may be an aptamer or its variant, an oligonucleotide, an antisense oligonucleotide, a CpG oligonucleotide, and a therapeutic RNA such as mRNA, siRNA, shRNA, microRNA, lncRNA, saRNA, and circular RNA.A method for producing the conjugate and its use are also provided.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 242,679, filed September 10, 2021, the entire contents of which are incorporated herein by reference. (Reference to sequence listing) This application has been filed with a Sequence Listing submitted electronically in XML format. Said XML copy, created on September 8, 2022, is named GBT-003WO.XML and is 17,707 bytes in size, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Nucleic acid-based therapeutics represent a novel category of drugs that target diseases and genes that are not targeted by classical small molecule approaches. One of the main challenges in developing nucleic acid-based drugs is to enhance their in vivo stability, half-life, clearance, and tissue distribution, thus increasing the therapeutic efficacy of the drug.

[0003] Chemically modified nucleosides are routinely used for incorporation into nucleic acid molecules to enhance one or more properties, such as, for example, nuclease resistance, pharmacokinetics, or affinity for a target. In some cases, additional modifications, such as chemical moieties, are conjugated to the nucleic acid therapeutic to improve the potency and efficacy of the nucleic acid compound.

[0004] The long blood circulation properties of human serum albumin provide an attractive technique for extending drug half-life. One strategy to increase albumin binding is to modify targeted drugs with fatty acid conjugates that can bind to albumin. One successful example is semaglutide, a glucagon-like peptide-1 receptor agonist (GLP-1 RA). Human GLP-1 is rapidly degraded by the dipeptidyl peptidase-4 (DPP-4) enzyme, resulting in a short half-life. Semaglutide, a human GLP-1 analog with 94% homology, contains two structural modifications, namely the substitution of the non-proteinogenic amino acid 2-aminoisobutyric acid (Aib) at position 2 of Gly, and the attachment of octadecanedioic acid to the side chain of Lys-26 via a short polyethylene glycol (PEG) spacer and a γ-glutamic acid linker. The presence of the 18-carbon fatty acid moiety confers high binding affinity to serum albumin, which translates into a half-life of approximately 7 days in humans (Non-Patent Document 1).

[0005] Similarly, lipid conjugation, including fatty acid conjugation, has been shown to affect therapeutic nucleic acids such as siNRA (eg, Non-Patent Document 2). The present disclosure relates to novel fatty acid conjugations to therapeutic nucleic acids, such as aptamers and small RNA molecules, where such fatty acid moieties affect in vivo efficacy, such as extending the half-life of the modified aptamer in the blood. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Witteloostuijn et al., Half-life extension of biopharmaceuticals using chemical methods: Alternatives to PEGylation. Chem Med Chem., 2016, 11(22):2474-2495 [Non-Patent Document 2] Biscans et al., The valency of fatty acid conjugates impact siRNA pharmacokinetics, distribution, and efficacy in vivo. J. Control Release, 2019, 302:116-125 Summary of the Invention

[0007] The present invention provides novel fatty acids that can be used to create nucleic acid-fatty acid conjugates to increase the performance of nucleic acid therapeutics, for example to extend in vivo half-life.

[0008] The present invention provides conjugates, compositions and methods of use thereof, comprising a nucleic acid moiety and at least one fatty acid moiety, wherein the fatty acid moiety is covalently attached to the nucleic acid moiety, wherein the fatty acid moiety modifies a characteristic, such as the half-life, of the nucleic acid moiety of the conjugate.

[0009] In some embodiments, the fatty acid moiety is -(CH) a -COOH, where a is an integer from 12 to 26. The conjugate may include at least one additional carboxyl group. In some examples, the conjugate may include one or two additional carboxyl groups. In one embodiment, the conjugate includes one additional carboxyl group. In another embodiment, the conjugate includes two additional carboxyl groups.

[0010] In some embodiments, the fatty acid moiety comprises at least one ethylene glycol group (-(OCH2CH2)-). In some examples, the fatty acid moiety comprises 1-10 ethylene glycol groups, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ethylene glycol groups.

[0011] In some embodiments, the fatty acid moiety comprises at least one polyethylene glycol (PEG) group. The fatty acid moiety may further comprise at least one amide group (-NH-CO-).

[0012] In one embodiment, the fatty acid moiety comprises at least one -(OCH2CH2-OCH2CH2-NH-CO)- group. In another embodiment, the fatty acid moiety comprises at least one -(CO-CH2-OCH2CH2-OCH2CH2-NH)- group.

[0013] In yet another embodiment, the fatty acid moiety comprises at least one glutamic acid group, in some embodiments, the glutamic acid group is a γ-glutamic acid group. In one embodiment, the fatty acid moiety is

[0014] [ka]

[0015] In another embodiment, the fatty acid moiety comprises:

[0016] [ka]

[0017] Includes. In some embodiments, the fatty acid moiety is a piperazine-2-carboxylic acid group (

[0018] [ka]

[0019] ),

[0020] [ka]

[0021] Groups, and

[0022] [ka]

[0023] The piperazine groups include the piperazine groups such as the group. In one embodiment, the conjugate comprises a fatty acid moiety comprising the structure:

[0024] [ka]

[0025] (Formula I), In the formula, n is an integer from 12 to 26, Block A is

[0026] [ka]

[0027] [ka]

[0028] or

[0029] [ka]

[0030] and na is 0 or 1, Block B is -(OCH2CH2)- or -(CO-CH2-OCH2CH2-OCH2CH2-NH)-; nb is an integer from 1 to 10; Block C is

[0031] [ka]

[0032] and nc is 0 or 1. As a non-limiting example, the conjugate comprises a fatty acid moiety selected from the group consisting of GTFA-1, GTFA-1', GTFA-2, GTFA-2', GTFA-3 and GTFA-3'.

[0033] The nucleic acid portion of the conjugate is a therapeutic nucleic acid, including, but not limited to, an aptamer or variants thereof, an oligonucleotide, an antisense oligonucleotide, a CpG oligonucleotide, an siRNA, a microRNA, an lncRNA, an mRNA, an antisense RNA, a saRNA, a circular RNA, and the like.

[0034] In some embodiments, the nucleic acid portion is an aptamer or a variant thereof, the aptamer comprising about 15-100 nucleotides, about 15-75 nucleotides, or about 15-50 nucleotides, or about 15-30 nucleotides, or about 20-50 nucleotides, or about 20-30 nucleotides.

[0035] In some embodiments, the aptamer comprises at least one chemical modification, such as a nucleoside modification and a backbone modification. In another aspect, the invention provides compositions comprising the nucleic acid-fatty acid conjugates and methods of using the compositions for therapeutic agents.

[0036] In some embodiments, the invention provides a method of increasing the half-life of a nucleic acid molecule, the method comprising modifying the nucleic acid molecule with a fatty acid moiety. In another aspect, the present invention provides a fatty acid moiety comprising the general formula:

[0037] [ka]

[0038] (Formula II), In the formula, n is an integer from 12 to 26, Block A is

[0039] [ka]

[0040] [ka]

[0041] or

[0042] [ka]

[0043] and na is 0 or 1, Block B is -(OCH2CH2)- or -(CO-CH2-OCH2CH2-OCH2CH2-NH)-; nb is an integer from 1 to 10; Block C is

[0044] [ka]

[0045] and nc is 0 or 1, Block D comprises a functional group capable of covalently binding to a nucleic acid moiety, nd is 0 or 1.

[0046] In some embodiments, the block D of the fatty acid moiety comprises an azide group, an alkyne group, a hydroxy group, a sulfhydryl group, or an amino group. d -N3, wherein R d In some embodiments, block D comprises an alkyl group, an aminoalkyl group, an amine group, and / or an alkoxyl group.

[0047] [ka]

[0048] [ka]

[0049] or

[0050] [ka]

[0051] As non-limiting examples, the fatty acid moiety is GTFA-1, GTFA-1', GTFA-2, GTFA-2', GTFA-3, or GTFA-3'. In another aspect, the present invention provides a conjugate comprising a polynucleotide comprising a nucleic acid sequence represented by SEQ ID NO: 1 and a fatty acid moiety conjugated to one end of SEQ ID NO: 1. As non-limiting examples, the conjugates are BT500 (SEQ ID NO: 3), BT600 (SEQ ID NO: 4), and BT700 (SEQ ID NO: 5). In some embodiments, a composition comprising a conjugate is provided, wherein the conjugate comprises BT500 (SEQ ID NO: 3), BT600 (SEQ ID NO: 4), or BT700 (SEQ ID NO: 5). [Brief description of the drawings]

[0052] [Figure 1] Figure 1 shows the fatty acid conjugates BT500, BT600, and BT700 binding to albumin. BT100 is tested as a control. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] The details of one or more embodiments of the present disclosure are described in the accompanying description below. Although any method and material similar or equivalent to the materials and methods described herein can be used to practice or test the present disclosure, the preferred materials and methods are now described. Other features, objects, and advantages of the present disclosure will be apparent from the description. In the description, the singular form includes the plural unless the context clearly dictates otherwise. All technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present disclosure belongs, unless otherwise defined. In case of discrepancy, the present description shall prevail.

[0054] Introduction Conjugation of moieties to nucleic acid molecules, such as small therapeutic nucleic acid molecules, is useful to modify the characteristics of the nucleic acid, for example to increase half-life in the body, stability, target binding, functional efficacy, etc. Great advances have been made in the technology of conjugating other functional molecules to nucleic acids directly or indirectly via post-synthetic labeling and various conjugation chemistries. For example, click chemistry is one of the most robust and efficient chemistries useful for biomolecular conjugation and is widely used in protein and DNA / RNA conjugation.

[0055] It has been observed that free fatty acids can bind to human serum albumin (HSA) (Curry S. et al., Fatty acid binding to human serum albumin: new insights from crystallographic studies. Biochim Biophys Acta, 1999, 1441: 131-140). HSA is abundant in blood and plays an important role in drug metabolism (Bhattacharya, AA, et al., Crystallographic analysis reveals common modes of binding of medium and long-chain fatty acids to human serum albumin. J Mol Biol., 2000, 303: 721-732). Most drugs bind to HSA and are released for blood circulation and tissue implantation (Ghuman, J. et al. Structural basis of the drug-binding specificity of human serum albumin. J Mol Biol., 2005, 353:38-52). The function of binding to HAS makes fatty acid a very useful tool for extending the blood retention time, i.e., half-life, of drugs.Many studies have demonstrated that the conjugation of fatty acid with therapeutic agents, such as peptides and siRNA, can delay the absorption rate, extend the circulation period, and protect against proteolysis (e.g., Troiber, C. et al., Stabilizing effect of tyrosine trimers on pDNA and siRNA polyplexes. Biomaterials, 2013, 34: 1624-1633, and Hackett, MJ, et al., A dicarboxylic fatty acid derivative of paclitaxel for albumin-assisted drug delivery. J Pharm Sci., 2012, 101:3292-3304).It was reported that conjugation of C16 fatty acids resulted in a significant increase in the half-life of GLP-1 peptides (Madsen, K. et al. Structure-activity and protraction relationship of long-acting glucagon-like peptide-1 derivatives: importance of fatty acid length, polarity and bulkiness. J Med Chem, 2007, 50, doi: 10.1021 / jm070861j). Due to its efficiency and clinically non-toxic effects, fatty acid conjugation has been developed as a useful strategy to extend the half-life and stability of therapeutic agents (e.g., peptides, DNA, and RNA).

[0056] The length and structure of fatty acid may affect different therapeutic agents. Conjugation of fatty acid with active therapeutic agent requires complicated process. In many cases, functional group in therapeutic agent is limited by its chemical and physical properties, such as its structural conformation. Active site in fatty acid may also limit their application. To overcome this obstacle, active group can be introduced into fatty acid and / or therapeutic agent. Modification can be achieved through molecular modification or linker to conjugate drug and fatty acid.

[0057] The present disclosure provides modified fatty acid analogs and optimized conjugation strategies for fatty acid conjugation to nucleic acid molecules, particularly small nucleic acid molecules, such as aptamers, oligonucleotides, antisense oligonucleotides, CpG oligonucleotides, siRNA, siRNA, microRNA, lncRNA, mRNA, antisense RNA, saRNA, etc. The fatty acid-like molecules of the present disclosure (referred to herein as "fatty acid moieties") contain one or more functional groups, such as two or more carboxyl groups, as well as additional active groups, such as azide groups, alkyne groups, hydroxy groups, sulfhydryl groups, and amino groups.

[0058] definition In order to more clearly and concisely describe the claimed subject matter of the present disclosure, the following definitions are provided for specific terms, which are used in the following description and the appended claims. Throughout this specification, illustrations of specific terms should be considered as non-limiting examples.

[0059] As used herein, the term "aliphatic" means a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is fully saturated or contains one or more units of unsaturation, or a substituted or unsubstituted monocyclic, bicyclic, or polycyclic hydrocarbon ring that is fully saturated or contains one or more units of unsaturation (but is not aromatic), or a combination thereof. In some cases, an aliphatic group contains 2-50 aliphatic carbon atoms, or 2-20 aliphatic carbon atoms, or 8-30 aliphatic carbon atoms, or 10-20 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 aliphatic carbon atoms. In accordance with the present disclosure, aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups, and hybrids thereof.

[0060] As used herein, the term "binding" refers to a sequence-specific, non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid). Not all components of a binding interaction need be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), as long as the interaction as a whole is sequence-specific. Such interactions are generally characterized by a 10 6 M 1 The following dissociation constant (K d The term "affinity" refers to the strength of binding, and increased binding affinity is characterized by a lower K d Correlated with.

[0061] As used herein, the term "molecular weight" generally refers to the mass or average mass of a material. In the case of a polymer or oligomer, the molecular weight can refer to the relative average chain length or relative chain mass of the bulk polymer. The molecular weight of a molecule may be calculated as the sum of the atomic weights of each atom multiplied by the number of each atom in the formula of the conjugate. It may also be measured by mass spectrometry, NMR, chromatography, light scattering, viscosity, and / or any other method known in the art. It is known in the art that the units of molecular weight may be g / mol, Daltons (Da), or atomic mass units (amu), where 1 g / mol=1 Da=1 amu.

[0062] As used herein, the term "pharmaceutical composition" refers to a combination of an active agent (e.g., a therapeutic nucleic acid) with an inert or active carrier, making the composition particularly suitable for in vivo or ex vivo diagnostic or therapeutic applications. The carrier in a pharmaceutical composition must be acceptable in the sense that it is compatible with the active ingredient and can stabilize it. One or more solubilizing agents may be utilized as pharmaceutical carriers for the delivery of the active agent. Examples of pharmaceutical acceptable carriers include, but are not limited to, biocompatible vehicles, adjuvants, additives, and diluents to achieve a composition usable as a dosage form. Examples of other carriers include colloidal silicon oxide, magnesium stearate, cellulose, and sodium lauryl sulfate.

[0063] As used herein, the term "pharmaceutically acceptable carrier or excipient" refers to a carrier or excipient that is generally safe, non-toxic, and not biologically or otherwise undesirable, useful in the preparation of a pharmaceutical composition, including carriers or excipients acceptable for veterinary use as well as for human pharmaceutical use. As used in the specification and claims, "pharmaceutically acceptable carrier or excipient" includes both one and more than one such carrier or excipient. As used herein, the term "pharmaceutically acceptable carrier" encompasses any of the standard pharmaceutical carriers, such as phosphate buffered saline, water, and emulsions, such as oil / water or water / oil emulsions, as well as various types of wetting agents. In some examples, compositions and formulations may also include stabilizers and preservatives.

[0064] As used herein, the term "pharmaceutical acceptable" refers to molecular entities and compositions that are physiologically tolerable and typically do not produce undesirable reactions when administered to humans. Preferably, as used herein, the term "pharmaceutical acceptable" means approved by a federal or state government regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopoeias that are generally recognized as safe for use in animals, more specifically humans, or in parenteral products.

[0065] As used herein, the term "treat" or "treatment" refers to both therapeutic and prophylactic or preventative treatments, which refer to preventing, curing, reversing, attenuating, mitigating, minimizing, suppressing, or halting the deleterious effects of a pathology, disease progression, disease causative agent (e.g., bacteria or virus), or other abnormal condition.

[0066] As used herein, the term "therapeutic agent" refers to any agent that has a therapeutic effect and / or induces a desired biological and / or pharmacological effect when administered to a subject. In some embodiments, a therapeutic agent is any substance that can be used to alleviate, ameliorate, mitigate, inhibit, prevent, delay onset, reduce severity, and / or reduce incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition. Examples of therapeutic agents may include, but are not limited to, small molecule drugs, chemotherapeutic agents, immunotherapeutic agents, therapeutic antibodies and fragments thereof, toxins (e.g., immunotoxins), radioisotopes, enzymes (e.g., enzymes that cleave prodrugs to cytotoxic drugs at target sites), nucleases, hormones, immunomodulatory agents, aptamers, antisense oligonucleotides, CpG oligodeoxynucleotides (or CpG ODNs), nucleic acid molecules (e.g., mRNA molecules, cDNA molecules, microRNA molecules, RNAi molecules such as siRNA or shRNA, saRNA, or lncRNA molecules), chelators, boron compounds, photoactive agents, and dyes. The therapeutic agent may also include metal, metal alloy, intermetallic or core-shell nanoparticles bound to chelators that act as radiosensitizers to make targeted cells more sensitive to radiation therapy compared to healthy cells.

[0067] As used herein, the term "therapeutically effective amount" generally refers to the amount of an aptamer of the present disclosure to affect a desired biological response. Such a response may be a beneficial outcome, including, but not limited to, the improvement, reduction, prevention, or elimination of symptoms of a disease or disorder. Thus, the total amount of each active component of the aptamer or method is sufficient to show a meaningful benefit in a subject in need thereof, including, but not limited to, the treatment of cancer. A therapeutically effective amount may be administered via one or more prophylactic or therapeutic administrations. When "therapeutically effective level" applies to a single component administered alone, the term refers to that composition alone. When applied to a combination, the term refers to the combined amount of active composition that produces a therapeutic effect, whether administered in combination, sequentially, or simultaneously. The exact amount required will vary from subject to subject, depending, for example, on the species, age, and general condition of the subject, the severity of the condition being treated, the particular target antigen, in the case of an immunological response, for example, the ability of the subject's immune system to synthesize antibodies, and the degree of protection desired, as well as the mode of administration. The appropriate "effective" amount in any individual case may be determined by one of skill in the art. Thus, a "therapeutically effective amount" will typically fall in a relatively broad range that can be determined through routine trials.

[0068] As used herein, the terms "patient", "individual" or "subject" are used interchangeably and are intended to include human and non-human animals. Exemplary human subjects include human patients suffering from cancer, particularly HCC. The term "non-human animal" includes all vertebrates, such as non-mammals (chickens, amphibians, reptiles, etc.), and mammals (non-human primates, livestock animals and / or animals useful for agriculture (sheep, dog, cat, rabbit, cow, pig, etc.), and rodents (mouse, rat, hamster, guinea pig, etc.)).

[0069] composition Fatty Acid-Nucleic Acid Conjugates The present disclosure relates to a fatty acid-nucleic acid conjugate. The conjugate disclosed herein comprises at least one fatty acid moiety and a nucleic acid moiety, and the fatty acid moiety is conjugated to a nucleic acid. The fatty acid moiety is preferably conjugated to one end of the nucleic acid. The conjugate has an extended half-life compared to the nucleic acid alone without the fatty acid moiety. Also provided are compositions comprising the fatty acid-nucleic acid conjugates described herein.

[0070] As used herein, the term "conjugate" is used to refer to two or more entities (e.g., moieties) that are linked by direct or indirect covalent or non-covalent interactions. In some embodiments, the interaction is covalent. In some embodiments, the covalent interaction is mediated by a linker moiety. In some embodiments, the interaction is non-covalent (e.g., charge interactions, affinity interactions, metal coordination, physical adsorption, host-guest interactions, hydrophobic interactions, stacking interactions, hydrogen bonding interactions such as through "sticky sequences", van der Waals interactions, magnetic interactions, electrostatic interactions, dipole-dipole interactions, etc.).

[0071] As used herein, the term "moiety" refers to a specific portion or functional group of a molecule or compound (e.g., a conjugate) that lacks one or more atoms compared to a corresponding reagent, such as when a reagent of the formula "H--X--H" reacts with another reagent and becomes part of the reaction product.

[0072] According to the present disclosure, the terms "nucleic acid", "oligonucleotide", and "polynucleotide" should not be considered limiting in any way and may be used interchangeably herein. A nucleic acid molecule is a polymer of nucleotides consisting of at least two nucleotides covalently linked together. Nucleic acid molecules are DNA (deoxyribonucleotides), RNA (ribonucleotides), and recombinant RNA and DNA molecules, or analogs of DNA or RNA produced using nucleotide analogs. Nucleic acids may be single-stranded or double-stranded, linear or circular. The term also includes fragments of nucleic acids, such as naturally occurring RNA or DNA that may be recovered using the disclosed extraction methods, or artificial DNA or RNA molecules artificially synthesized in vitro (i.e., synthetic polynucleotides). The molecular weight of the nucleic acid is also not limited and may be optionally within the range of a few base pairs (bp) to several hundred base pairs, for example, from about 2 nucleotides to about 1,0000 nucleotides, or from about 10 nucleotides to 5,000 nucleotides, or from about 10 nucleotides to about 1,000 nucleotides. "Oligonucleotide" is used when the nucleic acid molecule concerned typically contains less than about 100 bases. "Polynucleotide" is used when the nucleic acid molecule concerned typically contains more than about 100 bases. All terms are used to mean DNA, RNA, modified or synthetic DNA or RNA (including but not limited to nucleic acids containing synthetic and naturally occurring base analogs, dideoxy or other sugars, thiols or other non-natural or natural polymer backbones), or other nucleic acid base containing polymers that can hybridize to DNA and / or RNA. Thus, this term should not be construed to define or limit the length of the nucleic acid referred to and used herein, nor should this term be used to limit the nature of the polymer backbone to which the nucleic acid base is attached.Types of nucleic acids include, but are not limited to, oligonucleotides, such as antisense oligonucleotides and CpG oligonucleotides, aptamers and variants thereof, and small RNA molecules, such as microRNA, siRNA, shRNA, lncRNA and saRNA, mRNA, and cDNA.

[0073] As used herein, the term "fatty acid moiety" refers to a molecule or radical in the context of a conjugate, where the molecule or radical comprises a fatty acid. In some embodiments, the fatty acid moiety comprises at least one additional carboxyl group in addition to the carboxyl group of the fatty acid. In some embodiments, the fatty acid moiety comprises one additional carboxyl group in addition to the carboxyl group of the fatty acid. In some embodiments, the fatty acid moiety comprises two additional carboxyl groups in addition to the carboxyl group of the fatty acid.

[0074] In some embodiments, the molecular weight of the conjugate is at least 5KDa, for example, between 5K and 10KDa, between 10K and 20KDa, between 20K and 30KDa, between 30K and 40KDa, or between 40K and 50KDa.

[0075] In some embodiments, the conjugate has at least one carboxyl group (-COOH), for example, one carboxyl group, two carboxyl groups, or three carboxyl groups.

[0076] Nucleic acid molecules such as aptamers can be conjugated to any suitable position of fatty acid moiety, except terminal -COOH, via optional linker, to form conjugate, as long as the function of nucleic acid, such as binding of aptamer to its target, is not adversely affected.In some embodiments, nucleic acid, for example, aptamer, is attached to the end of fatty acid moiety.After nucleic acid is conjugated to fatty acid moiety, hydrogen or functional group at the end of fatty acid moiety is replaced with nucleic acid.

[0077] In some embodiments, the fatty acid moiety is conjugated to one end of the nucleic acid molecule. In some examples, the fatty acid moiety is conjugated to the 5' end of the nucleic acid. In other examples, the fatty acid moiety is conjugated to the 3' end of the nucleic acid.

[0078] In some embodiments, the fatty acid moiety is conjugated to the nucleic acid of interest via a linker. The fatty acid moiety described herein has been found to increase the half-life of said biomolecule much more than more commonly used fatty acid residues. In some embodiments, the fatty acid moiety binds to albumin. In some embodiments, the conjugate comprising the fatty acid moiety and nucleic acid has low renal clearance. In some embodiments, the conjugate has lower renal clearance than nucleic acid alone.

[0079] In some embodiments, the fatty acid moiety is covalently linked to the nucleic acid molecule described herein. The method of linking the fatty acid to the nucleic acid is not particularly limited. The fatty acid to the nucleic acid may be linked directly or via a linker (linkage region). In some embodiments, the linker used to link the fatty acid to the nucleic acid comprises a nucleic acid. In some embodiments, the linker used to link the fatty acid to the nucleic acid does not comprise a nucleic acid. Exemplary linkers include -OP(=O)(OH)-O-, -O-CO-O-, -NH-CO-O-, -NH-CO-NH-, -NH-(CH2) n1 -, -S-(CH2) n1 -, -CO-(CH2) n1 -CO-, -CO-(CH2) n1 -NH-, -NH-(CH2) n1 -NH-, -CO-NH-(CH2) n1 -NH-CO-, -C(=S)-NH-(CH2) n1 -NH-CO-, -C(=S)-NH-(CH2) n1 -NH-C-(=S)-, -CO-O-(CH2) n1 -O-CO-, -C(=S)-O-(CH2) n1 -O-CO-, -C(=S)~O-(CH2) n1-OC-(=S)-, -CO-NH-(CH2) n1 -O-CO-, -C(=S)-NH-(CH2) n1 -O-CO-, -C(=S)-NH-(CH2) n1 -OC-(=S)-, -CO-NH-(CH2) n1 -O-CO-, -C(=S)-NH-(CH2) n1 -CO-, -C(=S)-O-(CH2) n1 -NH-CO-, -C(=S)-NH-(CH2) n1 -OC-(=S)-, -NH-(CH2CH2O) n3 CH(CH20H)-, -NH-(CH2CH2O) n2 -CH2-, -NH-(CH2CH2O) n2 CO-, -O-(CH2) n3 -SS-(CH2)n4-OP( = 0)2-, -CO-(CH2)n3-O-CO-NH-(CH2) n4 - and -CO-(CH2)n3-CO-NH-(CH2) n 4-, but are not limited to these.

[0080] According to the present disclosure, the nucleic acid of the conjugate may be an oligonucleotide (e.g., antisense oligonucleotide (ASO) and CpG oligodeoxynucleotide (CpG ODN)), an aptamer or variants thereof, antisense RNA, small RNA, RNAi agent, small interfering nucleic acid (siRNA), small hairpin RNA (shRNA) molecule, long non-coding RNA (long ncRNA, lncRNA), small activating RNA (SaRNA), microRNA (miRNA), messenger RNA (mRNA), double-stranded RNA (dsRNA), circular RNA, etc. The nucleic acid molecule is a therapeutic nucleic acid.

[0081] "Oligonucleotide" refers to a short polymer of nucleotides and / or nucleotide analogs. Oligonucleotides can be 10-200 nucleotides in length, or 10-100 nucleotides in length, or 10-50 nucleotides in length, or 50-100 nucleotides in length. In some embodiments, the oligonucleotide may comprise at least 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 40 nucleotides, 41 nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48 ​​nucleotides, 49 nucleotides, or 50 nucleotides. "CpG oligodeoxynucleotides (or CpG ODNs)" are short, single-stranded DNA molecules that contain a CpG motif consisting of a cytosine triphosphate deoxynucleotide ("C") followed by a guanine triphosphate deoxynucleotide ("G"). The "p" refers to the phosphodiester link between consecutive nucleotides. CpG ODNs are a new class of Th-1 type immune stimulants that bind to and activate Toll-like receptor 9 (TLR-9). Antisense oligonucleotides (ASOs) are short, synthetic, single-stranded oligodeoxynucleotides that can alter RNA and reduce, restore, or modify protein expression through several distinct mechanisms.

[0082] "RNAi agent" refers to a nucleic acid molecule that contains a sequence that recognizes target mRNA sequence and induces target-specific RNA interference (RNAi). RNAi agent may be a single-stranded oligonucleotide or a double-stranded oligonucleotide. RNAi agent may be siRNA (short inhibitory RNA), shRNA (short or small hairpin RNA), dsRNA (double-stranded RNA), microRNA, etc.

[0083] "Small RNAs" are short (approximately 18-30 nucleotides) non-coding RNA molecules that can regulate gene expression in both the cytoplasm and nucleus via post-transcriptional gene silencing (PTGS), chromatin-dependent gene silencing (CDGS), or RNA activation (RNAa). There are three main classes of small RNAs: microRNAs (miRNAs), siRNAs, and Piwi-interacting RNAs (piRNAs) (e.g., Farazi et al., the growing catalog of small RNAs and their association with distinct Argonaute / Piwi family members. Development. 2008;135(7):1201-1214).

[0084] "mRNA" refers to a single-stranded RNA molecule that is complementary to one strand of a gene's DNA strand. It provides the genetic code and a template for translating the genetic code into their corresponding proteins.

[0085] "Aptamer" refers to a biomolecule that binds to a specific target molecule and modulates the activity, structure, or function of the target. Aptamers may be nucleic acid or amino acid based. In the context of this disclosure, the aptamer is a nucleic acid aptamer.

[0086] In some embodiments, the nucleic acid of the conjugate is therapeutic nucleic acid.As used herein, the term "therapeutic nucleic acid" refers to the nucleic acid molecule that is used as therapeutic agent.Exemplary therapeutic nucleic acids include aptamers, antisense oligonucleotides, mRNA, cDNA, RNAi molecules such as siRNA or shRNA, and saRNA.

[0087] In some embodiments, the nucleic acid moiety is an activating nucleic acid moiety or an antisense nucleic acid moiety. In some embodiments, the nucleic acid of the conjugate may be modified to promote enhanced efficacy and specificity, and / or to improve stability.Various combinations of modifications (e.g., chemical modifications) and / or conjugates may be used to modify the nucleic acid of the conjugate.Nucleotide analogs may be used to modify the nucleic acid, including sugar and / or backbone modified ribonucleotides, such as phosphothioate groups, sugar modifications at the 2'OH group, such as 2'-fluoro, 2'-amino and / or 2'-thio modifications. Particularly exemplary modifications include 2'-fluoro-cytidine, 2'-fluoro-uridine, 2'-fluoro-adenosine, 2'-fluoro-guanosine, 2'-amino-cytidine, 2'-amino-uridine, 2'-amino-adenosine, 2'-amino-guanosine, 2-aminopurine, 2'-amino-butyryl-pyrene-uridine, and 2,6-diaminopurine, at the 4' position, e.g., 4-thio-uridine, and / or at the 5' position, e.g., 5-amino-allyl-uridine, 5-bromo-uridine, 5-iodo-uridine, 5-methyl-cytidine, 5-fluoro-cytidine, 5-fluoro-uridine and 5-ribo-thymidine. Additional modified residues include deoxyabasic, inosine, N3-methyl-uridine, N6,N6-dimethyl-adenosine, pseudouridine, purine ribonucleosides, ribavirin, locked nucleic acid (LNA), and / or peptide nucleic acid (PNA).

[0088] Aptamers In a preferred embodiment, the nucleic acid of the conjugate is an aptamer, e.g., an aptamer identified by SELEX that binds with high affinity to a specific target, which may be a protein, peptide, nucleic acid molecule, lipid, sugar, chemical compound, cell, tissue, bacteria, and other analytes.

[0089] Aptamers refer to biomolecules that bind to specific target molecules and often modulate the activity, structure, or function of the target. Aptamers often have similar characteristics to antibodies and are referred to as "chemical antibodies." Aptamers can be either nucleic acid-based or amino acid-based, i.e., nucleic acid aptamers or peptide aptamers. Nucleic acid aptamers have specific binding affinity for target molecules through interactions other than classical Watson-Crick base pairing. Nucleic acid aptamers can specifically bind to selected targets with high affinity. Some aptamers can interfere with the function of the target through binding.

[0090] The aptamers of the present disclosure are synthetic oligonucleotides.Typical nucleic acid aptamers are approximately 10-15 kDa in size, bind to their targets with nanomolar to sub-nanomolar affinity, and discriminate against closely related targets.The targets of nucleic acid aptamers can be, but are not limited to, proteins, nucleic acid molecules, peptides, small molecules, and whole cells.

[0091] Nucleic acid aptamers may be ribonucleic acid (RNA), deoxyribonucleic acid (DNA), or mixed ribonucleic acid and deoxyribonucleic acid (DNA / RNA hybrid). Aptamers may be single stranded. Suitable nucleotide lengths for aptamers range from about 15 to about 150 nucleotides, and in various other preferred embodiments have a length of any of 15-30 nucleotides, 20-25 nucleotides, 20-45 nucleotides, 30-100 nucleotides, 30-60 nucleotides, 25-70 nucleotides, 25-60 nucleotides, 40-60 nucleotides, 25-40 nucleotides, 30-40 nucleotides, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides, or 30-50 nucleotides, 40-70 nucleotides, or 50-100 nucleotides. In some embodiments, the aptamer may be 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 nucleotides in length. In other embodiments, the aptamer may be 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides in length. However, the sequence can be designed with sufficient flexibility to accommodate the interaction of the aptamer with the target.

[0092] The term "nucleotide" refers to a compound consisting of a nucleic acid monomer, a heterocyclic base, a sugar, and one or more phosphate groups. The bases are derivatives of purines and pyrimidines, and the sugars are pentoses, either deoxyribose or ribose.

[0093] As used herein, the term "modification" refers to techniques that chemically react a nucleic acid, e.g., an oligonucleotide, with a chemical reagent. The nucleic acid may be modified at the base moiety, sugar moiety, or phosphate backbone. Modifications include, but are not limited to, 2'-position sugar modification, 5-position pyrimidine, modification, 8-position purine modification, modification with exocyclic amine, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil, backbone modification, phosphorothioate or alkyl phosphate modification, methylation, unusual base pair combinations such as isobases isocytidine and isoguanidine, and the like. Modifications may also include 3' and 5' modifications such as capping. Nucleic acid molecules may also be modified by conjugation to moieties with desired biological properties. Such moieties may include, but are not limited to, compounds, peptides and proteins, carbohydrates, antibodies, enzymes, polymers, drugs, and fluorophores. In some examples, the polynucleotides are conjugated with lipophilic compounds such as cholesterol, dialkylglycerol, diacylglycerol, or non-immunogenic high molecular weight compounds or polymers such as PEG (polyethylene glycol), or other water-soluble pharma- ceutically acceptable polymers, including, but not limited to, polyaminoamines (PAMAM), and polysaccharides such as dextran or polyoxazolines (POZ).Modifications may be intended, for example, to increase the in vivo stability of the nucleic acid molecule, or to enhance or mediate delivery of the molecule.

[0094] Aptamers may be either monovalent or multivalent. Aptamers may be monomeric, dimeric, trimeric, tetrameric, or other higher multimers. Individual aptamer monomers may be linked to form multimeric aptamer fusion molecules. As a non-limiting example, a binding oligonucleotide (i.e., a linker) may be designed to contain sequences complementary to both the 5'-arm and 3'-arm regions of a random aptamer to form a dimeric aptamer. For trimeric or tetrameric aptamers, small trimeric or tetrameric (i.e., Holliday junction-like) DNA nanostructures are engineered to contain sequences complementary to the 3'-arm region of a random aptamer, thus generating a multimeric aptamer fusion via hybridization. Additionally, 3-5 or 5-10 dT-rich nucleotides can be engineered into the linker polynucleotide as single-stranded regions between the aptamer-binding motifs, providing flexibility and freedom for multiple aptamers to coordinate and synergize multivalent interactions with cellular ligands or receptors. Alternatively, multimeric aptamers can also be formed by mixing biotinylated aptamers with streptavidin.

[0095] As used herein, the term "multimeric aptamer" or "multivalent aptamer" refers to an aptamer that includes multiple monomeric units, each of which may itself be an aptamer. A multivalent aptamer has multivalent binding properties. A multimeric aptamer may be a homomultimer or a heteromultimer. The term "homomultimer" refers to a multimeric aptamer that includes multiple binding units of the same type, i.e., each unit binds to the same binding site of the same target molecule. The term "heteromultimer" refers to a multimeric aptamer that includes multiple binding units of different types, i.e., each binding unit binds to a different binding site of the same target molecule, or each binding unit binds to a binding site on a different target molecule. Thus, a heteromultimer can refer to a multimeric aptamer that binds to one target molecule at different binding sites, or a multimeric aptamer that binds to different target molecules. A heteromultimer that binds to different target molecules may also be referred to as a multispecific multimer.

[0096] Nucleic acid aptamers contain a series of linked nucleosides or nucleotides. The term "nucleic acid" in its broadest sense includes any compound and / or substance that contains a polymer of nucleotides. These polymers are often referred to as polynucleotides. Exemplary nucleic acid molecules or polynucleotides of the present invention include, but are not limited to, either D-nucleic acid or L-nucleic acid, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, including LNA with β-D-ribo configuration, α-LNA (a diastereomer of LNA) with α-L-ribo configuration), 2'-amino-LNA with 2'-amino functionalization, and 2'-amino-α-LNA with 2'-amino functionalization) or hybrids thereof.

[0097] Nucleic acid aptamers may be ribonucleic acid, deoxyribonucleic acid, or mixed ribonucleic acid and deoxyribonucleic acid. Aptamers may be single-stranded ribonucleic acid, deoxyribonucleic acid, or mixed ribonucleic acid and deoxyribonucleic acid.

[0098] Aptamers can be generated against target molecules (e.g., proteins of interest) using a process called either in vitro selection (Ellington and Szostak, In vitro selection of RNA molecules that bind specific ligands. Nature. 1990; 346: 818-822) or SELEX (Tuerk and Gold, Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase; Science, 1990, 249: 505-510). This method allows for the in vitro evolution of nucleic acid molecules with highly specific binding target molecules. The SELEX method is described, for example, in U.S. Patent No. 7,087,735, U.S. Patent No. 5,475,096, and U.S. Patent No. 5,270,163, the contents of each of which are incorporated herein by reference in their entirety. Nucleic acid aptamers can be synthesized using methods known in the art. For example, the disclosed aptamers may be synthesized using standard oligonucleotide synthesis techniques known in the art.

[0099] In some embodiments, the aptamer comprises at least one chemical modification. In some embodiments, the chemical modification is selected from chemical substitution of the nucleic acid at the sugar position, chemical substitution at the phosphate position, and chemical substitution at the base position. In other embodiments, the chemical modification is selected from incorporation of modified nucleotides, 3'-capping, conjugation to a high molecular weight non-immunogenic compound, conjugation to a lipophilic compound, and incorporation of phosphorothioates into the phosphate backbone. In a preferred embodiment, the high molecular weight non-immunogenic compound is a polyalkylene glycol, more preferably polyethylene glycol (PEG). The process of covalent attachment of PEG to another molecule, usually a drug or therapeutic protein, is known as PEGylation. PEGylation is routinely achieved by incubation of a reactive derivative of PEG with the target molecule. Covalent attachment of PEG to a drug or therapeutic protein can increase the hydrodynamic size (size of solution) of the drug, masking the drug from the host's immune system, thereby providing reduced immunogenicity and antigenicity, and extending its circulation time by reducing renal clearance. PEGylation can also provide water solubility for hydrophobic drugs and proteins.

[0100] In some embodiments, nucleic acid aptamers are provided in which the P(O)O group is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), P(O)NR2 ("amidate"), P(O)R, P(O)OR', CO or CH2 ("formacetal"), or 3'-amine (-NH-CH2-CH2-), where each R or R' is independently H or substituted or unsubstituted alkyl. Linkage groups can be attached to adjacent nucleotides via -O-, -N-, or -S- linkages. Not all linkages in a nucleic acid aptamer need be identical.

[0101] As non-limiting examples, nucleic acid aptamers can include D-ribose or L-ribose nucleic acid residues and can include at least one modified ribonucleoside including, but not limited to, a 2'-O-methyl modified nucleoside, a nucleoside containing a 5' phosphorothioate group, a terminal nucleoside linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, a locked nucleoside and an abasic nucleoside, an inverted deoxynucleoside or an inverted ribonucleoside, a 2'-deoxy-2'-fluoro modified nucleoside, a 2'-amino modified nucleoside, a 2'-alkyl modified nucleoside, a morpholino nucleoside, a phosphoramidate containing nucleoside or a non-natural base, or any combination thereof. Alternatively, a nucleic acid aptamer can contain at least 2 modified ribonucleosides, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or more modified ribonucleosides, up to the entire length of the molecule. The modifications need not be the same for each of such multiple modified deoxynucleosides or ribonucleosides in a nucleic acid molecule.

[0102] The aptamers of the invention may include modifications or substitutions of nucleobases (often referred to in the art simply as "bases"). As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxylanal, other 8-substituted adenines and guanines, 5-halo, Specifically included are 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and adenine, 8-azaguanine and adenine, 7-deazaguanine and adenine, and 3-deazaguanine and adenine.Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; The Concise Encyclopedia of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press, 1993.

[0103] In some embodiments, the nucleic acid aptamer comprises one or more regions of double-stranded character. Such double-stranded regions may result from internal self-complementarity or complementarity with a second or further aptamer or oligonucleotide molecule. In some embodiments, the double-stranded region may be 4-12, 4-10, 4-8 base pairs in length. In some embodiments, the double-stranded region may be 5, 6, 7, 8, 9, 10, 11, or 12 base pairs in length. In some embodiments, the double-stranded region may form a stem region. Such extended stem regions with double-stranded character may function to stabilize the nucleic acid aptamer. As used herein, the term "double-stranded character" means that over any length of two nucleic acid molecules, their sequences form more than 50 percent base pairs (canonical or non-canonical) of their length.

[0104] Aptamers may be further modified to provide protection from nucleases and other enzymatic activity. Aptamer sequences can be modified by any suitable method known in the art. For example, phosphorothioates can be incorporated into the backbone, and 5' modified pyrimidines can be included at the 5' end of the ssDNA of DNA aptamers. For RNA aptamers, modified nucleotides, such as the replacement of the 2'-OH group of the ribose backbone with 2'-deoxy-NTPs or 2'-fluoro-NTPs, can be incorporated into the RNA molecule using T7 RNA polymerase mutants. The resistance of these modified aptamers to nucleases can be tested by incubating either purified nucleases from mouse serum or nucleases, and the integrity of the aptamer can be analyzed by gel electrophoresis.

[0105] In some embodiments, such modified nucleic acid aptamers may be synthesized entirely of modified nucleotides or with a subset of modified nucleotides. The modifications may be the same or different. All nucleotides may be modified, or all may contain the same modification. All nucleotides may be modified, but may contain different modifications, for example, all nucleotides containing the same base may have one type of modification, while nucleotides containing other bases may have a different type of modification. For example, all purine nucleotides may have one type of modification (or no modification), while all pyrimidine nucleotides may have another different type of modification (or no modification). In this way, oligonucleotides, or libraries of oligonucleotides, may be generated using any combination of modifications disclosed herein.

[0106] According to certain embodiments of the present invention, aptamer variants and derivatives are provided. The term "derivative" is used synonymously with the term "variant" and refers to a molecule that is modified or altered in some way relative to a reference or starting aptamer. The nucleic acid sequence of an aptamer variant may have substitutions, deletions, and / or insertions at specific positions in the nucleotide sequence compared to the reference or starting sequence. Typically, a variant has at least about 50% identity (homology) with the reference sequence, preferably at least about 80%, more preferably at least about 90% identity (homology) with the reference sequence.

[0107] In some embodiments, the aptamer is modified with a functional group that can be used to covalently bind to fatty acid moiety. The functional group can be added to the 3'-end or 5'-end of the aptamer. In some embodiments, additional linker groups or nucleotide substitutions can be added to the aptamer for conjugation.

[0108] In some embodiments, the 5' end of the aptamer has a dibenzocyclooctyne (DBCO) group:

[0109] [ka]

[0110] The DBCO group may undergo copper-free click chemistry reactions with azide groups. In some embodiments, variant mimics of the aptamers of the present disclosure are provided.As used herein, the term "variant mimic" refers to one or more nucleic acids that will mimic the activation sequence.The nucleic acid sequence of the variant mimic may comprise a naturally occurring nucleic acid, or alternatively a non-naturally occurring nucleic acid.

[0111] fatty acid part According to the present disclosure, the fatty acid moiety is conjugated to a nucleic acid, such as an aptamer. The fatty acid moiety may be a naturally occurring fatty acid or a fatty acid-like molecule.

[0112] In some embodiments, the molecular weight of the fatty acid or fatty acid moiety is at least 500 Da, for example, between 500 and 1 KDa, between 1 K and 1.5 KDa, between 1.5 K and 2 KDa, between 2 K and 2.5 KDa, or between 2.5 K and 3 KDa.

[0113] Fatty acids are carboxylic acids with the typical RCOOH structure consisting of a methyl end (-CH3), a hydrocarbon chain (R) (also called the "aliphatic tail"), and a terminal carboxyl group (-COOH). The hydrocarbon chain may contain 4-30 carbon atoms, may be saturated or unsaturated (having at least one double or triple bond), and may have a straight or branched hydrocarbon chain. They are often shown as schematic formulas such as CN:p nx, where CN represents the total number of carbon atoms, p is the number of double bonds, and x indicates the position of the first double bond from the methyl end (n) (IUPAC-IUB Commission, Eur J Biochem 1977, 79:11-21). In some embodiments, fatty acids consist of a straight chain of an even number of carbon atoms, with a hydrogen atom at one end of the chain and a carboxyl group (-COOH) at the other end along the length of the chain.

[0114] Fatty acids can be divided into several groups according to their structure, physiological role, and biological effect. Fatty acids can be classified as saturated and unsaturated fatty acids according to their structure. Fatty acid-like molecules refer to modified fatty acids in which certain carbon atoms can and may be replaced by other atoms or groups of atoms. Fatty acid-like molecules are modified to better facilitate conjugation strategies. As a non-limiting example, natural fatty acids can be modified to contain an amino terminus. In other examples, fatty acid molecules can include one or more hydrophilic groups to facilitate conjugation reactions.

[0115] In some embodiments, the fatty acid moiety comprises an alkyl, alkenyl, alkynyl, aryl, or aralkyl group, which may contain about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more carbon atoms.

[0116] In some embodiments, an alkyl group contains about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more carbon atoms.

[0117] In certain embodiments, the fatty acid moiety may comprise an aliphatic chain that is saturated or unsaturated, linear or branched, substituted or unsubstituted. In some embodiments, the fatty acid moiety may contain one or more unsaturated carbon bonds, in some embodiments, all of the unsaturated bonds are contained within the same chain.

[0118] As used herein, the term "alkyl" may include saturated aliphatic groups, including straight chain alkyl groups, branched chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In some embodiments, alkyl has from 2 to 100 carbon atoms, or from 2 to 50 carbon atoms, or from 10 to 50 carbon atoms, or from 2 to 30 carbon atoms, or from 10 to 30 carbon atoms, or from 12 to 20 carbon atoms.

[0119] As used herein, the term "alkenyl" refers to a branched or unbranched hydrocarbon having at least one carbon-carbon double bond. As used herein, the term "alkynyl" refers to a branched or unbranched hydrocarbon having at least one carbon-carbon triple bond.

[0120] As used herein, the term "aryl," used alone or as part of a larger moiety such as "aralkyl," "aralkoxy," or "aryloxyalkyl," refers to a monocyclic, bicyclic, or polycyclic ring system having a total of five to thirty ring members, wherein at least one ring in the system is aromatic.

[0121] In some embodiments, the fatty acid moiety contains 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more carbon atoms. In some embodiments, the fatty acid moiety contains 10 or more carbon atoms. In some embodiments, the fatty acid moiety contains 11 or more carbon atoms. In some embodiments, the fatty acid moiety contains 12 or more carbon atoms. In some embodiments, the fatty acid moiety contains 13 or more carbon atoms. In some embodiments, the fatty acid moiety contains 14 or more carbon atoms. In some embodiments, the fatty acid moiety contains 15 or more carbon atoms. In some embodiments, the fatty acid moiety contains 16 or more carbon atoms. In some embodiments, the fatty acid moiety contains 17 or more carbon atoms. In some embodiments, the fatty acid moiety contains 18 or more carbon atoms. In some embodiments, the fatty acid moiety contains 19 or more carbon atoms. In some embodiments, the fatty acid moiety contains 20 or more carbon atoms. In some embodiments, the fatty acid moiety contains 25 or more carbon atoms, hi some embodiments, the fatty acid moiety contains 30 or more carbon atoms.

[0122] In some embodiments, the fatty acid moiety is -(CH) a It contains -COOH, where a=12 to 26. In some embodiments, a=16. In other embodiments, a=18.

[0123] In some embodiments, the fatty acid moiety comprises at least one ethylene glycol group: -(OCH2CH2). In some embodiments, the fatty acid moiety comprises 1-10 ethylene glycol groups. In some embodiments, the fatty acid moiety comprises 1, 2, 3, 4, or 5 ethylene glycol groups.

[0124] In some embodiments, the fatty acid moiety comprises at least one polyethylene glycol (PEG) group: -(OCH2CH2) b -, where b=1 to 10. In some embodiments, b=4.

[0125] In some embodiments, the fatty acid moiety comprises at least one amide group (-NH-CO-). In some embodiments, the fatty acid moiety comprises at least one -(OCH2CH2-OCH2CH2-NH-CO)- group. In some embodiments, the fatty acid moiety comprises 1, 2, 3, 4 or 5 -(OCH2CH2-OCH2CH2-NH-CO)- groups.

[0126] In some embodiments, the fatty acid moiety contains at least one -(CO-CH2-OCH2CH2-OCH2CH2-NH)- group. In some embodiments, the fatty acid moiety contains 1 to 10 (CO-CH2-OCH2CH2-OCH2CH2-NH)- groups. In some embodiments, the fatty acid moiety contains two -(CO-CH2-OCH2CH2-OCH2CH2-NH)- groups.

[0127] In some embodiments, the fatty acid moiety comprises at least an amino acid group, or a derivative / analog thereof. In some embodiments, the fatty acid moiety comprises at least one glutamic acid group, or a derivative / analog thereof, e.g.

[0128] [ka]

[0129] In some embodiments, the glutamic acid group comprises a γ-glutamic acid group.

[0130] [ka]

[0131] It is. In some embodiments, the fatty acid moiety comprises a piperazine group. In some embodiments, the fatty acid moiety comprises a piperazine-2-carboxylic acid group.

[0132] [ka]

[0133] In some embodiments, the fatty acid moiety comprises:

[0134] [ka]

[0135] In some embodiments, the fatty acid moiety comprises a group:

[0136] [ka]

[0137] Contains a group. In some embodiments, the fatty acid moiety comprises an azide (-N3) group. In some embodiments, the fatty acid moiety comprises the general formula:

[0138] [ka]

[0139] (Formula I), In the formula, n is an integer from 12 to 26, Block A is

[0140] [ka]

[0141] [ka]

[0142] or

[0143] [ka]

[0144] and na is 0 or 1, Block B is -(OCH2CH2)- or -(CO-CH2-OCH2CH2-OCH2CH2-NH)-; nb is an integer from 1 to 10; Block C is

[0145] [ka]

[0146] and nc is 0 or 1. In some embodiments, the fatty acid moiety comprises the general formula:

[0147] [ka]

[0148] (Formula II), In the formula, n is an integer from 12 to 26, Block A is

[0149] [ka]

[0150] [ka]

[0151] or

[0152] [ka]

[0153] and na is 0 or 1, Block B is -(OCH2CH2)- or -(CO-CH2-OCH2CH2-OCH2CH2-NH)-; nb is an integer from 1 to 10; Block C is

[0154] [ka]

[0155] and nc is 0 or 1, Block D comprises a functional group capable of covalently binding to a nucleic acid, nd is 0 or 1.

[0156] In some embodiments, block D comprises an azide group, an alkyne group, a hydroxy group, a sulfhydryl group, or an amino group. d -N3, R d comprises an alkyl group, an aminoalkyl group, an amine group, and / or an alkoxyl group. In some embodiments, block D is

[0157] [ka]

[0158] [ka]

[0159] or

[0160] [ka]

[0161] It is. In some embodiments, the fatty acid moiety is -(CH) 16 In some embodiments, the fatty acid moiety further comprises at least one -(OCH2CH2-OCH2CH2-NH-CO-CH2)- group. In some embodiments, the fatty acid moiety further comprises at least one γ-glutamic acid group. Non-limiting examples include:

[0162] [ka]

[0163] GTFA-1, which has the structure

[0164] [ka]

[0165] GTFA-1' having the structure

[0166] [ka]

[0167] or GTFA-3 having the structure and

[0168] [ka]

[0169] An example of such a peptide is GTFA-3' having the structure: In some embodiments, the fatty acid moiety is -(CH) 16In some embodiments, the fatty acid moiety further comprises a polyethylene glycol (PEG) group. In some embodiments, the fatty acid moiety further comprises a piperazine-2-carboxylic acid group. Non-limiting examples include those having the following structure:

[0170] [ka]

[0171] or GTFA-2, having the structure

[0172] [ka]

[0173] An example of such a peptide is GTFA-2', which has the structure: Synthesis of fatty acid conjugates The nucleic acid molecules disclosed herein include natural nucleic acids and synthetic or modified nucleic acids. Modified nucleic acids have one or more modifications, such as base modifications, backbone modifications, etc., to provide new or enhanced characteristics (e.g., improved stability) to the nucleic acid. The nucleic acid molecule and the fatty acid can be synthesized by any suitable method known in the art. The nucleic acid and the fatty acid can then be linked via any suitable method known in the art. In a non-limiting example, the nucleic acid and the fatty acid are linked via click chemistry, such as azide-alkyne cycloaddition.

[0174] The term "click chemistry" encompasses a variety of reactions that are selective and proceed in high yields with few or no by-products under simple reaction conditions and solvents. As used herein, the term "click chemistry" refers to the Huisgen cycloaddition or 2,3-dipolar cycloaddition between an azide and a terminal alkyne to form a 1,2,4-triazole. As used herein, the term "cycloaddition" refers to a chemical reaction that combines two or more π-electron systems (e.g., unsaturated molecules or unsaturated portions of the same molecule) to form a cyclic product with a net reduction in bond multiplicity. In a cycloaddition, π electrons are used to form new sigma bonds. The product of a cycloaddition is called an "adduct" or "cycloadduct." Different types of cycloadducts are known in the art, including but not limited to [3+2] cycloadducts and Diels-Alder reactions. [3+2] cycloadditions, also called 2,3-dipolar cycloadditions, occur between 1,3-dipoles and dipolophiles and are typically used to construct five-membered heterocycles. The term "[3+2] cycloaddition" also encompasses the "copper-free" [3+2] cycloadditions between azides and cyclooctynes ​​and difluorocyclooctynes, described by Bertozzi et al. J. Am. Chem. Soc., 2004, 126:15046-15047. Any reagent that can be used to promote Huisgen cycloadditions can be used as a click chemistry reagent. In some embodiments, the click chemistry reagent includes a pyridyl azide. In some embodiments, the click chemistry reagent includes a picolyl azide. Without being limited thereto, any isomer of picolyl azide can be used.

[0175] In some embodiments, the fatty acid moiety is covalently attached to the 5' end of the nucleic acid sequence. In other embodiments, the fatty acid moiety is covalently attached to the 3' end of the nucleic acid sequence. In some embodiments, the conjugate comprises:

[0176] [ka]

[0177] having a general formula of (Formula III), where n is an integer from 12 to 26; Block A is

[0178] [ka]

[0179] [ka]

[0180] or

[0181] [ka]

[0182] and na is 0 or 1, Block B is -(OCH2CH2)- or -(CO-CH2-OCH2CH2-OCH2CH2-NH)-; nb is an integer from 1 to 10; Block C is

[0183] [ka]

[0184] and nc is 0 or 1, Block E is an optional linker, ne is an integer from 1 to 10.

[0185] In some embodiments, block E is a group formed by a click chemistry reaction between DBCO and an azide. In some embodiments, block E is

[0186] [ka]

[0187] Includes. In some embodiments, fatty acid-nucleic acid conjugates may be produced according to the general steps of 1) modifying a nucleic acid to add an active group, such as, for example, adding a DBCO group to one end of a nucleotide sequence, 2) synthesizing a fatty acid moiety with one or more functional groups, such as the fatty acid moieties discussed in Example 1 of the present disclosure, and 3) synthesizing the fatty acid-nucleic acid conjugate via a chemical reaction between the functional group of the fatty acid moiety and the nucleic acid. The reaction mixture may be further processed to isolate the fatty acid-nucleic acid conjugate.

[0188] In some embodiments, the present disclosure provides a fatty acid conjugation of an anti-VWF aptamer. The anti-VWF aptamer may comprise the polynucleotide sequence: NH2-mGmCmCmAmGmGmGmAmCmCmUmAmAmGmAmCmAmCmAmUmGmUmCmCmCmUmGmGmC-idT (SEQ ID NO: 1, BT100), where "NH" is a 5'-hexylamine linker phosphoramidite, "idT" is an inverted deoxythymidine, and "mN" is a 2'-O-methyl-containing residue. As a non-limiting example, the fatty acid conjugation of BT100 comprises the polynucleotide sequence: (5'-) palmitic acid-linker-mGmCmCmAmGmGmGmAmCmCmUmAmAmGmAmCmAmCmAmUmGmUmCmCmCmUmGmGmCidT (-3') (SEQ ID NO: 3, BT500).

[0189] As another example, the fatty acid conjugation of BT100 comprises the polynucleotide sequence: (5'-)GTFA-1-linker-mGmCmCmAmGmGmGmAmCmCmUmAmAmGmAmCmAmCmAmUmGmUmCmCmCmUmGmGmCidT (-3') (SEQ ID NO: 4, BT600).

[0190] As another example, the fatty acid conjugation of BT100 comprises the polynucleotide sequence: (5'-)GTFA-3-linker-mGmCmCmAmGmGmGmAmCmCmUmAmAmGmAmCmAmCmAmUmGmUmCmCmCmUmGmGmCidT (-3') (SEQ ID NO:5, BT700).

[0191] Pharmaceutical Compositions and Methods of Administration In another aspect, the present disclosure provides a pharmaceutical composition comprising the nucleic acid-fatty acid conjugate of the present disclosure. The nucleic acid of the conjugate is a therapeutic nucleic acid. In some embodiments, the pharmaceutical composition further comprises at least one pharma- ceutically acceptable carrier, diluent, or excipient.

[0192] The composition may be formulated for a particular route of administration, such as parenteral, enteral, oral, or other suitable route. Parenteral administration may be performed by injection or by insertion of an implanted catheter, including, but not limited to, intravenous (IV), intramuscular (IM), subcutaneous (SC), transdermal, epidural, intracerebral (intracerebral), intraventricular (intracerebroventricular), extraamniotic, intranasal, intraarterial, intracardiac, intraosseous (IO), intraperitoneal infusion or injection, transdermal diffusion, enteral and gastrointestinal routes, topical administration, and oral routes.

[0193] Furthermore, the pharmaceutical compositions of the present disclosure may be made in solid forms, including but not limited to capsules, tablets, pills, granules, lyophilisates, powders, or suppositories, or in liquid forms, including but not limited to solutions, suspensions, or emulsions. The pharmaceutical compositions may be subjected to conventional pharmaceutical operations such as aseptic manufacture, sterilization, and / or may contain conventional inert diluents, cake-forming agents, isotonicity agents, lubricants, or buffers, as well as adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, and buffers.

[0194] In some embodiments, pharmaceutical compositions suitable for injectable use typically include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.

[0195] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS). In all cases, the composition is sterile for injection. Preferred pharmaceutical preparations are stable under the conditions of manufacture and storage. In general, relevant carriers can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerin, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents in the composition, for example, sugars, polyalcohols such as mannitol, amino acids, sorbitol, or sodium chloride. Prolonged absorption of injectable compositions can be brought about by including agents that delay absorption, for example, aluminum monostearate and gelatin in the composition. In some embodiments, multifunctional excipients such as recombinant albumin may be incorporated into the formulation process to improve solubility, aid in administration and release of the active ingredient, and promote stabilization of the conjugate product from degradation or aggregation. (BioPharm International, 2012, 23(3):40-44).

[0196] Certain injectable compositions are isotonic aqueous solutions or suspensions, and suppositories are advantageously prepared from fatty emulsions or suspensions. The compositions may be sterilized and / or contain adjuvants, such as preservatives, stabilizing agents, wetting agents or emulsifying agents, solution promoters, salts for regulating the osmotic pressure, and / or buffers.

[0197] Sterile injection solution can be prepared by incorporating the active compound in the required amount with one or combination of the above-listed components in a suitable solvent, followed by sterilization by filtration.Generally, dispersion is prepared by combining the active compound with a sterile vehicle containing a basic dispersion medium and other components required from the above-listed substances.For the preparation of sterile powder for sterile injection solution, the preferred preparation method is vacuum drying and freeze-drying.By this method, the powder of active ingredient and any additional desired ingredient can be obtained from the solution already sterilized and filtered.

[0198] Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound may be incorporated with an excipient and used in the form of tablets, troches, or capsules (e.g., gelatin capsules). The tablets, pills, capsules, troches, etc. may contain any of the following ingredients, or compounds of a similar nature: binders, such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients, such as starch or lactose; disintegrating agents, such as alginic acid, Primogel, or corn starch; lubricants, such as magnesium stearate or Sterotes; glidants, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; or flavoring agents, such as peppermint, methyl salicylate, or orange flavoring. Formulations for oral delivery may advantageously incorporate agents to improve stability in the gastrointestinal tract and / or to enhance absorption.

[0199] For administration by inhalation, the compositions are preferably delivered in the form of an aerosol spray from a pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. It is noted that the lungs offer a large surface area for systemic delivery of therapeutic agents.

[0200] In some embodiments, the pharmaceutical compositions are formulated for systemic administration, for example, by intravenous, transmucosal, or transdermal means. The composition suitable for transdermal application comprises an effective amount of the conjugate of the present disclosure with a suitable carrier.Carriers suitable for transdermal delivery include absorbent pharmacologically acceptable solvents to aid in passing through the host's skin.For example, the transdermal device is in the form of a dressing that comprises a backing member, a reservoir that contains the compound, optionally with a carrier, and optionally a rate controlling barrier for delivering the compound to the host's skin at a controlled and predetermined rate over a long period of time, and a means for fixing the device to the skin.

[0201] In an embodiment, the pharmaceutical composition is formulated for subcutaneous administration. How to use In another aspect, provided herein is a method for treating a disease or disorder in a patient in need of such treatment, comprising administering to the patient a conjugate of the present disclosure.

[0202] The fatty acid conjugated nucleic acid and compositions comprising the fatty acid conjugated nucleic acid can be used in a variety of different pharmaceutical, therapeutic, diagnostic, and biomedical applications. The fatty acid modification improves the potency and efficacy of nucleic acids (e.g., aptamers), revealing the possibility of oral delivery, as well as enhancing subcutaneous and intravenous administration. The fatty acid conjugation may increase the potency of therapeutic nucleic acids, allowing for the administration of lower doses, which reduces the possibility of toxicity and immunogenicity. The fatty acid conjugation may increase stability, allowing for less frequent administration.

[0203] As a non-limiting example, fatty acid conjugated nucleic acids and compositions comprising fatty acid conjugated nucleic acids find use in therapeutic applications. Non-limiting examples of diseases or disorders include angiogenic disorders, cardiovascular disorders, stroke, neurodegenerative disorders, cancer, genetic disorders, and rare diseases.

[0204] In some embodiments, the method of treatment may alter the onset of symptoms of a disease or disorder. In one aspect, the present disclosure provides a method for preventing the above-mentioned disease or disorder in a subject by administering to the subject a therapeutically effective amount of a conjugate of the present disclosure.Subjects at risk of disease can be identified, for example, by either or a combination of diagnostic or prognostic assays.Administration of the prophylactic agent can occur prior to the onset of symptoms characteristic of the disease or disorder, such that the disease or disorder is prevented or, alternatively, its progression is delayed.

[0205] In one embodiment, the subject is administered an initial dose of the conjugate of the present disclosure and one or more maintenance doses. The maintenance dose is generally lower than the initial dose, e.g., half the amount of the initial dose. The maintenance regimen may include treating the subject with a dose ranging from 0.01 mg to 100 mg per kg of body weight per day, e.g., 100, 10, 1, 0.1, 0.01, 0.001, or 0.00001 mg per kg of body weight per day. The maintenance dose is preferably administered no more than once every 2, 5, 10, or 30 days. Furthermore, the treatment regimen may continue for various periods of time, depending on the nature of the particular disease, its severity, and the overall condition of the patient. In a preferred embodiment, the dosage may be delivered no more than once a day, e.g., every 24, 36, 48 hours, or more, e.g., once every 5 or 8 days. Following treatment, the patient may be monitored for changes in condition and relief of symptoms of the condition. The dosage of the compound may be increased if the patient does not respond significantly to the current dosage level, or may be decreased if a reduction in the symptoms of the condition is observed, if the condition is eliminated, or if undesirable side effects are observed.

[0206] In some embodiments, fatty acid conjugation of BT100 (e.g., BT500, BT600 and BT700) can be used to bind to VWF antigen. In some embodiments, the conjugates can be used to treat and / or prevent stroke and transient ischemic attack (TIA) in patients with primary ischemic stroke and TIA, and stroke recurrence. For example, the treatment and prevention methods use BT500, BT600, or BT700 in combination with one or more antithrombotic agents.

[0207] Equivalents and Scope Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the present disclosure described herein. The scope of the present disclosure is not intended to be limited to the above description, but is as set forth in the appended claims.

[0208] In the claims, articles such as "a," "an," and "the" may mean one or more unless specifically stated otherwise or clear from the context. A claim or description including "or" between one or more members of a group is deemed to be satisfied when one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process, unless the context indicates to the contrary or otherwise. The present disclosure includes embodiments in which exactly one member of a group is present in, employed in, or otherwise relevant to a given product or process. The present disclosure also includes embodiments in which two or more, or the entire group member is present in, employed in, or otherwise relevant to a given product or process.

[0209] It should also be noted that the term "comprising" is intended to be open-ended, permitting but not requiring the inclusion of additional elements or steps. When the term "comprising" is used herein, therefore, the term "consisting of" is also included and disclosed.

[0210] Where ranges are given, the endpoints are included. Furthermore, unless otherwise indicated or clear from the context and the understanding of one of ordinary skill in the art, it should be understood that values ​​expressed as ranges can assume from any particular value or subrange within the ranges set forth in different embodiments of this disclosure, down to one tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0211] Furthermore, it should be understood that any particular embodiment of the present disclosure that falls within the prior art may be expressly excluded from any one or more of the claims. Such embodiments may be excluded even if the exclusion is not expressly set forth herein, since they are deemed known to those skilled in the art. Any particular embodiment of the composition of the present disclosure (e.g., any antibiotic, therapeutic or active ingredient, any method of manufacture, any method of use, etc.) may be excluded from any one or more of the claims for any reason, whether related to the existence of prior art or not.

[0212] The words that have been used are words of description rather than of limitation, and it is to be understood that changes may be made within the purview of the appended claims without departing from the true scope and spirit of the present disclosure in its broader aspects.

[0213] While the present disclosure has been described at some length and in some detail with respect to certain illustrated embodiments, it should not be limited to any such details, or embodiments, or to any particular embodiment, but rather should be construed with reference to the appended claims, which are to be given the broadest interpretation in view of the prior art, and therefore effectively encompass the intended scope of the present disclosure. EXAMPLES

[0214] The following examples are offered by way of illustration and not by way of limitation. Example 1: Exemplary Methods for the Synthesis of Fatty Acid Moieties GTFA-1 synthesis

[0215] [ka]

[0216] Step 1: To a solution of compound A1 (2.46 g, 9.4 mmol) and compound A2 (940 mg, 9.4 mmol) in anhydrous THF (25 ml) was added T3P (9 ml, 50% in EA, 14 mmol) and DIPEA (3.3 ml, 18.8 mmol) at 0° C. The reaction mixture was stirred for 1 h and diluted with EtOAc. The mixture was washed with water, brine, dried over Na2SO4 and concentrated. The residue was purified by preparative HPLC to give compound A3 (2.8 g, 8.1 mmol). LCMS: 346 [M+H] + .

[0217] Step 2: Compound A3 (2.7 g, 7.8 mmol) was dissolved in 50 ml of methanol and HCl (20 ml, 4N in dioxane, 80 mmol) was added at 0° C. The reaction mixture was stirred at room temperature for 3 hours and concentrated to give compound A4 (2.2 g, 7.8 mmol). LCMS: 246 [M+H] + .

[0218] Step 3: To a solution of compound A4 (1.5 g, 5.2 mmol) and compound A1 (1.4 g, 5.3 mmol) in anhydrous THF (30 ml) was added T3P (4.6 ml, 50% in EA, 7.8 mmol) and DIPEA (3.6 ml, 20.8 mmol) at 0° C. The reaction mixture was stirred for 1 h and diluted with EtOAc. The mixture was washed with water, brine, dried over Na2SO4 and concentrated. The residue was purified by preparative HPLC to give compound A5 (2.2 g, 4.5 mmol). LCMS: 491 [M+H] + .

[0219] Step 4: Compound A5 (2.2 g, 4.5 mmol) was dissolved in 30 ml of methanol and HCl (12 ml, 4N in dioxane, 48 mmol) was added at 0° C. The reaction mixture was stirred at room temperature for 4 hours and concentrated to give compound A6 (1.7 g, 4 mmol). LCMS: 391 [M+H] + .

[0220] Step 5: To a solution of compound A6 (1.4 g, 3.3 mmol) and compound A7 (1.1 g, 2.6 mmol) in anhydrous THF (15 ml) was added T3P (3 ml, 50% in EA, 5 mmol) and DIPEA (2.3 ml, 13.2 mmol) at 0° C. The reaction mixture was stirred for 1.5 h and diluted with EtOAc. The mixture was washed with water, brine, dried over Na2SO4 and concentrated to give crude compound A8 (2.3 g), which was used in the next step without further purification. LCMS: 798 [M+H] + .

[0221] Step 6: To a solution of compound A8 (2.3 g) in 30 ml of DCM was added DBU (1 mL). The resulting mixture was stirred at room temperature for 2 h, then diluted with DCM (100 ml) and washed with water (50 mL*2). The organic phase was concentrated, and the residue was dissolved in water (20 mL) and extracted with MTBE (50 mL). The aqueous phase was purified by preparative HPLC to give compound 9 (1.2 g, 2.1 mmol). LCMS: 576 [M+H] + .

[0222] Step 7: To a solution of compound A9 (1.0 g, 1.74 mmol) and compound A10 (640 mg, 1.73 mmol) in anhydrous DMF (10 ml) was added T3P (1.5 ml, 50% in EA, 2.6 mmol) and DIPEA (605 μ, 3.48 mmol) at 0° C. The reaction mixture was stirred for 1 h and diluted with EtOAc. The mixture was washed with water, brine, dried over Na2SO4 and concentrated. The residue was purified by preparative HPLC to give compound 11 (1.4 g, 1.5 mmol). LCMS: 928 [M+H] + .

[0223] Step 8: Compound A11 (1.4 g, 1.5 mmol) was dissolved in 10 ml of DCM and TFA (5 ml) was added at 0° C. The reaction mixture was stirred at room temperature for 3 h and concentrated. The residue was purified by preparative HPLC to give the final product GTFA-1 (850 mg, 1.04 mmol). C38H69N7O12 LCMS: 816 [M+H] + . 1 H NMR(400MHz,DMSO-d6)δ:13.00-11.00(br,2H),8.00-8.02(m,1H),7.88-7.85( m,1H),7.77-7.74(m,1H),7.66-7.63(m,1H),4.15-4.09(m,1H),3.88(s,2H),3. 86(s,2H),3.59-3.51(m,8H),3.44-3.43(m,2H),3.41-3.38(m,2H),3.33-3.25 (m,4H),3.21-3.13(m,4H),2.17-2.06(m,6H),1.95-1.88(m,1H),1.75-1.70(m, 1H), 1.68-1.63(m, 2H), 1.47-1.45(m, 4H), 1.22(s, 24H). GTFA-2 synthesis

[0224] [ka]

[0225] [ka]

[0226] [ka]

[0227] Step 1: To a solution of compound B1 (160 mg, 0.5 mmol) and compound B2 (150 mg, 0.6 mmol) in anhydrous DMF (4 ml) was added T3P (500 μl, 50% in EA, 0.84 mmol) and DIPEA (260 μl, 1.5 mmol) at 0° C. The reaction mixture was stirred for 30 min and diluted with EtOAc. The mixture was washed with water, brine, dried over Na2SO4 and concentrated. The residue was purified by preparative HPLC to give compound B3 (200 mg, 0.37 mmol). LCMS: 548 [M+H] + .

[0228] Step 2: Compound 3 (200 mg, 0.37 mmol) was dissolved in 2 ml of dioxane and HCl (2 ml, 4N in dioxane, 8 mmol) was added. The reaction mixture was stirred at room temperature for 40 minutes and concentrated to give compound B4 (180 mg, 0.36 mmol). LCMS: 448 [M+H] + .

[0229] Step 3: To a solution of compound 4 (180 mg, 0.36 mmol) and compound B5 (135 mg, 0.36 mmol) in anhydrous DMF (4 ml) was added T3P (400 μl, 50% in EA, 0.67 mmol) and DIPEA (260 μl, 1.5 mmol) at 0° C. The reaction mixture was stirred for 1 h and diluted with EtOAc. The mixture was washed with water, brine, dried over Na2SO4 and concentrated. The residue was purified by preparative HPLC to give compound B6 (135 mg, 0.17 mmol). LCMS: 800 [M+H] + .

[0230] Step 4: Compound B6 (135 mg, 0.17 mmol) was dissolved in 4 ml of MeOH and NaOH (1 ml, 4N in water, 4.0 mmol) was added. The reaction mixture was stirred at room temperature for 1 h and MeOH was removed under reduced pressure. The residue was diluted with water, cooled to 0° C. and HCl (2 ml, 2N, 4.0 mmol) was added. The mixture was extracted with DCM, dried over Na2SO4 and concentrated to give compound B7 (130 mg, 0.16 mmol). LCMS: 786 [M+H] + .

[0231] Step 5: Compound B7 (130 mg, 0.16 mmol) was dissolved in 2 ml of DCM and TFA (1 ml) was added. The reaction mixture was stirred at room temperature for 1 h and concentrated to give the final product GTFA-2 (110 mg, 0.15 mmol). C35H63N5O11 LCMS: 730 [M+H] + . 1 H NMR(400MHz,CDCl3)δ:4.60-4.35(m,2H),4.21-4.15(m,1H),3.79-3.61(m,20H),3.54-3.45(m,1H),3.41-3.38(m,2H),3.35-3.28(m, 1H),3.05-2.93(m, 1H),2.80-2.70(m,1H),2.43-2.33(m,4H),1.68-1.60(m,4H),1.22(m,24H). GTFA-3 synthesis

[0232] [ka]

[0233] [ka]

[0234] Step 1: To a solution of compound C1 (3.83 g, 9 mmol) and compound C2 (1.5 mg, 9 mmol) in anhydrous THF (50 ml) was added T3P (7.5 ml, 50% in EA, 1.3 eq.) and DIPEA (5 ml, 3 eq.) at 0° C. The reaction mixture was stirred for 30 min and compound C2 (100 mg) was added. The reaction mixture was stirred for 50 min and diluted with EtOAc. The mixture was washed with water, brine, dried over Na2SO4 and concentrated to give crude compound C3 (approximately 4.2 g) for use in the next step. LCMS: 538 [M+H] + .

[0235] Step 2: To a solution of crude compound C3 (ca. 4.2 g) in DCM (20 mL) solution at 0° C., DBU (3 mL, 19.7 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 30 min and diluted with water. The aqueous layer was extracted with DCM. The combined DCM layers were dried over Na2SO4 and concentrated. Brine was added to the residue and washed with MTBE. The brine layer was extracted with DCM. The combined DCM layers were dried over Na2SO4 and concentrated to give crude compound C4 (ca. 2 g) used in the next step. LCMS: 316 [M+H] + .

[0236] Step 3: To a solution of crude compound C4 (ca. 2 g) and compound C5 (1.7 g, 4.4 mmol) in EtOAc (150 ml) was added T3P (7.5 ml, 50% in EA) and DIPEA (7 mL) at 0° C. The reaction mixture was stirred for 30 min and diluted with EtOAc. The mixture was washed with water, brine, dried over Na2SO4 and concentrated to give crude compound C6 (ca. 3.6 g) for use in the next step. LCMS: 683 [M+H] + .

[0237] Step 4: To a solution of crude compound C6 (ca. 3.6 g) in DCM (6 mL) at 0° C. was added DBU (1 mL, 6.5 mmol) dropwise. The reaction mixture was stirred at room temperature for 10 min and diluted with DCM (100 mL) and brine (40 mL). The brine was extracted with DCM and the combined DCM layers were dried over Na2SO4 and concentrated. Water was added to the residue and washed with hexane. The aqueous layer was saturated with NaCl and extracted with EtOAc. The combined EtOAc layers were dried over Na2SO4 and concentrated to give crude compound C7 (ca. 1.6 g) used in the next step. LCMS: 461 [M+H] + .

[0238] Step 5: To a solution of crude compound C7 (ca. 1.6 g) and compound C5 (1.18 g, 3.06 mmol) in EtOAc (200 ml) was added T3P (4 mL, 50% in EA) and DIPEA (3 mL) at 0° C. The reaction mixture was stirred for 180 min and diluted with EtOAc. The mixture was washed with water, brine, dried over Na2SO4 and concentrated to give crude compound C8 (ca. 2.5 g) for use in the next step. LCMS: 828 [M+H] + .

[0239] Step 6: To a solution of crude compound C8 (ca. 2.5 g) in DCM (3 mL) at 0° C. was added DBU (0.3 mL, 1.98 mmol) dropwise. The reaction mixture was stirred at room temperature for 2 h and diluted with DCM (100 mL) and brine (40 mL). The brine was extracted with DCM and the combined DCM layers were dried over Na2SO4 and concentrated. Water was added to the residue and washed with hexane. The aqueous layer was saturated with NaCl and extracted with EtOAc. The combined EtOAc layers were dried over Na2SO4 and concentrated to give crude compound C9 (ca. 1.5 g) used in the next step. LCMS: 606 [M+H] + .

[0240] Step 7: To a solution of crude compound C9 (ca. 1.5 g) and compound C1 (0.89 g, 2.1 mmol) in DCM (60 ml) was added T3P (3 mL, 50% in EA) and DIPEA (2 mL) at 0° C. The reaction mixture was stirred for 120 min and diluted with EtOAc. The mixture was washed with water, brine, dried over Na2SO4 and concentrated to give crude compound C10 (ca. 2.4 g) used in the next step. LCMS: 1013 [M+H] + .

[0241] Step 8: To a solution of crude compound C10 (ca. 2.4 g) in DCM (2 mL) at 0° C. was added DBU (0.3 mL, 1.98 mmol) dropwise. The reaction mixture was stirred at room temperature for 2 h and diluted with DCM (100 mL) and brine (40 mL). The brine was extracted with DCM and the combined DCM layers were dried over Na2SO4 and concentrated. Water was added to the residue and washed with hexane. The aqueous layer was saturated with NaCl and extracted with EtOAc. The combined EtOAc layers were dried over Na2SO4 and concentrated to give crude compound C11 (ca. 1.49 g) used in the next step. LCMS: 780 [M+H] + .

[0242] Step 9: To a solution of crude compound C9 (ca. 1.49 g) and compound C12 (0.584 g, 2.1 mmol) in DCM (30 ml) was added T3P (2 mL, 50% in EA) and DIPEA (2 mL) at 0° C. The reaction mixture was stirred for 60 min and diluted with EtOAc. The mixture was washed with water, brine, dried over Na2SO4 and concentrated. The residue was purified by silica gel column chromatography with EtOAc / methanol (20:1) to give compound C13 (1.58 g) as a colorless oil. LCMS: 572 [M+2H] + .

[0243] Step 10: Compound C13 (807 mg, 0.7 mmol) was dissolved in dioxane (3 mL) and HCl (6 ml, 4N in dioxane, 2.4 mmol) was added. The reaction mixture was stirred at room temperature for 6 hours and concentrated to give compound GTFA-3 (576 mg). C44H78N8O16 LCMS: 975 [M+H] + . 1 H NMR(400MHz,DMSO-d6)δ:13.00-11.00(br,3H),8.01-8.03(m,1H),7.90-7.85(m,3H),7.68-7.65(m,1H),4.25- 4.21(m,1H),4.14-4.09(m,1H),3.91(s,2H),3.86(s,2H),3.57-3.52(m,10H),3.46-3.36(m,8H),3.29-3.25(m, 2H),3.21-3.16(m,4H),2.18-2.05(m,8H),2.04-1.7(m,4H),1.47-1.45(m,4H),1.22(s,24H). Example 2: Fatty acid conjugation to BT100 The core sequence of BT100 (SEQ ID NO:1) was conjugated with three different fatty acids to generate BT500, BT600, and BT700, respectively.

[0244] The DBCO moiety was first incorporated at the amino terminus of BT100. HPLC purified BT100 bearing a 5' amino group (amino modifier C6) was reacted with 2.5 molar equivalents of DBCO-Sulfo-NHS ester (CAS#1400191-52-7, Broadpharm, catalog no. BP-22289) in 100 mM sodium borate buffer pH 8.0 overnight at room temperature.

[0245] After purification by anion exchange HPLC, DBCO-labeled BT100 was reacted with 2.5 molar equivalents of palmitic acid, GTFA1, or GTFA3 overnight at room temperature without pH adjustment. BT500 (palmitic acid conjugate to BT100), BT600 (GTFA1 conjugate to BT100), or BT700 (GTFA3 conjugate to BT100) were obtained after anion exchange HPLC purification.

[0246] BT500: The DBCO-labeled aptamer sequence (BT100) was interacted with azidopalmitic acid, a saturated C16 fatty acid with a nitrogen at one end and one free carboxyl group (-COOH) at the other end. The DBCO moiety reacts with the azide to generate a stable triazole, forming a C16 fatty acid conjugate aptamer with one free COOH group at one end (BT500, SEQ ID NO:3).

[0247] [ka]

[0248] BT600: DBCO-labeled BT100 was conjugated to octadecanedioic acid through a short polyethylene glycol (PEG) spacer and a gamma-glutamic acid linker (GTFA-1) to form a bicarboxyl fatty acid-containing conjugate (BT600, SEQ ID NO: 4). The resulting fatty acid BT100 aptamer compound contains two free COOH groups.

[0249] [ka]

[0250] BT700: DBCO-labeled BT100 is conjugated with a tricarboxylic fatty acid (GTFA-3) (chemical formula: C44H78N8O16) containing three free COOH to form BT700 (SEQ ID NO:5).

[0251] [ka]

[0252] [Table 1]

[0253] Example 3: PK-PD exposure of fatty acid conjugates in cynomolgus monkeys Pharmacokinetic and pharmacodynamic (PK-PD exposure) parameters will be examined to define the impact of fatty acid conjugation on aptamer distribution (half-life) and efficacy in vivo.

[0254] All pharmacokinetic (PK) studies were performed in cynomolgus monkeys. In addition to the PK readout, we also have a PFA100 (PD) readout that correlates well with the PK readout, suggesting that fatty acid conjugation does not alter BT100 activity. BT200, a 40k pegylated BT100 (SEQ ID NO: 2), was used for comparison.

[0255] Animal experiments were performed in accordance with animal care ethics approval and guidelines. BT500 was administered to animals at 1 mg / kg (n=3) via intravenous injection (iv) or alternatively at 1 mg / kg via subcutaneous injection (sc) (n=3). REAADS vWF activity and vWF antigen levels were measured before dosing and at 48, 72, 96, and 168 hours after dosing (Tables 2 and 3). Platelet Function Analyzer 100 (PFA100) assessed primary hemostasis under shear stress of blood samples collected at these time points as a PD outcome for BT500 functionality (Table 4). The readouts indicate that BT500 is able to block vWFA1 binding to Gp1B and inhibit platelet function, thus increasing the PFA100 readout from normal values ​​of <100 seconds up to approximately 300 seconds. The readouts are comparable to those observed with BT100 and BT200 administration.

[0256] [Table 2]

[0257] [Table 3]

[0258] [Table 4]

[0259] BT600 was administered to animals at 1 mg / kg (n=2) via intravenous injection (iv) or alternatively at 3 mg / kg via subcutaneous injection (sc) (n=2). REAADS vWF activity and vWF antigen levels were measured pre-dose and at 48, 72, 96, and 168 hours post-dose (Tables 5 and 6).

[0260] [Table 5]

[0261] [Table 6]

[0262] BT600 can be administered either intravenously or subcutaneously for at least 24 hours (Table 7).

[0263] [Table 7]

[0264] Compared to BT100 administration (Table 8), the half-life of BT600 is extended.

[0265] [Table 8]

[0266] In contrast to the increases in FVIII and vWF antigen levels in both monkeys and humans administered the 40k PEG-conjugated BT100 aptamer, BT200, BT600 administration (e.g., sc) had no effect on circulating FVIII or vWF antigen levels (Zhu et al., The development and characterization of a long acting anti-thrombotic von Willebrand factor (VWF) aptamer, Thromb Haemost., 2020, 18(5): 1113-1123; https: / / isth2021.abstractserver.com / program / # / details / presentations / 2661; and https: / / isth2021.abstractserver.com / program / # / details / presentations / 1327) (Table 9).

[0267] [Table 9]

[0268] BT700 was administered to animals via intravenous injection (iv) at 1 mg / kg (n=2) or alternatively via subcutaneous injection (sc) at 2 mg / kg (n=2). BT700 activity was measured by UV-HPLC before administration and at 0.5, 1, 2, 4, 8, 24, 48, and 72 hours after administration (Tables 10 and 11).

[0269] [Table 10]

[0270] [Table 11]

[0271] Platelet Function Analyzer 100 (PFA100) assessed primary hemostasis under shear stress of blood samples collected at these time points as a PD outcome for BT700 functionality (Table 12). The readouts indicate that BT700 is also able to block vWFA1 binding to Gp1B and inhibit platelet function, thus increasing the PFA100 readout from normal values ​​of <100 seconds up to approximately 300 seconds. The readouts are comparable to those observed with administration of BT100 and BT200.

[0272] [Table 12]

[0273] BT100 and BT200 were administered to animals at 3 mg / kg via intravenous injection (iv) for comparison (Tables 13 and 14).

[0274] [Table 13]

[0275] [Table 14]

[0276] Example 4: BT600 Oral Bioavailability Cynomolgus monkeys were fed the BT600 conjugate directly via oral administration (po) at a dose of 5 mg / kg. BT600 was co-formulated with the absorption enhancer sodium N-(8-[2-hydroxybenzoyl]amino)caprylate (SNAC) for oral ingestion (5 mg / kg BT600 + 150 mg SNAC). Oral administration of BT600 resulted in systemic exposure by bioanalytical assay using a fluorescent hybridization HPLC method capable of detecting as little as 1 ng / mL of BT600 in the animals tested (Tables 15 and 16).

[0277] [Table 15]

[0278] [Table 16]

[0279] A low bioavailability (0.2%) was detected, with the plasma concentration of BT600 being too low to affect the PFA100 assay. However, although tissue distribution is low, a long half-life of BT600 is observed in the animals tested.

[0280] Example 5: Fatty acid conjugates that bind to albumin A CM5 chip coated with albumin via amine coupling was used. The sensor chip was coated with FC-2 human albumin, FC-3 mouse albumin, and FC-4 bovine albumin. FC-1 was used as a control. Aptamer and fatty acid conjugates BT500, BT600, and BT700 were flowed over the surface of the chip. BT100 was used as a control (as shown in Figure 1).

[0281] Based on this simple 1:1 Langmuir interaction model, the association constant (ka), dissociation constant (kd), and equilibrium dissociation constant (KD) were calculated and compared. No significant binding was observed and curve fitting was applied to BT100, while all three fatty acid conjugates BT500, BT600, and BT700 showed binding to human albumin (Table 17). BT100 does not bind to albumin and therefore has the shortest half-life in monkeys. Although BT500 has a low affinity for albumin (Figure 1), the low binding of albumin still results in a longer half-life for BT500 compared to BT100. BT600 and BT700 have a high affinity for albumin and therefore have a much longer half-life (Figure 1).

[0282] [Table 17]

Claims

1. A conjugate comprising a nucleic acid moiety and at least one fatty acid moiety, wherein the fatty acid moiety is conjugated to one end of the nucleic acid moiety, and the fatty acid moiety is -(CH ２ ) ａ A conjugate comprising -COOH, wherein a is an integer from 12 to 26.

2. The conjugate of claim 1 , comprising at least one additional carboxyl group.

3. A conjugate as described in claim 1, which contains one or two additional carboxyl groups.

4. The fatty acid moiety has at least one ethylene glycol group (-(OCH ２ CH ２ 2. The conjugate of claim 1, comprising:

5. The conjugate of claim 4, wherein the fatty acid moiety comprises 1 to 10 ethylene glycol groups.

6. The conjugate of claim 1 , wherein the fatty acid moiety comprises at least one polyethylene glycol (PEG) group.

7. The conjugate of claim 1, wherein the fatty acid moiety comprises at least one amide group (-NH-CO-).

8. The fatty acid moiety has at least one —(OCH ２ CH ２ -OCH ２ CH ２ 8. The conjugate of claim 7, comprising a -NH-CO)- group or at least one -(CO-CH 2 -OCH 2 CH 2 -OCH 2 CH 2 -NH)- group.

9. The conjugate of claim 1 , wherein the fatty acid moiety comprises at least one glutamic acid group.

10. The fatty acid moiety is 【Chemical 1】 or 【Chemistry 2】 10. The conjugate of claim 9, comprising:

11. The conjugate of claim 9, wherein the glutamic acid group is a γ-glutamic acid group.

12. The conjugate of claim 1 , wherein the fatty acid moiety comprises a piperazine group.

13. The fatty acid moiety is a piperazine-2-carboxylic acid group ( 【Chemistry 3】 ）、 【Chemistry 4】 Group, or 【Chemistry 5】 13. The conjugate of claim 12, comprising a group.

14. The fatty acid moiety is 【Chemistry 6】 comprising the structure of (Formula I): In the formula, n is an integer from 12 to 26; Block A is 【Chemistry 7】 【Chemistry 8】 or 【Chemistry 9】 and na is 0 or 1, Block B is -(OCH ２ CH ２ ) - or -(CO-CH ２ -OCH ２ CH ２ -OCH ２ CH ２ —NH)—, nb is an integer from 1 to 10, Block C is 【Chemistry 10】 and The conjugate of claim 1, wherein n c is 0 or 1.

15. 2. The conjugate of claim 1, wherein the fatty acid moiety comprises GTFA-1', GTFA-2', or GTFA-3'.

16. 2. The conjugate of claim 1, wherein the nucleic acid molecule is a therapeutic nucleic acid, including an aptamer or a variant thereof, an oligonucleotide, an antisense oligonucleotide, a CpG oligonucleotide, an siRNA, an shRNA, a microRNA, an lncRNA, an mRNA, an antisense RNA, a saRNA, a circular RNA, or the like.

17. The conjugate of claim 16 , wherein the therapeutic nucleic acid is an aptamer or a variant thereof.

18. 18. The conjugate of claim 17, wherein the aptamer comprises at least one modification, which may be a nucleoside modification or a backbone modification.

19. 19. The conjugate of claim 18, wherein the aptamer comprises at least one nucleoside modification, optionally wherein the modification is a 2'-O-methyl modification.

20. A composition comprising the conjugate of any one of claims 1 to 19 and one or more therapeutically acceptable carriers.

21. A composition comprising a nucleic acid sequence and an aptamer comprising at least one fatty acid moiety, wherein the 5' end of the nucleic acid sequence of the aptamer has a DBCO group. 【Chemistry 11】

22. 22. The composition of claim 21, wherein the fatty acid moiety is covalently attached to the 5' end of the nucleic acid sequence of the aptamer.

23. 22. The composition of claim 21, wherein the fatty acid moiety comprises GTFA-1, GTFA-2, or GTFA-3.

24. The composition of any one of claims 21 to 23, wherein the aptamer comprises a modified nucleoside.

25. 25. The composition of claim 24, wherein the aptamer comprises at least one 2'-O-methyl modified nucleoside.

26. general formula 【Chemistry 12】 A fatty acid moiety comprising (Formula II): In the formula, n is an integer from 12 to 26; Block A is 【Chemistry 13】 【Chemistry 14】 or 【Chemistry 15】 and na is 0 or 1, Block B is -(OCH ２ CH ２ ) - or -(CO-CH ２ -OCH ２ CH ２ -OCH ２ CH ２ —NH)—, nb is an integer from 1 to 10, Block C is 【Chemistry 16】 and n c is 0 or 1, Block D comprises a functional group capable of covalently binding to a nucleic acid, nd is 0 or 1, a fatty acid moiety.

27. 27. The fatty acid moiety of claim 26, wherein block D comprises an azide group, an alkyne group, a hydroxy group, a sulfhydryl group, or an amino group.

28. Block D is R ｄ -N ３ wherein R ｄ 27. The fatty acid moiety of claim 26, wherein comprises an alkyl group, an aminoalkyl group, an amine group, and / or an alkoxyl group.

29. Block D is 【Chemistry 17】 【Chemistry 18】 or 【Chemistry 19】 29. The fatty acid moiety of claim 28, wherein:

30. 27. The fatty acid moiety of claim 26, wherein the fatty acid moiety is GTFA-1, GTFA-2, or GTFA-3.

31. 1. A polynucleotide comprising the nucleic acid sequence represented by SEQ ID NO:1 and a fatty acid moiety conjugated to one end of the sequence of SEQ ID NO:1, wherein the fatty acid moiety is selected from the group consisting of octadecanedioic acid, GTFA-1, GTFA-2 and GTFA-3.

32. 32. The polynucleotide of claim 31, comprising the nucleic acid sequence represented by SEQ ID NO:3 and palmitic acid conjugated to the 5' end of SEQ ID NO:

3.

33. 32. The polynucleotide of claim 31, comprising the nucleic acid sequence represented by SEQ ID NO:4 and GTFA-1 conjugated to the 5' end of SEQ ID NO:

4.

34. 32. The polynucleotide of claim 31, comprising the nucleic acid sequence represented by SEQ ID NO:5 and GTFA-3 conjugated to the 5' end of SEQ ID NO:

5.

35. A pharmaceutical composition comprising the polynucleotide of any one of claims 31 to 34 and a pharmaceutically acceptable carrier.

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