Glycan conjugate compositions and methods

Abstract

The present disclosure provides methods and compositions for using a novel class of glycan conjugates for modulating cell surface proteins and receptor complexes that can be used to engage signaling pathways within desired cell types. Such defined cell-targeting bioactive glycoligands are directed to cell engagement and activation in therapeutic applications.

Description

[0001] Related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 579,419, filed August 29, 2023, the entire teachings of which are incorporated herein by reference. Technical Field

[0003] The disclosures herein generally pertain to the fields of RNA therapeutics and glycobiology. More specifically, the embodiments described herein relate to glycosylated ligand compositions, their production, and their therapeutic administration in subjects. Background Technology

[0004] Glycans have numerous applications in nanomedicine, including the creation of biomaterials, the coating of nanoparticles to evade the immune system, and the initiation of cell signaling in specific cell types. (Sampaolesi et al., (2019), Future Med. Chem. 11(1): 43-60). For example, glycans can be used to target macrophages, B cells, or hepatocytes. (Sampaolesi et al., “Glycans in nanomedicine, impact and perspectives” (2019), Future Med. Chem. 11(1): 43-60).

[0005] In one study, certain polysaccharide residues exhibited signal transduction capabilities in dendritic cells. Exposure to nanoparticles functionalized through covalent bonding with disaccharide and lactose residues increased the cell surface expression of MHC II, CD86, CD40, the C-type lectin receptor CIRE, and the mannose receptor CD206. (Brenda et al., “Mannose-functionalized ‘pathogen-like’ polyanhydride nanoparticles target C-typelectin receptors on dendritic cells.” Molecular pharmaceutics, Vol. 8, 5 (2011): 1877-86). Exposure to unfunctionalized nanoparticles did not increase the expression of these cell surface markers. (Brenda et al., 2011). Both functionalized and unfunctionalized nanoparticles were internalized into dendritic cells, and blocking the mannose and CIRE receptors prior to exposure to functionalized nanoparticles prevented the increase of cell surface MHC II, CD40, and CD86. (Brenda et al., 2011). Therefore, the interaction of functionalized nanoparticles with mannose and CIRE receptors, as well as their internalization into dendritic cells, is essential for the upregulation of cell surface MHC II, CD40, and CD86 expression. (Brenda et al., 2011)

[0006] Recently, RNA therapeutics have been targeted at hepatocytes by utilizing the ability of specific polysaccharides, N-acetylgalactosamine (GalNAc), to bind to desialized glycoprotein receptors (ASGPR). Hu, B., Zhong, L., Weng, Y. et al., (2020), Sig. Transduct. Target Ther. 5(101). Unlike other cell types, each hepatocyte contains approximately 500,000 ASGPR receptors, allowing GalNAc-containing ligands to target them with high specificity. Hu, B., Zhong, L., Weng, Y. et al., (2020), Sig. Transduct. Target Ther. 5(101). For example, Alnylam Pharmaceuticals, Inc. has targeted hepatocytes by conjugating siRNA therapy with tetravalent and trivalent GalNAc ligands. Hu, B., Zhong, L., Weng, Y. et al., (2020), Sig. Transduct. Target Ther. 5(101). In 2019, Alnylam received the first FDA approval for its GalNAc-conjugated RNAi therapy, GIVLAARI® (givosiran), for the treatment of acute hepatic porphyria (AHP) in adults. The approved drug is a double-stranded siRNA that causes degradation of aminolevulinic acid synthase 1 (ALAS1) mRNA in hepatocytes through RNA interference, resulting in decreased levels of the neurotoxic intermediates aminolevulinic acid (ALA) and bile pigmentogen (PBG) in circulation, factors associated with the onset of AHP and other disease manifestations [Product insert 12 / 2020].

[0007] Recent advances have expanded the endogenous scaffold library of glycans to include RNA in addition to classic proteins and lipids. Flynn et al., (2019), bioRxiv: 787614. They found that sialylated glycans attached to RNA are displayed on the cell surface and interact with members of the Siglec receptor family. Flynn et al., (2021), Cell 184(12): 3109-3124. This evidence of RNA glycosylation suggests that glycosylation may play an important role in cell signaling.

[0008] Therefore, there is a need for a new class of cell signaling molecules that can be used to target specific cell types and mediate desired biological functions. Summary of the Invention

[0009] This document describes methods and compositions for producing pharmaceutical compositions comprising one or more glycans operably linked to one or more sites on a synthetic scaffold structural domain. The present invention provides methods for developing targeted therapeutic agents for pharmacological intervention against multiple targets mediated by glycan-mediated interactions. Such compositions exhibiting desired biophysical and pharmacodynamic properties are intended for the treatment of a variety of conditions, including cancer, inflammatory disorders, and autoimmune diseases. Therefore, the glycan-mediated compositions and methods of the present invention provide novel classes of therapeutic agents and new modalities of treatment.

[0010] In one aspect of this disclosure, a polysaccharide-polynucleotide conjugate of formula (A-1) is provided:

[0011] (KX A -V 1 -X B ) m -X 1 -V 2 -X 2 -W A-1,

[0012] Or its pharmaceutically acceptable salt.

[0013] In another respect, this disclosure provides a pharmaceutical composition comprising a polynucleotide conjugate of formula (A-1).

[0014] In various aspects, the present invention provides one or more glycans operably linked to one or more modification sites on a synthetic scaffold domain. Such synthetic scaffold domains include, but are not limited to, one or more nucleic acid sequences wherein at least one nucleotide site is modified, for example, the modification sequence on the scaffold operably links to signaling molecules, such as one or more glycans. Click chemistry has recently demonstrated methods for covalently conjugating glycans to RNA. Dong et al., Nature 2020 demonstrated that converting terminal amines to azides provides a chemical handle to the glycan, which can react with alkynes on nucleic acids, thereby resulting in covalent conjugation. Preferably, such methods operably link one or more desired glycans to RNA, thereby generating various combinations of therapeutic glycosylated RNA molecules.

[0015] Preferred synthetic scaffold domains include one or more nucleic acids selected from DNA, RNA, γ RNA, miRNA, mRNA, siRNA, antisense oligonucleotides (ASO), circRNA, ribosomal RNA, small RNA fragments (e.g., transfer RNA fragments), and related RNA types.

[0016] In other aspects of the invention, the glycans conjugated to the synthetic scaffold domains comprise one or more N-linked or O-linked glycans. Such glycans include, but are not limited to, one or more glycans selected, for example, from Tables 1A-1F. Preferably, the glycans conjugated to the synthetic scaffold domains contain autoantigens that are not readily recognized as foreign antigens by the host immune system or do not trigger unwanted immune responses. Exemplary embodiments of the invention demonstrate glycan-conjugated synthetic scaffold domains, for example, where glycoligands mediate desired cell signaling, receptor-mediated signaling cascades target cells of interest, or interact with specific carbohydrate receptors.

[0017] Methods and compositions for site-specific modification of target regions of synthetic scaffold domains are also provided, the methods further comprising contacting one or more glycans with a defined region of a target nucleic acid molecule, thereby stably attaching one or more desired glycans to the synthetic scaffold domain.

[0018] In various respects, pharmaceutical compositions comprising synthetic scaffold domains are characterized by a glycan site occupancy rate on a specific scaffold target greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or higher. Preferably, glycoligand compositions are described herein comprising: one or more glycans; and a ribonucleic acid sequence operably linked via covalent bonds to one or more glycans. More preferably, such pharmaceutical compositions comprising glycoligands are characterized by a glycan site occupancy rate greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or higher.

[0019] In related aspects, pharmaceutical compositions containing the desired glycoligand regulate cell surface proteins on target cells. In other aspects, pharmaceutical compositions exhibiting the desired glycoligand regulate the activity of target cells via cell surface proteins. Preferably, the pharmaceutical composition exhibits features associated with one or more of the following characteristics:

[0020] Stable glycoligands that mediate required biological functions;

[0021] Configurable and programmable sugar ligands for regulating biology;

[0022] Glycoligands that deliver or enhance other bioactive molecules to specific target cells.

[0023] In other respects, glycoligand compositions exhibit improved stability properties. For example, RNA-conjugated glycans modulate physicochemical properties such as conformational stability and interactions with cell surface proteins.

[0024] This article also describes a method for modulating the activation or inhibition of cell surface proteins on the surface of a cell population present in a subject, comprising contacting a pharmaceutical composition comprising a glycosylated synthetic scaffold. Therefore, this article provides methods and compositions for contacting glycoligands on cell surface proteins to transduce cell signaling. Preferably, the glycans on the glycoligands initiate receptor-mediated signaling cascades to target cells of interest, or by interacting with specific carbohydrate receptors such as lectins. Lectins have binding affinity for carbohydrates ranging from mM to nM. [Cummings RD, Darvill AG, Etzler ME et al. Glycan-Recognizing Probes as Tools. 2017. Source: Varki A, Cummings RD, Esko JD et al., eds. Essentials of Glycobiology [Internet]. 3rd ed. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2015-2017. Chapter 48]. For example, lectins have binding affinities in the mM range for monosaccharides, in the μM range for complex glycans, and in the nM range for multivalent complex glycan conjugates. [Cummings RD, Darvill AG, Etzler ME et al. Glycan-Recognizing Probes as Tools. 2017. Source: Varki A, Cummings RD, Esko JD et al., eds. Essentials of Glycobiology [Internet]. 3rd ed. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2015-2017. Chapter 48. More preferably, carbohydrate receptors recognize certain glycan structures or a limited number of sugar residues (e.g., even terminal sugar residues), and receptor-glycoligand interactions induce more robust responses upon repeated presentation (e.g., cluster effects). In various aspects, the glycan ligands of the present invention may be characterized by positive or negative regulators of the target receptor.

[0025] This document also describes a method for formulating glycoligand compositions. In some aspects, the method further includes a lipid formulation. In other aspects, the method includes a non-lipid formulation. In a preferred aspect, the glycoligand composition does not contain a lipid or non-lipid formulation. In some aspects, the formulation includes stabilizers and excipients.

[0026] A method for administering the pharmaceutical composition is also provided. Preferably, the pharmaceutical composition is administered subcutaneously or intradermally via microneedles.

[0027] In various aspects, this disclosure provides methods and compositions for administering one or more pharmaceutical compositions to a subject, said pharmaceutical compositions comprising one or more glycans linked to one or more sites on a synthetic scaffold structural domain. Preferably, the pharmaceutical compositions are for alleviating certain diseases, including, for example, cancer, inflammatory disorders, and autoimmune diseases. Detailed Implementation

[0028] This disclosure provides methods and compositions for modulating cell surface proteins and receptor complexes using novel classes of glycan conjugates, which can be used to bind signaling pathways within desired cell types. These defined cell-targeting bioactive glycoligands are directed for cell binding and activation in therapeutic applications.

[0029] definition

[0030] Unless otherwise defined herein, scientific and technical terms used in connection with this invention shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include plural terms and plural terms shall include singular terms. Generally, the nomenclature used in connection with biochemistry, enzymology, molecular and cell biology, microbiology, genetics and protein and nucleic acid chemistry, and hybridization techniques described herein is well known and commonly used in the art.

[0031] Unless otherwise indicated, the methods and techniques of the present invention are generally carried out according to conventional methods well known in the art, and as described in the various general and more specific references cited and discussed throughout this specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and 2002 Supplementary Edition); Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. (1990); Taylor and Drickamer, Introduction to Glycobiology, Oxford Univ. Press (2003); Worthington EnzymeManual, Worthington Biochemical Corp., Freehold, NJ; Handbook ofBiochemistry: Section A Proteins, Volume I, CRC Press (1976); Handbook ofBiochemistry: Section A Proteins, Volume II, CRC Press (1976); Essentials of Glycobiology, Cold Spring Harbor Laboratory Press (1999).

[0032] All publications, patents and other references mentioned herein are incorporated herein by reference in their entirety.

[0033] Unless otherwise instructed, the following terms shall be understood to have the following meanings:

[0034] In this document, numerical values ​​preceded by the term "approximately" are used to represent certain ranges. The term "approximately" provides textual support for precise figures that follow it, as well as for figures that are close to or approximate to those following the term "approximately." In determining whether a number is close to or approximates a specifically listed number, a number that is close to or approximates an unlisted number may be a number that provides substantially equal value to the specifically listed number in the context in which it is presented.

[0035] It should be noted that, unless the context clearly specifies otherwise, the singular forms “a / an” and “the” as used herein and in the appended claims include multiple indicators. It should be further noted that claims may be drafted to exclude any optional elements. Therefore, this description is intended to serve as a basis for using exclusive terms such as “only”, “merely”, or antecedents with “negative” restrictions related to reference to elements of the claims.

[0036] Throughout this specification and claims, the word “comprise” or variations such as “comprises” or “comprising” will be understood to imply inclusion of the stated integer or group of integers, but not to exclude any other integer or group of integers.

[0037] As used herein, the term “synthetic scaffold domain” refers to, but is not limited to, DNA, RNA, cellulose, chitosan, glycosaminoglycans (GAG), hyaluronic acid, chondroitin sulfate, alginate, polycaprolactone, and collagen, including nanoparticles or nanostructures.

[0038] As used herein, the term “modification site” refers to one or more sites on a synthetic scaffold domain or a location on the scaffold, such as a polymer containing reactive functional groups suitable for glycan conjugation, or more specifically, a conjugation site of one or more glycans.

[0039] As used herein, the term "polymer" refers to a substance composed of natural or synthetic monomers such as ribonucleotides.

[0040] As used herein, the term “bioactive” refers to molecules that are biologically active. For example, in the context of assays for “bioactive polymers,” receptor binding, as demonstrated by SPR, can detect biomolecular interactions, including interactions between carbohydrates and proteins, thereby indicating the presence of biologically active molecules [Nguyen HH, Park J, Kang S, Kim M. Surface plasmon resonance: a versatile technique for biosensor rapplications. Sensors (Basel). 2015 May 5;15(5):10481-510.].

[0041] As used herein, the term "part" refers to a molecule. For example, "carbohydrate part" or "oligosaccharide part" typically refers to a polysaccharide composition.

[0042] "Modified sequences" are nucleic acid molecules that differ from naturally occurring nucleic acid molecules by at least one factor. Modified sequences include all exogenously modified and unmodified heterologous sequences (i.e., sequences derived from organisms or cells other than those containing the modified sequence), as well as endogenous genes, operons, coding sequences, or non-coding sequences that have been modified, mutated, or contain deletions or insertions compared to naturally occurring sequences. Such sequences also include all sequences linked to an inducible promoter or another control sequence not naturally associated with it, regardless of their origin. These sequences further include all sequences that can be used to downregulate or knock out the expression of endogenous genes. These sequences include antisense molecules, RNAi molecules, constructs for generating homologous recombination, cre-lox constructs, etc.

[0043] The terms "polynucleotide," "nucleic acid molecule," or "nucleotide sequence" refer to a polymeric form of nucleotides with a length of at least 10 bases. These terms include DNA molecules (e.g., cDNA, genomic, or synthetic DNA) and RNA molecules (e.g., mRNA or synthetic RNA), as well as DNA or RNA analogs containing non-natural nucleotide analogs, non-natural nucleoside bonds, or both. Nucleic acids can be in any topological conformation. For example, nucleic acids can be single-stranded, double-stranded, triple-stranded, quadruple-stranded, partially double-stranded, branched, hairpin, circular, or lock-like conformations.

[0044] Unless otherwise indicated, and as an example of all sequences described herein with the general formula “SEQ ID NO:”, “a nucleic acid containing SEQ ID NO:1” means a nucleic acid having at least a portion of either of the following: (i) the sequence of SEQ ID NO:1 or (ii) a sequence complementary to SEQ ID NO:1. The choice between the two is determined by the context. For example, if the nucleic acid is used as a probe, the choice between the two is determined by the requirement that the probe is complementary to the desired target.

[0045] "Separated" RNA, DNA, or mixed polymers are RNA, DNA, or mixed polymers that are substantially separated from other cellular components (such as ribosomes, polymerases, and their naturally associated genomic sequences) that are naturally associated with the natural host cell.

[0046] As used herein, an "isolated" composition (e.g., a glycoligand) is a composition substantially isolated from the cellular components (membrane lipids, chromosomes, proteins) of its source host cell or the culture medium in which the host cell is cultured. The term does not require that the biomolecule has been isolated from all other chemical substances, although some isolated biomolecules may be purified to a near-homogeneous state.

[0047] The term “recombinant” refers to a biomolecule, such as a gene or protein, that (1) has been removed from its naturally occurring environment, (2) is not associated with all or part of a polynucleotide found in nature, (3) is operatively linked to a polynucleotide not linked to it in nature, or (4) is not found in nature. The term “recombinant” can also refer to cloned DNA isolates, chemically synthesized polynucleotide analogs or polynucleotide analogs biosynthesized through heterologous systems, and proteins and / or mRNA encoded by such nucleic acids.

[0048] As used herein, an endogenous nucleic acid sequence (or the protein product encoded by that sequence) in an organism's genome is considered "recombinant" if placing a heterologous sequence adjacent to an endogenous nucleic acid sequence alters the expression of that endogenous nucleic acid sequence. In this context, a heterologous sequence is a sequence that is not naturally adjacent to an endogenous nucleic acid sequence, regardless of whether the heterologous sequence itself is endogenous (originating from the same host cell or its descendants) or exogenous (originating from different host cells or their descendants). For example, a promoter sequence can replace (e.g., through homologous recombination) the natural promoter of a gene in a host cell's genome, thereby altering the expression pattern of that gene. Because this gene is separated from at least some naturally flanked sequences, the gene will now become "recombinant."

[0049] Nucleic acids are also considered "recombinant" if they contain any modifications that are not naturally present in the corresponding nucleic acid in the genome. For example, if an endogenous coding sequence contains artificially introduced insertions, deletions, or point mutations (e.g., through human intervention), the endogenous coding sequence is considered "recombinant." "Recombinant nucleic acids" also include nucleic acids integrated into heterologous sites on the host cell chromosome and nucleic acid constructs existing as episomes.

[0050] As used herein, a “degenerate variant” of a phrase reference nucleic acid sequence encompasses a nucleic acid sequence that can be translated according to the standard genetic code to provide the same amino acid sequence translated from the reference nucleic acid sequence. The terms “degenerate oligonucleotide” or “degenerate primer” are used to refer to oligonucleotides capable of hybridizing with target nucleic acid sequences that are not necessarily identical but are homologous to each other within one or more specific segments.

[0051] In the context of nucleic acid sequences, the term "sequence identity percentage" or "identity" refers to the same residues in two sequences when aligned to obtain the maximum correspondence. The length of a sequence identity comparison can be more than at least about 9 nucleotides, typically at least about 20 nucleotides, more typically at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably an extension of at least about 36 or more nucleotides. Many different algorithms are known in the art for measuring nucleotide sequence identity. For example, FASTA, Gap, or Bestfit can be used to compare polynucleotide sequences; these programs are all found in WisconsinPackage version 10.0 of the Genetics Computer Group (GCG), Madison, Wis. FASTA provides the alignment of the best overlapping region between the query and search sequences and the sequence identity percentage. Pearson, Methods Enzymol. 183:63-98 (1990) (incorporated hereinth by reference in its entirety). For example, FASTA and its default parameters (word length of 6 and NOPAM factor of the scoring matrix) or Gap and its default parameters can be used to determine the percentage of sequence identity between nucleic acid sequences, as provided in GCG version 6.1, which is incorporated herein by reference. The computer program BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993); Madden et al., Meth. Enzymol. 266:131-141 (1996); Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res. 7:649-656 (1997)) can be used to compare sequences, especially blastp or tblastn (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)).

[0052] The terms “substantially homology” or “substantially similarity” when referring to nucleic acids or fragments thereof indicate that, when optimally aligned with another nucleic acid (or its complementary strand) for appropriate nucleotide insertions or deletions, at least about 76%, 80%, 85%, preferably at least about 90%, and more preferably at least about 95%, 96%, 97%, 98%, or 99% of the nucleotide bases have nucleotide sequence identity, as measured by any well-known sequence identity algorithm (such as FASTA, BLAST, or Gap, as discussed above).

[0053] Under stringent hybridization conditions, when a nucleic acid or a fragment thereof hybridizes with another nucleic acid, a strand of another nucleic acid, or its complementary strand, there is substantial homology or similarity. In the context of nucleic acid hybridization experiments, "stringent hybridization conditions" and "stringent washing conditions" depend on many different physical parameters. Nucleic acid hybridization is affected by a variety of conditions, such as salt concentration, temperature, solvent, base composition of the hybrid species, length of the complementary region, and the number of nucleotide base mismatches between the hybrid nucleic acids, which is obvious to those skilled in the art. Those skilled in the art know how to modify these parameters to achieve a specific degree of hybridization stringency.

[0054] Typically, "strict hybridization" occurs under specific conditions at a temperature higher than the melting point (T0) of a specific DNA heterozygote. m The washing was performed at a temperature approximately 25°C lower than that of a specific DNA heterozygote. "Strict washing" refers to washing under specific conditions, at a temperature lower than that of a specific DNA heterozygote. m It was carried out at a temperature approximately 5°C lower. m This is the temperature at which 50% of the target sequence hybridizes with a perfectly matched probe. See Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), p. 9.51, which is incorporated herein by reference. For the purposes of this paper, “strict conditions” are defined as aqueous hybridization (i.e., formamide-free) in solution phase hybridization at 65°C for 8–12 h in 6xSSC (of which 20xSSC contains 3.0 M NaCl and 0.3 M sodium citrate) and 1% SDS, followed by two washes at 65°C for 20 min each in 0.2xSSC and 0.1% SDS. Those skilled in the art will understand that the rate of hybridization at 65°C will vary depending on a number of factors, including the length of the hybridized sequence and the percentage of identity.

[0055] The nucleic acids (also known as polynucleotides) of the present invention may include the sense and antisense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers thereof. They may be chemically or biochemically modified, or may contain non-natural or derived nucleotide bases, as will be apparent to those skilled in the art. Such modifications include, for example, labeling, methylation, substitution of one or more naturally occurring nucleotides with analogs, internucleotide modifications such as uncharged linkages (e.g., methylphosphonates, triphosphates, phosphoramides, carbamates, etc.), charged linkages (e.g., thiophosphates, dithiophosphates, etc.), side chain portions (e.g., polypeptides), intercalating agents (e.g., acridine, psoralen, etc.), chelating agents, alkylating agents, and modified linkages (e.g., alpha anomeric nucleic acids, etc.). Synthetic molecules are also included that mimic the ability of a polynucleotide to bind to a specified sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, molecules in which peptide linkages replace phosphate linkages in the molecular backbone. Other modifications may include, for example, ribose rings containing bridging portions or other structural analogues, such as those found in "lock" nucleic acids.

[0056] When the term “mutation” is applied to a nucleic acid sequence, it means that nucleotides in the nucleic acid sequence may be inserted, deleted, or altered compared to a reference nucleic acid sequence. A single alteration (point mutation) can be made at a locus, or multiple nucleotides may be inserted, deleted, or altered at a single locus. Additionally, one or more alterations may be made at any number of loci within the nucleic acid sequence. Nucleic acid sequences can be mutated by any method known in the art, including but not limited to mutagenesis techniques such as “error-prone PCR” (a method of performing PCR under conditions of low DNA polymerase replication fidelity, resulting in a high rate of point mutations across the entire length of the PCR product; see, for example, Leung et al., Technique, 1:11-15 (1989) and Caldwell and Joyce, PCR Methods Applic. 2:28-33 (1992)); and “oligonucleotide directed mutagenesis” (a method capable of generating site-specific mutations in any cloned DNA segment of interest; see, for example, Reidhaar-Olson and Sauer, Science 241:53-57 (1988)).

[0057] The term "downregulation," such as "downregulation signal," refers to a process that compares the expression levels of a target gene before and after contact with a glycoligand, for example, at the mRNA or protein level. If it is determined that the amount of RNA or protein expressed by the target gene decreases after contact with a glycoligand, it can be concluded that the glycoligand downregulates the target gene expression. The levels of target RNA or protein in cells can be determined by any desired method. For example, the level of target RNA can be determined by Northern blot analysis, reverse transcription polymerase chain reaction (RT-PCR), or RNase protection assay. For example, the level of protein can be determined by Western blot analysis.

[0058] The term "silencing," such as "silencing a target gene," refers to a process in which a cell, when not in contact with a glycoligand, contains and / or secretes a product of a target gene, but when in contact with a glycoligand, the amount of such gene product contained and / or secreted by the cell is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to similar cells not in contact with a glycoligand. Such products of the target gene can be, for example, messenger RNA (mRNA), proteins, or regulatory elements.

[0059] As used herein, the term “attenuation” generally refers to a loss of function, including mutations, partial or complete deletions, insertions, or other variations in a gene sequence or a sequence that controls the transcription of a gene sequence, thereby reducing or inhibiting the production of the gene product, or rendering the gene product nonfunctional. In some cases, loss of function is described as a knockout mutation. Attenuation also includes altering the amino acid sequence by changing the nucleic acid sequence, placing the gene under the control of a promoter with lower activity, downregulating the expression of interfering RNA, ribozymes, or antisense sequences targeting the gene of interest, or by any other technique known in the art. In one instance, a particular enzyme is less sensitive to feedback inhibition or inhibition caused by a combination of non-products or reactants (non-pathway-specific feedback), such that the enzyme activity is unaffected by the presence of the compound. In other cases, an enzyme whose activity is reduced by alteration may be referred to as attenuated. As used herein, with respect to a gene sequence, the term “deletion” generally refers to the removal of one or more nucleotides from a nucleic acid molecule, or the removal of one or more amino acids from a protein, with the regions on either side joined together. As used herein, with respect to a gene sequence, the term “knockout” generally refers to a gene whose expression level or activity is reduced to zero. In some instances, a gene is knocked out by deleting part or all of its coding sequence. In other instances, a gene is knocked out by introducing one or more nucleotides into its open reading frame, resulting in the translation of a meaningless or otherwise nonfunctional protein product.

[0060] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is the "plasmid," which generally refers to a circular double-stranded DNA loop into which an additional DNA segment can be linked, but also includes linear double-stranded molecules, such as those produced by polymerase chain reaction (PCR) amplification or by treating circular plasmids with restriction enzymes. Other vectors include granules, bacterial artificial chromosomes (BACs), and yeast artificial chromosomes (YACs). Another type of vector is the viral vector, in which an additional DNA segment can be linked to a viral genome (discussed in more detail below). Some vectors are capable of autonomous replication in the host cell to which they are introduced (e.g., vectors with an origin of replication that functions in the host cell). Other vectors can integrate into the host cell's genome after introduction into the host cell and thereby replicate along with the host genome. Furthermore, certain preferred vectors are capable of guiding the expression of genes operatively linked to them. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors").

[0061] "Operationally linked" or "operably linked" expression control sequences refer to expression control sequences that are contiguous with the gene of interest to control the expression of the gene of interest, as well as trans- or long-range acting expression control sequences to control the expression of the gene of interest. The terminology is also used herein to refer to the glycan portion conjugated to the synthetic scaffold domain, as described herein.

[0062] As used herein, the term "expression control sequence" refers to a polynucleotide sequence essential to the expression of a coding sequence to which it is operably linked. Expression control sequences are sequences that control transcription, post-transcriptional events, and translation of nucleic acid sequences. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance protein stability; and sequences that enhance protein secretion when needed. The nature of such control sequences varies from host organism to host; in prokaryotes, such control sequences typically include promoters, ribosome binding sites, and transcription termination sequences. The term "control sequence" is intended to include at least all components whose presence is essential for expression, and may also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences.

[0063] As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell in which a recombinant vector has been introduced. It should be understood that this term is intended not only to refer to a specific subject cell, but also to refer to the progeny of such cells. Certain modifications may occur during successive passages due to mutations or environmental influences, and therefore such progeny may not be substantially identical to the parent cells, but they are still included within the scope of the term "host cell" as used herein. Recombinant host cells can be isolated cells or cell lines grown in culture, or cells present in living tissues or organisms.

[0064] As used herein, the term "peptide" refers to a short polypeptide, for example, typically less than about 50 amino acids in length, and more typically less than about 30 amino acids in length. As used herein, the term encompasses analogs and mimics that imitate structures to mimic biological functions.

[0065] The term "peptide" encompasses both naturally occurring and non-naturally occurring proteins, as well as their fragments, mutants, derivatives, and analogs. Peptides can be monomers or polymers. Furthermore, peptides can contain multiple distinct domains, each possessing one or more different activities.

[0066] The terms “isolated protein” or “isolated polypeptide” refer to a protein or polypeptide that, due to its derived origin, is characterized by: (1) not being associated with the naturally occurring associated components that accompany it in its native state; (2) existing with a purity not found in nature, wherein the purity can be determined by the presence of other cellular material (e.g., not containing other proteins from the same species); (3) being expressed by cells from a different species; or (4) not existing in nature (e.g., it is a fragment of a polypeptide found in nature, or it contains amino acid analogs or derivatives not found in nature, or contains bonds other than standard peptide bonds). Thus, a polypeptide synthesized chemically or synthesized in a cellular system different from its native source cell will be “isolated” from its naturally occurring associated components. Isolation can also render a polypeptide or protein substantially free of its naturally occurring associated components using protein purification techniques well known in the art. By this definition, “isolation” does not necessarily require that the described protein, polypeptide, peptide, or oligopeptide has been physically removed from its native environment.

[0067] As used herein, the term "peptide fragment" refers to a polypeptide that has deletions (e.g., deletions of the N-terminus and / or C-terminus) compared to a full-length polypeptide. In a preferred embodiment, the polypeptide fragment is a continuous sequence in which the amino acid sequence of the fragment corresponds to the position in a naturally occurring sequence. The length of the fragment is typically at least 5, 6, 7, 8, 9, or 10 amino acids, preferably at least 12, 14, 16, or 18 amino acids, more preferably at least 20 amino acids, more preferably at least 25, 30, 35, 40, or 45 amino acids, even more preferably at least 50 or 60 amino acids, and even more preferably at least 70 amino acids.

[0068] "Modified derivatives" refer to polypeptides or fragments thereof that are substantially homologous in their primary structural sequence but include, for example, in vivo or in vitro chemical and biochemical modifications, or the incorporation of amino acids not found in the native polypeptide. Such modifications include, for example, acetylation, carboxylation, phosphorylation, glycosylation, ubiquitination, labeling (e.g., with radioactive isotopes), and various enzymatic modifications, which will be apparent to those skilled in the art. Labeling polypeptides and various methods of substituents or labeling for such purposes are well known in the art, and include radioactive isotopes (such as... 125 I, 32 P, 35 S and 3 H), ligands that bind to labeled anti-ligands (e.g., antibodies), fluorophores, chemiluminescent agents, enzymes, and anti-ligands that can specifically bind to the labeled ligand member. The choice of label depends on the required sensitivity, ease of primer conjugation, stability requirements, and available instruments. Methods for labeling peptides are well known in the art. See, for example, Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and 2002 ed.) (incorporated hereby by reference).

[0069] The term "fusion protein" refers to a polypeptide comprising a polypeptide or fragment coupled to a heterologous amino acid sequence. Fusion proteins are useful because they can be constructed to contain two or more desired functional elements from two or more different proteins. Fusion proteins comprise at least 10 consecutive amino acids from the polypeptide of interest, more preferably at least 20 or 30 amino acids, even more preferably at least 40, 50, or 60 amino acids, and even more preferably at least 75, 100, or 125 amino acids. Fusions comprising all the proteins of the present invention have particular utility. The heterologous polypeptide contained within the fusion protein of the present invention is at least 6 amino acids in length, typically at least 8 amino acids in length, and preferably at least 15, 20, and 25 amino acids in length. Fusions comprising larger polypeptides (such as the IgG Fc region) and even entire proteins (such as proteins containing a green fluorescent protein (“GFP”) chromophore) have particular utility. Fusion proteins can be generated by constructing a nucleic acid sequence encoding a polypeptide or a fragment thereof, and then expressing the fusion protein recombinantly, said nucleic acid sequence being in the same frame as nucleic acid sequences encoding different proteins or peptides. Fusion proteins can be chemically produced by crosslinking a polypeptide or a fragment thereof with another protein.

[0070] The term "non-peptide analog" refers to a compound whose properties are similar to those of a reference peptide. Non-peptide compounds may also be called "peptide mimetic" or "peptidomimetic". See, for example, Jones, Amino Acid and Peptide Synthesis, Oxford University Press (1992); Jung, Combinatorial Peptide and Nonpeptide Libraries: A Handbook, John Wiley (1997); Bodanszky et al., Peptide Chemistry—A Practical Textbook, Springer Verlag (1993); Synthetic Peptides: A Users Guide, (Grant ed., WH Freeman and Co., 1992); Evans et al., J. Med. Chem. 30:1229 (1987); Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber and Freidinger, Trends Neurosci., 8:392-396 (1985); and references cited in each of the above-mentioned works, which are incorporated herein by reference. Such compounds are typically developed using computer molecular modeling. Peptide mimics with structures similar to the useful peptides of the present invention can be used to produce equivalent effects, and are therefore envisioned as part of the present invention.

[0071] A "peptide mutant" or "mutant protein" is a polypeptide whose sequence, compared to that of a native or wild-type protein, contains one or more insertions, duplications, deletions, rearrangements, or substitutions of amino acids. Mutant proteins may have one or more amino acid substitutions, where a single amino acid at a position is changed to another amino acid; one or more insertions and / or deletions, where one or more amino acids are inserted or deleted in the sequence of a naturally occurring protein; and / or truncation of the amino acid sequence at any one or both of the amino or carboxyl termini. Mutant proteins may have the same biological activity as naturally occurring proteins, but preferably have different biological activities.

[0072] The mutant protein has at least 85% overall sequence homology with its wild-type counterpart. Even more preferably, the mutant protein has at least 90% overall sequence homology with the wild-type protein.

[0073] In even more preferred embodiments, the mutant protein exhibits at least 95% sequence identity, even more preferably 98%, even more preferably 99%, and even more preferably 99.9% overall sequence identity.

[0074] Sequence homology can be measured using any common sequence analysis algorithm, such as Gap or Bestfit.

[0075] Amino acid substitution may include the following substitutions: (1) reducing susceptibility to protein hydrolysis, (2) reducing susceptibility to oxidation, (3) altering the binding affinity for forming protein complexes, (4) altering binding affinity or enzyme activity, and (5) conferring or altering other physicochemical or functional properties of such analogues.

[0076] As used herein, the twenty common amino acids and their abbreviations follow conventional usage. See Immunology-ASynthesis (Golub and Gren, eds., Sinauer Associates, Sunderland, Mass., 2nd ed. 1991), which is incorporated herein by reference. Stereoisomers of the twenty common amino acids (e.g., D-amino acids), non-natural amino acids (such as α,α-disubstituted amino acids, N-alkyl amino acids, and other unconventional amino acids) may also be suitable components of the peptides of this invention. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamic acid, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In accordance with standard usage and convention, in the peptide representation used herein, the left end corresponds to the amino terminus, and the right end corresponds to the carboxyl terminus.

[0077] The protein is said to be "homological" or "homologous" to the second protein if the nucleic acid sequence encoding a protein has a similar sequence to that encoding a second protein. In an embodiment, the protein is homologous to the second protein if the two proteins have "similar" amino acid sequences. (Therefore, the term "homological protein" is defined as meaning that two proteins have similar amino acid sequences.) As used herein, homology between two regions of an amino acid sequence (particularly in relation to predicted structural similarity) is interpreted as implying functional similarity.

[0078] When “homologous” is used to refer to proteins or peptides, it is recognized that differences in residue positions are usually due to conserved amino acid substitutions. A “conserved amino acid substitution” is an amino acid substitution in which another amino acid residue in a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity) replaces an amino acid residue. Generally, conserved amino acid substitutions do not substantially alter the functional properties of a protein. If two or more amino acid sequences differ from each other due to conserved substitutions, the percentage of sequence identity or degree of homology can be adjusted upwards to correct for the conservation of the substitution. Methods for making such adjustments are well known to those skilled in the art. See, for example, Pearson, 1994, Methods Mol. Biol. 24:307-31 and 25:365-89 (incorporated herein by reference).

[0079] The following six groups each contain amino acids that are conserved substitutes for each other: 1) serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), alanine (A), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W).

[0080] Sequence homology (also known as sequence identity percentage) of peptides is typically measured using sequence analysis software. See, for example, the sequence analysis software package from the Genetic Computing Group (GCG), University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wis. 53705. Protein analysis software uses measurements assigned to homology-matching sequences for various substitutions, deletions, and other modifications, including conserved amino acid substitutions. For example, GCG contains programs such as “Gap” and “Bestfit”, which can be used with default parameters to determine sequence homology or sequence identity between closely related peptides (such as homologous peptides from different biological species) or between wild-type proteins and their mutant counterparts. See, for example, GCG version 6.1.

[0081] When comparing a specific polypeptide sequence with a database containing a large number of sequences from different organisms, the preferred algorithm is the computer program BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993); Madden et al., Meth. Enzymol. 266:131-141 (1996); Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res. 7:649-656 (1997)), especially blastp or tblastn (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)).

[0082] The preferred parameters for BLASTp are: Expected value: 10 (default); Filter: seg (default); Cost of opening gaps: 11 (default); Cost of expanding gaps: 1 (default); Maximum number of alignments: 100 (default); Word length: 11 (default); Number of descriptions: 100 (default); Penalty matrix: BLOWSUM62.

[0083] The length of peptide sequences used for homology comparison is typically at least about 16 amino acid residues, typically at least about 20 residues, more typically at least about 24 residues, typically at least about 28 residues, and preferably more than about 35 residues. When searching a database containing sequences from a large number of different organisms, it is preferable to compare amino acid sequences. Database searches using amino acid sequences can be measured using algorithms other than blastp, which are known in the art. For example, peptide sequences can be compared using FASTA (the procedure in GCG version 6.1). FASTA provides alignment of the best overlapping regions between the query and search sequences and the percentage of sequence identity. Pearson, Methods Enzymol. 183:63-98 (1990) (incorporated hereby by reference). For example, the percentage of sequence identity between amino acid sequences can be determined using FASTA and its default parameters (word length of 2 and PAM250 scoring matrix), as provided in GCG version 6.1, which is incorporated hereby by reference.

[0084] "Specific binding" refers to the ability of two molecules to preferentially bind to each other rather than to other molecules in the environment. Typically, "specific binding" is distinguished from accidental binding by at least two times, more often at least 10 times, and usually at least 100 times, in a reaction. The affinity or cohesion (quantified by the dissociation constant) of a specific binding reaction is typically around 10. -7 M or stronger (e.g., about 10) -8 M, 10 -9 M or even stronger).

[0085] As used herein, the term "region" refers to a physically continuous portion of the primary structure of a biomolecule. For proteins, a region is defined by a continuous portion of the protein's amino acid sequence.

[0086] As used herein, the term "domain" refers to a structure in a biomolecule that contributes to the known or suspected function of the biomolecule. A domain may extend along with its regions or parts; a domain may also include different, discontinuous regions of the biomolecule. Examples of protein domains include, but are not limited to, Ig domains, extracellular domains, transmembrane domains, and cytoplasmic domains.

[0087] As used herein, the term "molecule" means any compound, including but not limited to small molecules, peptides, proteins, sugars, nucleotides, nucleic acids, lipids, etc., and such compounds may be natural or synthetic.

[0088] The term "N-linked glycan" or "N-glycan" refers to an N-linked oligosaccharide structure covalently bonded to a nitrogen atom, optionally via an amide bond, optionally as an N-glycan, and conjugated at asparagine or arginine residues via N-acetylglucosamine residues on the glycan, typically via glycosyltransferases. These "N-linked glycosylation sites" are present in the primary structure of peptides, which, for example, contain the classic amino acid sequence asparagine-X-serine / threonine, where X is any amino acid residue other than proline and aspartic acid. "N-linked glycan" refers to an N-linked oligosaccharide structure. N-glycans can be attached to proteins or scaffolds that can be further manipulated in vitro or in vivo. Common N-linked glycans typically include complex, mixed, high-mannose, branched, and multi-tentacle structures. In the case of glycans, the term "N-linked" can refer to a scaffold having attached N-acetylglucosamine (GlcNAc) residues that are linked to the amide nitrogen (N-linked) of asparagine residues on a protein or scaffold, which is similar to or even the same as human-produced glycans.

[0089] "O-glycan" or "O-linked glycan" refers to an O-linked oligosaccharide structure. O-glycans can be attached to proteins or scaffolds that can be further manipulated in vitro or in vivo. Common O-GalNAc core structures typically include a core 1, a core 2, and a poly-N-acetylgalactosamine (LacNAc) structure. In some embodiments, the O-linked oligosaccharide is covalently linked via an oxygen atom on a serine residue. In the context of glycans, the term "O-linked" can refer to a conjugate having attached N-acetylgalactosamine (GalNAc) residues linked to an oxygen atom on a serine or threonine residue on a protein or scaffold, similar to or even identical to human-derived glycans.

[0090] In the case of glycans, the term "N-linked" refers to a scaffold having attached N-acetylglucosamine (GlcNAc) residues that are linked to the amide nitrogen (N-linked) of asparagine residues on a protein or scaffold, which is similar to or even the same as glycans produced by humans.

[0091] In the case of glycans, the term "O-linked" refers to a conjugate having attached N-acetylgalactosamine (GalNAc) residues that are linked to the oxygen atom of a serine or threonine residue on a protein or scaffold, which is similar to or even identical to glycans produced by humans.

[0092] As used herein, the term "monosaccharide" refers to a carbohydrate molecule that cannot be hydrolyzed into two or more simpler carbohydrates. Examples of monosaccharides include, but are not limited to, GlcNAc, mannose, fucose, glucose, fructose, and galactose.

[0093] The term "glycan" refers to oligosaccharide structures, the main oligosaccharide structures found on glycoproteins, including glucose (Glu), galactose (Gal), mannose (Man), fucose (Fuc), N-acetylglucosamine (GlcNAc), N-acetylglucosamine (GalNAc), glucosamine (GlcN), galactosamine (GalN), glucuronic acid (GlcA), iduronic acid (IdoA), and sialic acid (e.g., N-acetyl-neuraminic acid (NeuNAc or NANA)). Hexoses, belonging to the six-carbon monosaccharides such as glucose, galactose, and mannose, are not easily distinguishable by mass spectrometry and may also be present. N-glycans differ in the number of their branches ("tentacles" or "arms"), which contain peripheral sugars (e.g., GlcNAc, galactose, fucose, and sialic acid) added to the "trimannosyl core". The term "trimannose core," also known as "M3," "M3GN2," "trimannose core," "pentasaccharide core," or "paucimannose core," reflects the oligosaccharide structure of Man3GlcNAc2, in which the Manα1,3 and Manα1,6 arms extend from the di-GlcNAc structure (GlcNAc2): β1,4GlcNAc-β1,4GlcNAc. N-glycans are classified according to their branched components (e.g., high-mannose, complex, or mixed).

[0094] "High-mannose" type N-glycans contain four or more mannose residues in their di-GlcNAc oligosaccharide structure. "M9" reflects Man9GlcNAc2. "M5" reflects Man5GlcNAc2.

[0095] "Mixed" N-glycans have at least one GlcNAc residue at the end of the α1,3-mannose (Man α1,3) arm of the trimannose core, and zero or more mannose residues on the α1,6-mannose (Man α1,3) arm of the trimannose core. An example of a mixed-type glycan is GlcNAcMan3GlcNAc2.

[0096] "Complex" N-glycans typically have at least one GlcNAc residue on the Manα1,3 arm attached to the trimannose core (sometimes referred to as "G0" or "G0F" fucosylation), and at least one GlcNAc residue on the Manα1,6 arm attached to the trimannose core. Complex N-glycans may also have galactose or N-acetylgalactosamine residues ("G2" or "G2F" fucosylation), which are optionally modified with sialic acid ("G2S2" or "G2FS2" fucosylation) or derivatives (e.g., "Neu" refers to neuraminic acid and "Ac" refers to acetyl). Complex N-glycans may also have intrachain substitutions comprising a "dichotomous" GlcNAc and a core fucose. Complex N-glycans can also have multiple tentacles on a trimannose core, and are often referred to as "multi-tentacle glycans" or "multi-branched glycans", which can be tritentacle, tetratentacle or pentatentacle glycans.

[0097] The term "glycoform" generally refers to an isoform of oligosaccharides attached to a protein or scaffold (such as an RNA molecule), differing only in the number and / or type of one or more glycans attached. Glycoligands can contain one or more different or identical glycoforms. Based on the presence or absence of one or more isoforms of oligosaccharides attached to or conjugated to a protein or scaffold, glycoforms can be described as homogeneous, dominant, or heterogeneous, which is typically measured using analytical techniques.

[0098] As used herein, the term “major” or variations such as “primary” or “is major” should be understood to mean the glycan species that has the highest molar percentage (%) in total N-glycans after removal of the glycoligands (e.g., treatment with PNGase and release of the glycan) and as measured by mass spectrometry (e.g., MALDI-TOF MS). In other words, the phrase “major” is defined as an individual entity (such as a particular glycotype) that exists at a molar percentage greater than that of any other individual entity. For example, if a composition consists of 40 molar percentages of species A, 35 molar percentages of species B, and 25 molar percentages of species C, then the composition primarily contains species A. The terms “enriched,” “homogeneous,” “homogeneous,” and “consistently of” are also synonymous with “major” when referring to one or more glycans.

[0099] The molar percentage of N-glycans measured by MALDI-TOF-MS in positive ion mode refers to the molar percentage of sugar transfer relative to the molar percentage of total N-glycans. Certain cationic adducts, such as K+ and Na+, are typically correlated with the elution peaks, increasing the mass of the N-glycan by the molecular weight of the corresponding adduct.

[0100] The term "effective dose" or "therapeutic effective dose" refers to a dose sufficient to produce the desired result, such as an amount sufficient to achieve beneficial or desired (including preventative and / or therapeutic) outcomes, such as reducing symptoms of a medical condition (e.g., cancer, infectious diseases, immune-mediated conditions (e.g., autoimmune diseases, inflammatory diseases), etc.) compared to a control. In the case of cancer, in some implementations, the therapeutic effective dose is sufficient to slow tumor growth, shrink tumor size, etc. The effective dose can be administered in one or more applications.

[0101] When listing a range of values, it is intended to cover every value within that range and its subranges. For example, "C 1-6 "Alkyl" is intended to encompass C1, C2, C3, C4, C5, C6, C 1-6 C 1-5 C 1-4 C 1-3 C 1-2 C 2-6 C 2-5 C 2-4 C 2-3 C 3-6 C 3-5 C 3-4 C 4-6 C 4-5 and C 5-6 alkyl.

[0102] The term "alkyl" refers to a group having one to ten carbon atoms, either a straight-chain or branched saturated hydrocarbon group ("C"). 1-10 Alkyl group (“alkyl”). In some embodiments, the alkyl group has 1 to 9 carbon atoms (“C”). 1-9 Alkyl group (“alkyl”). In some embodiments, the alkyl group has 1 to 8 carbon atoms (“C”). 1-8 Alkyl group (“alkyl”). In some embodiments, the alkyl group has 1 to 7 carbon atoms (“C”). 1-7 Alkyl group (“alkyl”). In some embodiments, the alkyl group has 1 to 6 carbon atoms (“C”). 1-6 Alkyl group (“alkyl”). In some embodiments, the alkyl group has 1 to 5 carbon atoms (“C”). 1-5 Alkyl group (“alkyl”). In some embodiments, the alkyl group has 1 to 4 carbon atoms (“C”). 1-4 Alkyl group (“alkyl”). In some embodiments, the alkyl group has one to three carbon atoms (“C”). 1-3 Alkyl group (“alkyl”). In some embodiments, the alkyl group has one to two carbon atoms (“C”). 1-2 Alkyl group (“C1 alkyl”). In some embodiments, the alkyl group has one carbon atom (“C1 alkyl”). In some embodiments, the alkyl group has two to six carbon atoms (“C1 alkyl”). 2-6 Alkyl group). C1-6 Examples of alkyl groups include methyl (C1), ethyl (C2), propyl (C3) (e.g., n-propyl, isopropyl), butyl (C4) (e.g., n-butyl, tert-butyl, sec-butyl, isobutyl), pentyl (C5) (e.g., n-pentyl, 3-pentyl, amyl, neopentyl, 3-methyl-2-butyl, tert-pentyl), and hexyl (C6) (e.g., n-hexyl). Additional examples of alkyl groups include n-heptyl (C7), n-octyl (C8), etc. Unless otherwise specified, each example of an alkyl group is independently unsubstituted (“unsubstituted alkyl”) or substituted with one or more substituents (e.g., halogens such as F) (“substituted alkyl”). In some embodiments, the alkyl group is an unsubstituted C1 alkyl group. 1-10 Alkyl groups (such as unsubstituted C4) 1-6 Alkyl groups, such as -CH3(Me), unsubstituted ethyl groups (Et), unsubstituted propyl groups (Pr, such as unsubstituted n-propyl (n-Pr), unsubstituted isopropyl (i-Pr)), and unsubstituted butyl groups (Bu, such as unsubstituted n-butyl (n-Bu), unsubstituted tert-butyl (tert-Bu or t-Bu), unsubstituted sec-butyl (sec-Bu), or unsubstituted isobutyl (i-Bu)). In some embodiments, the alkyl group is a substituted C. 1-10 Alkyl groups (such as substituted C4 groups) 1-6 Alkyl groups, such as -CF3, Bn).

[0103] The term "heteroalkyl" refers to an alkyl group that further comprises at least one heteroatom selected from oxygen, nitrogen, or sulfur (e.g., one, two, three, or four heteroatoms), said heteroatom being located within the parent chain (i.e., inserted between adjacent carbon atoms) and / or situated at one or more end positions of the parent chain. In some embodiments, a heteroalkyl group refers to a saturated group ("heteroalkyl") having one to 20 carbon atoms and one or more heteroatoms within the parent chain. 1-20 Alkyl group (“alkyl”). In some embodiments, the heteroalkyl group is a saturated group having 1 to 18 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroalkyl”). 1-18 Alkyl group (“alkyl”). In some embodiments, the heteroalkyl group is a saturated group having 1 to 16 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroalkyl”). 1-16 Alkyl group (“alkyl”). In some embodiments, the heteroalkyl group is a saturated group having 1 to 14 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroalkyl”). 1-14 Alkyl group (“alkyl”). In some embodiments, the heteroalkyl group is a saturated group having 1 to 12 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroalkyl”). 1-12Alkyl group (“alkyl”). In some embodiments, the heteroalkyl group is a saturated group having 1 to 10 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroalkyl”). 1-10 Alkyl group (“alkyl”). In some embodiments, the heteroalkyl group is a saturated group having one to eight carbon atoms and one or more heteroatoms within the parent chain (“heteroalkyl”). 1-8 Alkyl group (“alkyl”). In some embodiments, the heteroalkyl group is a saturated group having one to six carbon atoms and one or more heteroatoms within the parent chain (“heteroalkyl”). 1-6 Alkyl group (“alkyl”). In some embodiments, the heteroalkyl group is a saturated group having one to four carbon atoms and one or two heteroatoms within the parent chain (“heteroalkyl”). 1-4 Alkyl group (“alkyl”). In some embodiments, the heteroalkyl group is a saturated group having one to three carbon atoms and one heteroatom in the parent chain (“heteroalkyl”). 1-3 Alkyl group (“alkyl”). In some embodiments, the heteroalkyl group is a saturated group having one to two carbon atoms and one heteroatom in the parent chain (“heteroalkyl”). 1-2 Alkyl group (“heteroalkyl”). In some embodiments, a heteroalkyl group is a saturated group having one carbon atom and one heteroatom (“heteroC1 alkyl”). In some embodiments, a heteroalkyl group as defined herein is a partially unsaturated group having one or more heteroatoms and at least one unsaturated carbon atom in the parent chain, such as a carbonyl group. For example, a heteroalkyl group may contain amide or ester functional groups in its parent chain such that one or more carbon atoms are unsaturated carbonyl groups. Unless otherwise specified, each instance of a heteroalkyl group is independently unsubstituted (“unsubstituted heteroalkyl”) or substituted with one or more substituents (“substituted heteroalkyl”). In some embodiments, a heteroalkyl group is an unsubstituted heteroC1 alkyl group. 1-20 Alkyl group. In some embodiments, the heteroalkyl group is an unsubstituted heteroalkyl group. 1-10 Alkyl group. In some embodiments, the heteroalkyl group is a substituted heteroalkyl group. 1-20 Alkyl group. In some embodiments, the heteroalkyl group is an unsubstituted heteroalkyl group. 1-10 alkyl.

[0104] The term "alkenyl" refers to a straight-chain or branched hydrocarbon group having 2 to 10 carbon atoms and one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds). In some embodiments, the alkenyl group has 2 to 9 carbon atoms ("C... 2-9 Alkenyl group (“Alkenyl”). In some embodiments, the alkenyl group has 2 to 8 carbon atoms (“C”). 2-8 Alkenyl group (“Alkenyl”). In some embodiments, the alkenyl group has 2 to 7 carbon atoms (“C”). 2-7 Alkenyl group (“Alkenyl”). In some embodiments, the alkenyl group has 2 to 6 carbon atoms (“C”). 2-6Alkenyl group (“Alkenyl”). In some embodiments, the alkenyl group has 2 to 5 carbon atoms (“C”). 2-5 Alkenyl group (“Alkenyl”). In some embodiments, the alkenyl group has 2 to 4 carbon atoms (“C”). 2-4 Alkenyl group (“Alkenyl”). In some embodiments, the alkenyl group has 2 to 3 carbon atoms (“C”). 2-3 The alkenyl group has two carbon atoms (“C2 alkenyl”). One or more carbon-carbon double bonds may be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). 2-4 Examples of alkenyl groups include vinyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), etc. 2-6 Examples of alkenyl groups include the C mentioned above. 2-4 Alkenyl groups include pentenyl (C5), pentadienyl (C5), hexenyl (C6), etc. Additional examples of alkenyl groups include heptenyl (C7), octenyl (C8), octetrinyl (C8), etc. Unless otherwise specified, each example of an alkenyl group is independently unsubstituted (“unsubstituted alkenyl”) or substituted with one or more substituents (“substituted alkenyl”). In some embodiments, the alkenyl group is an unsubstituted C5 group. 2-10 Alkenyl group. In some embodiments, the alkenyl group is a substituted C- group. 2-10 Alkenyl group. In the alkenyl group, the stereochemical C=C double bond is not specified (e.g., -CH=CHCH3 or...). It can be an (E)-double bond or a (Z)-double bond.

[0105] The term "alkynyl" refers to a straight-chain or branched hydrocarbon group having 2 to 10 carbon atoms and one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds). 2-10 The alkynyl group (“C”) has 2 to 9 carbon atoms in some embodiments. 2-9 The alkynyl group (“C”) has 2 to 8 carbon atoms in some embodiments. 2-8 The alkynyl group (“C”) has 2 to 7 carbon atoms in some embodiments. 2-7 The alkynyl group (“C”) has 2 to 6 carbon atoms in some embodiments. 2-6 The alkynyl group (“C”) has 2 to 5 carbon atoms in some embodiments. 2-5 The alkynyl group (“C”) has 2 to 4 carbon atoms in some embodiments. 2-4 The alkynyl group (“C”) has two to three carbon atoms in some embodiments. 2-3The alkynyl group has two carbon atoms (“C2 alkynyl”). One or more carbon-carbon triple bonds may be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl). 2-4 Examples of alkynyl groups include, but are not limited to, ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), and 2-butynyl (C4). 2-6 Examples of alkenyl groups include the C mentioned above. 2-4 The alkynyl group includes pentynyl (C5), hexynyl (C6), etc. Additional examples of alkynyl groups include heptynyl (C7), octyynyl (C8), etc. Unless otherwise specified, each example of an alkynyl group is independently unsubstituted (“unsubstituted alkynyl”) or substituted with one or more substituents (“substituted alkynyl”). In some embodiments, the alkynyl group is an unsubstituted C5 group. 2-10 Alkynyl group. In some embodiments, the alkynyl group is a substituted C- group. 2-10 Alkyne group.

[0106] As used herein, the term "aliphatic" or "aliphatic group" means a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is fully saturated or contains one or more unsaturated units, or a monocyclic or bicyclic hydrocarbon (also referred to herein as "carbocyclic," "cycloaliphatic," or "cycloalkyl") that is fully saturated or contains one or more unsaturated units but is not aromatic, having a single attachment point to the rest of the molecule. Unless otherwise specified, an aliphatic group contains 1 to 6 aliphatic carbon atoms. In some embodiments, the aliphatic group contains 1 to 5 aliphatic carbon atoms. In other embodiments, the aliphatic group contains 1 to 4 aliphatic carbon atoms. In other embodiments, the aliphatic group contains 1 to 3 aliphatic carbon atoms, and in yet another embodiment, the aliphatic group contains 1 to 2 aliphatic carbon atoms. In some embodiments, "cycloaliphatic" (or "carbocyclic" or "cycloalkyl") means a monocyclic C3-C6 hydrocarbon that is fully saturated or contains one or more unsaturated units but is not aromatic, having a single attachment point to the rest of the molecule. Suitable aliphatic groups include, but are not limited to, straight-chain or branched substituted or unsubstituted alkyl, alkenyl, alkynyl groups and their hybrids, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

[0107] The term "carbocyclic group" or "carbocyclic ring" refers to a ring containing 3 to 14 carbon atoms ("C"). 3-14 A carbocyclic group (“C”) is a non-aromatic cyclic hydrocarbon group with zero heteroatoms in a non-aromatic ring system. In some embodiments, the carbocyclic group has 3 to 10 ring carbon atoms (“C”). 3-10 (Carbocyclic group). In some embodiments, the carbocyclic group has 3 to 8 cyclic carbon atoms (“C”). 3-8(Carbocyclic group). In some embodiments, the carbocyclic group has 3 to 7 cyclic carbon atoms (“C”). 3-7 (Carbocyclic group). In some embodiments, the carbocyclic group has 3 to 6 cyclic carbon atoms (“C”). 3-6 (Carbocyclic group). In some embodiments, the carbocyclic group has 4 to 6 cyclic carbon atoms (“C”). 4-6 (Carbocyclic group). In some embodiments, the carbocyclic group has 5 to 6 cyclic carbon atoms (“C”). 5-6 (Carbocyclic group). In some embodiments, the carbocyclic group has 5 to 10 cyclic carbon atoms (“C”). 5-10 (Carbocyclic group). Example C 3-6 The carbocyclic group includes, but is not limited to, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), and cyclohexadienyl (C6). Example C 3-8 Carbocyclic groups include, but are not limited to, the C groups mentioned above. 3-6 Carbocyclic groups, including cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptanetrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), bicyclo[2.2.1]heptyl (C7), bicyclo[2.2.2]octyl (C8), etc. Example C 3-10 Carbocyclic groups include, but are not limited to, the C groups mentioned above. 3-8 Carbocyclic groups and cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C9) 10 ), cyclodecenyl (C 10 ), octahydro-1H-indenyl (C9), decahydronaphthyl (C9) 10 ), spiro[4.5]decyl(C 10 As illustrated in the foregoing examples, in some embodiments, the carbocyclic group is monocyclic (“monocyclic carbocyclic”) or polycyclic (e.g., containing fused, bridged, or spirocyclic systems, such as bicyclic systems (“bicyclic carbocyclic”) or tricyclic systems (“tricyclic carbocyclic”)), and may be saturated or may contain one or more carbon-carbon double or triple bonds. “Carbocyclic” also includes ring systems in which the carbocyclic ring as defined above is fused with one or more aryl or heteroaryl groups (where the attachment point is on the carbocyclic ring), and in such instances, the number of carbons continues to specify the number of carbons in the carbocyclic ring system. Unless otherwise specified, each instance of a carbocyclic group is independently unsubstituted (“unsubstituted carbocyclic”) or substituted with one or more substituents (“substituted carbocyclic”). In some embodiments, the carbocyclic group is an unsubstituted C 3-14 Carbocyclic group. In some embodiments, the carbocyclic group is a substituted C 3-14 Carbon cyclic group.

[0108] In some embodiments, "carbocyclic group" is a monocyclic saturated carbocyclic group having 3 to 14 ring carbon atoms ("C"). 3-14 cycloalkyl group (“Cycloalkyl”). In some embodiments, the cycloalkyl group has 3 to 10 cyclic carbon atoms (“C”). 3-10 cycloalkyl group (“Cycloalkyl”). In some embodiments, the cycloalkyl group has 3 to 8 cyclic carbon atoms (“C”). 3-8 cycloalkyl group (“Cycloalkyl”). In some embodiments, the cycloalkyl group has 3 to 6 cyclic carbon atoms (“C”). 3-6 cycloalkyl group (“Cycloalkyl”). In some embodiments, the cycloalkyl group has 4 to 6 cyclic carbon atoms (“C”). 4-6 cycloalkyl group (“Cycloalkyl”). In some embodiments, the cycloalkyl group has 5 to 6 cyclic carbon atoms (“C”). 5-6 cycloalkyl group (“Cycloalkyl”). In some embodiments, the cycloalkyl group has 5 to 10 cyclic carbon atoms (“C”). 5-10 cycloalkyl). C 5-6 Examples of cycloalkyl groups include cyclopentyl (C5) and cyclohexyl (C5). 3-6 Examples of cycloalkyl groups include the C10+ groups mentioned above. 5-6 Cycloalkyl groups, as well as cyclopropyl (C3) and cyclobutyl (C4). 3-8 Examples of cycloalkyl groups include the C10+ groups mentioned above. 3-6 Cycloalkyl groups, including cycloheptyl (C7) and cyclooctyl (C8). Unless otherwise specified, each instance of a cycloalkyl group is independently unsubstituted (“unsubstituted cycloalkyl”) or substituted with one or more substituents (“substituted cycloalkyl”). In some embodiments, the cycloalkyl group is an unsubstituted C7 group. 3-14 Cycloalkyl. In some embodiments, the cycloalkyl group is a substituted C. 3-14 Cycloalkyl.

[0109] The term "heterocyclic group" or "heterocycle" refers to a group comprising a 3- to 14-membered non-aromatic ring system having a ring carbon atom and one to four ring heteroatoms, each heteroatom being independently selected from nitrogen, oxygen, and sulfur ("3- to 14-membered heterocyclic groups"). In heterocyclic groups containing one or more nitrogen atoms, the attachment point may be a carbon or nitrogen atom, where the valence allows. Heterocyclic groups may be monocyclic ("monocyclic heterocyclic group") or polycyclic (e.g., fused, bridged, or spirocyclic systems, such as bicyclic systems ("bicyclic heterocyclic group") or tricyclic systems ("tricyclic heterocyclic group")), and may be saturated or contain one or more carbon-carbon double or triple bonds. Heterocyclic polycyclic systems may contain one or more heteroatoms in one or two rings. "Heterocyclic group" also includes ring systems in which a heterocyclic ring as defined above is fused with one or more carbocyclic groups (where the attachment point is on the carbocyclic or heterocyclic ring), or ring systems in which a heterocyclic ring as defined above is fused with one or more aryl or heteroaryl groups (where the attachment point is on the heterocyclic ring), and in such instances, the number of ring members continues to specify the number of ring members in the heterocyclic ring system. Unless otherwise specified, each instance of a heterocyclic group is independently unsubstituted ("unsubstituted heterocyclic group") or substituted with one or more substituents ("substituted heterocyclic group"). In some embodiments, the heterocyclic group is an unsubstituted 3-14 membered heterocyclic group. In some embodiments, the heterocyclic group is a substituted 3-14 membered heterocyclic group.

[0110] In some embodiments, the heterocyclic group is a 5-10 membered non-aromatic ring system having a cyclic carbon atom and 1-4 cyclic heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-10 membered heterocyclic group”). In some embodiments, the heterocyclic group is a 5-8 membered non-aromatic ring system having a cyclic carbon atom and 1-4 cyclic heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heterocyclic group”). In some embodiments, the heterocyclic group is a 5-6 membered non-aromatic ring system having a cyclic carbon atom and 1-4 cyclic heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heterocyclic group”). In some embodiments, the 5-6 membered heterocyclic group has 1-3 cyclic heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclic group has 1-2 cyclic heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclic group has one cyclic heteroatom selected from nitrogen, oxygen, and sulfur.

[0111] Exemplary 3-membered heterocyclic groups containing one heteroatom include, but are not limited to, aziridinyl, ethylene oxide, and thiiranyl. Exemplary 4-membered heterocyclic groups containing one heteroatom include, but are not limited to, azirrobutyl, oxadiazolinyl, and thiohexacyclobutyl. Exemplary 5-membered heterocyclic groups containing one heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolyl, dihydropyrrolyl, and pyrrolyl-2,5-diketone. Exemplary 5-membered heterocyclic groups containing two heteroatoms include, but are not limited to, dioxopentyl, oxothiopentanyl, and dithiopentanyl. Exemplary 5-membered heterocyclic groups containing three heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclic groups containing one heteroatom include, but are not limited to, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclic groups containing two heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithiaalkyl, and dioxane. Exemplary 6-membered heterocyclic groups containing three heteroatoms include, but are not limited to, triazineyl. Exemplary 7-membered heterocyclic groups containing one heteroatom include, but are not limited to, azirheptyl, oxeheptyl, and thioheptyl. Exemplary 8-membered heterocyclic groups containing one heteroatom include, but are not limited to, azirheptyl, oxeheptyl, and thioheptyl. Exemplary bicyclic heterocyclic groups include, but are not limited to, indololinyl, isoindololinyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, tetrahydrobenzothiophenyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl, octahydroisochromenyl, decahydronaphridyl, decahydro-1,8-naphridyl, octahydropyrrolo[3,2-b]pyrrole, indololinyl, phthalimide, naphthimide, chromenyl, 1H-benzo[e][1,4]diazazolyl, 1,4,5,7-tetrahydropyranolo[3,4-b]pyrrole, 5, 6-Dihydro-4H-furano[3,2-b]pyrrolithyl, 6,7-Dihydro-5H-furano[3,2-b]pyrrolithyl, 5,7-Dihydro-4H-thieno[2,3-c]pyrrolithyl, 2,3-Dihydro-1H-pyrroli[2,3-b]pyridyl, 2,3-Dihydrofurano[2,3-b]pyridyl, 4,5,6,7-Tetrahydro-1H-pyrroli[2,3-b]pyridyl, 4,5,6,7-Tetrahydrofurano[3,2-c]pyridyl, 4,5,6,7-Tetrahydrothieno[3,2-b]pyridyl, 1,2,3,4-Tetrahydro-1,6-naphthidyl, etc.

[0112] The term "aryl" refers to a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., sharing 6, 10, or 14 π electrons in a cyclic arrangement), the aromatic ring system having 6-14 ring carbon atoms and zero heteroatoms provided by the aromatic ring system ("C").6-14 Aryl group (“C6 aryl”); in some embodiments, the aryl group has 6 ring carbon atoms (“C6 aryl”; for example, phenyl). In some embodiments, the aryl group has 10 ring carbon atoms (“C6 aryl”). 10 Aryl; for example, naphthyl, such as 1-naphthyl and 2-naphthyl). In some embodiments, the aryl group has 14 ring carbon atoms (“C14”). 14 "Aryl"; for example, anthracene. "Aryl" also includes ring systems in which an aryl ring as defined above is fused with one or more carbocyclic or heterocyclic groups (wherein the group or attachment site is on the aryl ring), and in such instances, the number of carbon atoms continues to specify the number of carbon atoms in the aryl ring system. Unless otherwise specified, each instance of an aryl group is independently unsubstituted ("unsubstituted aryl") or substituted with one or more substituents ("substituted aryl"). In some embodiments, the aryl group is an unsubstituted C 6-14 Aryl group. In some embodiments, the aryl group is a substituted C. 6-14 Aryl.

[0113] The term "heteroaryl" refers to a 5-14 membered monocyclic or polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic ring system (e.g., sharing 6, 10, or 14 π electrons in a cyclic arrangement), the aromatic ring system having a cyclic carbon atom and 1-4 cyclic heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-14 membered heteroaryl"). In heteroaryl groups containing one or more nitrogen atoms, the attachment site may be a carbon or nitrogen atom, where the valence allows. Heteroaryl polycyclic systems may contain one or more heteroatoms in one or two rings. "Heteroaryl" includes ring systems in which a heteroaryl ring as defined above is fused with one or more carbocyclic or heterocyclic groups (where the attachment site is on the heteroaryl ring), and in such instances, the number of ring members continues to specify the number of ring members in the heteroaryl ring system. "Heteroaryl" also includes ring systems in which a heteroaryl ring as defined above is fused with one or more aryl groups (where the attachment point is on the aryl or heteroaryl ring), and in such instances, the number of ring members specifies the number of ring members in the fused polycyclic (aryl / heteroaryl) ring system. A polycyclic heteroaryl group, in which one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, etc.), may have the attachment point located on either ring, i.e., a ring with a heteroatom (e.g., 2-indolyl) or a ring without a heteroatom (e.g., 5-indolyl).

[0114] In some embodiments, the heteroaryl group is a 5-10 membered aromatic ring system having a cyclic carbon atom and 1-4 cyclic heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”). In some embodiments, the heteroaryl group is a 5-8 membered aromatic ring system having a cyclic carbon atom and 1-4 cyclic heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”). In some embodiments, the heteroaryl group is a 5-6 membered aromatic ring system having a cyclic carbon atom and 1-4 cyclic heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”). In some embodiments, the 5-6 membered heteroaryl has 1-3 cyclic heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6-membered heteroaryl group has 1-2 cyclic heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6-membered heteroaryl group has 1 cyclic heteroatom selected from nitrogen, oxygen, and sulfur. Unless otherwise specified, each instance of a heteroaryl group is independently unsubstituted (“unsubstituted heteroaryl”) or substituted with one or more substituents (“substituted heteroaryl”). In some embodiments, the heteroaryl group is an unsubstituted 5-14-membered heteroaryl. In some embodiments, the heteroaryl group is a substituted 5-14-membered heteroaryl.

[0115] Exemplary 5-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyrroleyl, furanyl, and thiophenyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, but are not limited to, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, but are not limited to, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyridinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, but are not limited to, pyridinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, but are not limited to, triazinyl and tetraazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, but are not limited to, azirzolyl, oxazirzolyl, and thiazolyl. Exemplary 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazole, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzoimidazolyl, benzoxazolyl, benzoisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzoisothiazolyl, benzothiadiazolyl, indolazinyl, and purinel. Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthidyl, pteridyl, quinolinyl, isoquinolinyl, cinnamyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplary tricyclic heteroaryl groups include, but are not limited to, phenanthridine, dibenzofuranyl, carbazole, acridineyl, phenothiazinyl, phenotoxazinyl, and phenothiazinyl.

[0116] Adding the suffix "-ene" to a group indicates that the group is a divalent moiety. For example, alkylene is the divalent moiety of an alkyl group, alkenylene is the divalent moiety of an alkenyl group, ynynylene is the divalent moiety of an alkyne group, heteroalkylene is the divalent moiety of a heteroalkyl group, heteroalkenylene is the divalent moiety of a heteroalkenyl group, heteroynynylene is the divalent moiety of a heteroynyl group, carbocyclic is the divalent moiety of a carbocyclic group, heterocyclic is the divalent moiety of a heterocyclic group, arylene is the divalent moiety of an aryl group, and heteroarylene is the divalent moiety of a heteroaryl group.

[0117] As used herein, the term "substituted" refers to all permissible substituents in the compounds described herein. In the broadest sense, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. Illustrative substituents include, but are not limited to, halogens, hydroxyl groups, or any other organic group containing any number of carbon atoms (e.g., 1-14 carbon atoms), and optionally include one or more heteroatomic groups such as oxygen, sulfur, or nitrogen groups in linear, branched, or cyclic structural formats. Representative substituents include alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, hydroxyl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aryloxy, substituted aryloxy, alkylthio, substituted alkylthio, phenylthio, substituted phenylthio, arylthio, substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amide, substituted amide, sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl, polyaryl, substituted polyaryl, C3-C 20 cyclic, substituted C3-C 20 Cyclic, heterocyclic, substituted heterocyclic, amino acid, peptide, and polypeptide groups.

[0118] As described herein, the compounds of this disclosure may contain an "optionally substituted" portion. Generally, regardless of whether the term "optionally" is preceding it, the term "substituted" means that one or more hydrogens of the specified portion are replaced by suitable substituents. Unless otherwise indicated, the "optionally substituted" group may have suitable substituents at each substituted position of the group, and the substituents at each position may be the same or different when more than one position in any given structure is substituted by more than one substituent selected from the specified group. The combinations of substituents contemplated by this disclosure are preferably those substituents that result in the formation of a stable or chemically viable compound. As used herein, the term "stable" means a compound that remains substantially unchanged when subjected to conditions permissible for the production, detection, and, in some embodiments, recovery, purification, and use for one or more purposes disclosed herein.

[0119] Suitable monovalent substituents on the substituted carbon atoms of the "optionally substituted" group are independently halogens; —(CH2) 0-4 R ° ;—(CH2) 0-4 OR ° ;—O(CH2) 0-4 R ° —O—(CH2) 0-4 C(O)OR ° ;—(CH2) 0-4CH(OR ° )2;—(CH2) 0-4 SR ° ;—(CH2) 0-4 Ph, which can be R ° Substitution; —(CH2) 0-4 O(CH2) 0-1 Ph, which can be R ° Replacement; —CH═CHPh, which can be replaced by R ° Substitution; —(CH2) 0-4 O(CH2) 0-1 -pyridyl group, which can be R ° Substitution; —NO2; —CN; —N3; —(CH2) 0-4 N(R ° )2;—(CH2) 0-4 N(R ° )C(O)R ° ;—N(R ° )C(S)R ° ;—(CH2) 0-4 N(R ° )C(O)NR ° 2; —N(R) ° )C(S)NR ° 2; —(CH2) 0-4 N(R ° )C(O)OR ° ;—N(R ° )N(R ° )C(O)R ° ;—N(R ° )N(R ° )C(O)NR ° 2; —N(R) ° )N(R ° )C(O)OR ° ;—(CH2) 0-4 C(O)R ° ;—C(S)R ° ;—(CH2) 0-4 C(O)OR ° ;—(CH2) 0-4 C(O)SR ° ;—(CH2) 0-4 C(O)OSiR ° 3; —(CH2) 0-4 OC(O)R ° ;—OC(O)(CH2) 0-4 SR ° SC(S)SR ° ;—(CH2)0-4 SC(O)R ° ;—(CH2) 0-4 C(O)NR ° 2; —C(S)NR ° 2; —C(S)SR ° ;—SC(S)SR ° —(CH2) 0-4 OC(O)NR ° 2; —C(O)N(OR) ° )R ° ;—C(O)C(O)R ° ;—C(O)CH2C(O)R ° ;—C(NOR ° )R ° ;—(CH2) 0-4 SSR ° ;—(CH2) 0-4 S(O)2R ° ;—(CH2) 0-4 S(O)2OR ° ;—(CH2) 0-4 OS(O)2R ° ;—S(O)2NR ° 2; —(CH2) 0-4 S(O)R ° ;—N(R ° )S(O)2NR ° 2; —N(R) ° )S(O)2R ° ; —N(OR ° )R ° ;—C(NH)NR ° 2; —P(O)2R ° ;—P(O)R ° 2; —OP(O)R ° 2; —OP(O)(OR ° )2; SiR ° 3; —(C 1-4 (straight-chain or branched alkylene)O—N(R) ° )2; or —(C 1-4 (straight-chain or branched alkylene)C(O)O—N(R) ° )2, where each R ° It can be substituted as defined below and is independently hydrogen, C 1-6 Aliphatic, —CH2Ph, —O(CH2) 0-1Ph, —CH2- (5-6 membered heteroaryl ring), or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, although defined above, two independent R groups. ° Together with one or more intercalated atoms, they form 3-12 saturated, partially unsaturated, or aryl monocyclic or bicyclic rings having 0-4 independent heteroatoms selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.

[0120] In R ° Appropriate monovalent substituents (or two independent Rs) ° The ring formed together with the intercalated atoms is independently a halogen, —(CH2). 0-2 R • -(halogenated R) • ), —(CH2) 0-2 OH, —(CH2) 0-2 OR • —(CH2) 0-2 CH(OR • )2;—O(halogenated R) • —CN, —N3, —(CH2) 0-2 C(O)R • —(CH2) 0-2 C(O)OH、—(CH2) 0-2 C(O)OR • —(CH2) 0- 2SR • —(CH2) 0-2 SH、—(CH2) 0-2 NH2、—(CH2) 0-2 NHR • —(CH2) 0-2 NR • 2、—NO2、—SiR • 3. —OSiR • 3、—C(O)SR • —(C 1-4 (straight-chain or branched alkylene)C(O)OR • Or —SSR • , where each R • It is an unsubstituted or previously "halogenated" site that is substituted by only one or more halogens, and is independently selected from C. 1-4 Aliphatic, —CH2Ph, —O(CH2) 0-1 Ph, or a 5-6 membered saturated, partially unsaturated, or aryl ring having 0-4 independent heteroatoms selected from nitrogen, oxygen, or sulfur. In R °Suitable divalent substituents on saturated carbon atoms include ═O and ═S.

[0121] Suitable divalent substituents on the saturated carbon atom of the "optionally substituted" group include the following: ═O, ═S, ═NNR*2, ═NNHC(O)R*, ═NNHC(O)OR*, ═NNHS(O)2R*, ═NR*, ═NOR*, —O(C(R*2)) 2-3 O—or —S(C(R*2)) 2-3 S—, where each independently occurring R* is selected from hydrogen, C, which can be substituted as defined below. 1-6 Aliphatic, or having 0-4 unsubstituted 5-6 membered saturated, partially unsaturated, or aryl rings independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents attached to the adjacent substituted carbon of the "optionally substituted" group include: —O(CR*2). 2-3 O—, where each independently occurring R* is selected from hydrogen, C, which can be substituted as defined below. 1-6 Aliphatic, or having 0-4 unsubstituted 5-6 saturated, partially unsaturated or aryl rings independently selected from nitrogen, oxygen or sulfur.

[0122] Suitable substituents on the aliphatic group of R* include halogens, -R • -(halogenated R) • —OH, —OR • —O(halogenated R) • ), —CN, —C(O)OH, —C(O)OR • —NH2, —NHR • —NR • 2 or —NO2, where each R • It is an unsubstituted or previously "halogenated" site that is substituted by one or more halogens and is independently C. 1-4 Aliphatic, —CH2Ph, —O(CH2) 0-1 Ph, or a 5-6 member saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0123] Suitable substituents on the substituted nitrogen of the "optionally substituted" group include —R † —NR † 2、—C(O)R † —C(O)OR † —C(O)C(O)R † —C(O)CH2C(O)R † —S(O)2R † —S(O)2NR †2、—C(S)NR † 2、—C(NH)NR † 2 or —N(R) † )S(O)2R † ; where each R † C that is independently hydrogen and can be substituted as defined below 1-6 Aliphatic, unsubstituted -OPh, or unsubstituted 5-6 membered saturated, partially unsaturated, or aryl rings having 0-4 independently selected heteroatoms chosen from nitrogen, oxygen, or sulfur, or R appearing twice independently, although as defined above. † Together with one or more intercalated atoms, they form 3-12 saturated, partially unsaturated, or aryl monocyclic or bicyclic rings with 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0124] R † Suitable substituents on the aliphatic group can be halogens, -R • -(halogenated R) • —OH, —OR • —O(halogenated R) • ), —CN, —C(O)OH, —C(O)OR • —NH2, —NHR • —NR • 2 or —NO2, where each R • It is an unsubstituted or previously "halogenated" site that is substituted by one or more halogens and is independently C. 1-4 Aliphatic, —CH2Ph, —O(CH2) 0-1 Ph, or a 5-6 member saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0125] Heteroatoms such as nitrogen may have hydrogen substituents and / or any permissible substituents in the organic compounds described herein that satisfy the heteroatom valence. It should be understood that “substituted” or “substituted” includes the implicit condition that such substitution meets the permissible valence of the substituted atom and the substituent, and that said substitution produces a stable compound, i.e., a compound that does not spontaneously undergo transformation (e.g., by rearrangement, cyclization, or elimination).

[0126] In a broad sense, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. Illustrative substituents include, for example, those described herein. For a suitable organic compound, permissible substituents may be one or more and may be the same or different. Heteroatoms such as nitrogen may have hydrogen substituents and / or any permissible substituent of the organic compounds described herein that satisfies the heteroatom valence.

[0127] In various embodiments, the substituents are selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, aralkyl, carbamate, carboxyl, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclic, hydroxyl, ketone, nitro, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thione, and each of the substituents is optionally substituted by one or more suitable substituents. In some embodiments, the substituent is selected from alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, aralkyl, carbamate, carboxyl, cycloalkyl, ester, ether, formyl, haloalkyl, heteroaryl, heterocyclic, ketone, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thione, wherein each of the alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, aralkyl, carbamate, carboxyl, cycloalkyl, ester, ether, formyl, haloalkyl, heteroaryl, heterocyclic, ketone, phosphate, sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thione may be further substituted by one or more suitable substituents.

[0128] Examples of substituents include, but are not limited to, halogens, azides, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxy, amino, nitro, mercapto, imino, amide, phosphonate, phosphonite, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamide, ketone, aldehyde, thion, ester, heterocyclic, -CN, aryl, aryloxy, perhaloalkoxy, arylalkoxy, heteroaryl, heteroaryloxy, heteroarylalkyl, heteroarylalkoxy, azide, alkylthio, oxo, acylalkyl, carboxyl ester, formamide, acyloxy, aminoalkyl, alkylaminoaryl, alkylaryl, alkylaminoalkyl, alkoxyaryl, arylamino, aralkylalkylamino, alkylsulfonyl, formamidealkylaryl, formamidealkylaryl, hydroxyalkyl, haloalkyl, alkylaminoalkylcarboxyl, aminoformamidealkyl, cyano, alkoxyalkyl, perhaloalkyl, arylalkoxyalkyl, etc. In some implementations, the substituents are selected from cyano, halogen, hydroxyl, and nitro groups.

[0129] As used herein, the term "salt" means any and all salts and encompasses pharmaceutically acceptable salts. Salts include ionic compounds produced by the neutralization reaction of acids and bases. A salt consists of one or more cations (positively charged ions) and one or more anions (negatively charged ions), and is therefore electrically neutral (without a net charge). Salts of the compounds of this invention include salts derived from inorganic and organic acids and bases. Examples of acid addition salts are salts formed by an amino group with an inorganic acid (such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid) or an organic acid (such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid), or salts formed by other methods known in the art (such as ion exchange). Other salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucono-p-ethylhexanoate, glyceryl phosphate, gluconate, hemisulfate, heptanate, hexanoate, hydroiodate, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, dodecyl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, dihydroxynaphthalate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, neopentanoate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, hippurate, etc. Salts derived from suitable bases include alkali metal salts, alkaline earth metal salts, ammonium salts, and N+(C1–4 alkyl)4 salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium. Other salts include ammonium salts, quaternary ammonium salts, and amine cations, which are formed using counterions such as halide ions, hydroxide ions, carboxyl ions, sulfate ions, phosphate ions, nitrate ions, lower alkyl sulfonate ions, and aryl sulfonate ions.

[0130] The term "subject" considered for administration includes, but is not limited to, humans (i.e., males or females of any age group, such as pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged, or elderly)) and / or other non-human animals, such as mammals (e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys); commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, and / or dogs) and birds (e.g., commercially relevant birds such as chickens, ducks, geese, and / or turkeys). In some embodiments, the animal is a mammal. The animal may be male or female and at any developmental stage. Non-human animals may also be transgenic animals. "Patient" refers to a human subject who requires treatment for a disease.

[0131] The terms “administer,” “administering,” or “administration” refer to the introduction of the compound or pharmaceutical composition thereof by means of implantation, absorption, ingestion, injection, inhalation, or other means.

[0132] The terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the occurrence of, or inhibiting the progression of a “pathological condition” (e.g., a disease, symptom, or disorder, or one or more of its signs or symptoms) as described herein. In some embodiments, treatment may be administered after one or more signs or symptoms have been developed or observed. In other embodiments, treatment may be administered even in the absence of signs or symptoms of a disease or disorder. For example, treatment may be administered to a susceptible individual before symptoms appear (e.g., based on a history of symptoms and / or based on genetic or other susceptibility factors). Treatment may also continue after symptoms have subsided, for example, to delay or prevent recurrence.

[0133] The term "biological sample" refers to any sample, including tissue samples (such as tissue sections and tissue needle biopsies); cell samples (such as cell smears (such as Pap smears or blood smears) or samples of cells obtained through microdissection); samples of intact organisms (such as samples of yeast or bacteria); or cell fractions, fragments, or organelles (such as those obtained by lysing cells and separating their components by centrifugation or other means). Other examples of biological samples include blood, serum, urine, semen, feces, cerebrospinal fluid, tissue fluid, mucus, tears, sweat, pus, biopsy tissue (e.g., obtained by surgical or needle biopsy), nipple aspiration, breast milk, vaginal fluid, saliva, swabs (such as oral swabs), or any material containing biomolecules derived from the first biological sample.

[0134] As used herein, the terms “valence” or “multivalence” generally refer to one (monovalent) or multiple (multivalent) glycans on a scaffold that are capable of binding to the receptor or carbohydrate recognition domain of a target. Relatedly, as used herein, the term “heteropolyvalence” generally refers to different heteroglycans or mixtures of heteroglycans on a scaffold that are capable of binding to the receptor or carbohydrate recognition domain of a target.

[0135] As used herein, the terms "selective" or "selectively binding" refer to a ligand-receptor relationship in which the ligand binds to the receptor with at least 90% specificity, such that off-target binding is less than 10%. In some embodiments, the ligand binds to the receptor with at least 95%, at least 98%, at least 99%, or at least 99.9% specificity.

[0136] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although similar or equivalent methods and materials may be used in the practice of this invention, exemplary methods and materials are described below. All publications and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail. The materials, methods, and examples described are illustrative only and not intended to be limiting.

[0137] Nucleic acid sequence

[0138] In various aspects, the methods and compositions include one or more glycans operatively linked to one or more sites on a synthetic scaffold domain comprising a synthetic nucleic acid polymer containing RNA. The methods provide the synthesis of one or more glycan ligands to modulate desired receptors, thereby mediating biological effects.

[0139] In a preferred aspect, the method of the present invention for synthesizing glycoligands comprises conjugating a glycan with one or more short hairpin RNAs, double-stranded RNAs, long non-coding RNAs, circular RNAs (circRNAs), small nuclear RNAs (γNRAs), short interfering RNAs (siRNAs), antisense oligonucleotides (ASOs), messenger RNAs (mRNAs), guide RNAs on RNPs, aptamers, and other such nucleic acid molecules.

[0140] Preferably, the synthesized nucleic acid polymer comprises at least one nucleobase modification. More preferably, the method and composition comprise modifying one or more nucleic acid sequences by inserting, deleting, or altering one or more base pairs in a target region to achieve conjugation. In various embodiments, γ RNA, small nuclear RNA, and small nucleolar RNA are modified at guanosine residues.

[0141] In some embodiments, the polynucleotide is siRNA or ASO as described in Hu et al., Sig Transduct Target Ther 5, 101 (2020). In some embodiments, the siRNA or ASO of this disclosure comprises one or more phosphonate modifications selected from phosphate thioester (PS), phosphate dithioester (PS2), methylphosphonate (MP), methoxypropylphosphonate (MOP), 5'-(E)-vinylphosphonate (5'-(E)-VP), 5'-methylphosphonate (5'-MP), (S)-5'-C-methylphosphonate, 5'-phosphate thioester (5'-PS), and peptide nucleic acid (PNA). In some embodiments, the siRNA or ASO of this disclosure comprises one or more ribose modifications selected from 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-O-MOE), 2'-deoxy-2'-fluoro (2'-F), 2'-arabinose-fluorine (2'-Ara-F), 2'-O-benzyl, 2'-O-methyl-4-pyridine (2'-O-CH2Py(4)), locked nucleic acid (LNA), (S)-cET-BNA, tricyclic DNA (tcDNA), PMO, non-locked nucleic acid (UNA), and glycosyl nucleic acid (GNA). In some embodiments, the siRNA comprises a locked nucleic acid (LNA) comprising a methyl bridge, an ethyl bridge, a propyl bridge, a butyl bridge, or a variant of any of the above bridges optionally substituted. In some embodiments, the siRNA or ASO of this disclosure comprises one or more modified bases selected from pseudouridine (ψ), 2'-thiouridine (s2U), N6'-methyladenosine (m... 6 A), 5'-methylcytidine (m) 5 C), 5'-fluoro2'-deoxyuridine, N-ethylpiperidine 7'-EAA triazole-modified adenine, N-ethylpiperidine 6'-triazole-modified adenine, 6'-benzopyrrole-cytosine (PhpC), 2',4'-difluorotolyl ribonucleoside (rF) and 5'-nitroindole.

[0142] In this context, the substitution, modification, alteration, etc. of terms do not imply any process limitations. For example, modification does not mean that it is necessary to start with a reference or naturally occurring ribonucleic acid and modify it to produce a modified ribonucleic acid. Rather, modification simply indicates a difference from the naturally occurring molecule.

[0143] This document may use any of the cyclic polynucleotides taught, for example, in U.S. Provisional Application No. 61 / 873,010, filed September 3, 2013, or U.S. Patent No. 10,709,779. The contents of these references are incorporated herein by reference in their entirety. In addition, this document considers any of the circular RNAs, methods for manufacturing circular RNAs, and circular RNA compositions described in the following publications, which are incorporated herein by reference in their entirety as a part of this specification: U.S. Patents US 11,352,640, US 11,352,641, US 11,203,767, US 10,683,498, US 5,773,244, and US 5,766,903; U.S. Application Publications US 2022 / 0177540, US 2021 / 0371494, US 2022 / 0090137, US 2019 / 0345503, and US 2015 / 0299702; and PCT Application Publications WO 2021 / 226597, WO 2019 / 236673, WO 2017 / 222911, WO2016 / 187583, WO2014 / 082644 and WO 1997 / 007825.

[0144] In other respects, one or more nucleobases on the synthetic scaffold domain are modified, for example, as target sites for one or more glycans that can be operatively linked during assembly. Preferably, the nucleobase modification provides covalent bonding with one or more desired glycans, thereby producing a glycoligand composition. In some embodiments, the glycoligand composition comprises multiple modifications to the nucleic acid to make it suitable for better industrial applicability and application.

[0145] Some modified sequences are designed to alter the function of nucleic acid sequences. These modified sequences are unwanted, counterproductive, interfering, harmful, or unsuitable as glycoligand compositions.

[0146] In such embodiments where the synthetic scaffold domain comprises RNA, one or more nucleotides are modified at one or more guanosine sites. For certain types of RNA, such as siRNA, specific patterns of alternating 2'-O-methyl and 2'-O-fluoronucleotides can be formed by inserting phosphate-thioester (PS) bonds at the ends of the strand to enhance pharmacokinetic properties. In such embodiments where the synthetic scaffold domain comprises ASO, modification at the 2' position of the furanose can enhance metabolic stability and binding affinity to biological targets, as well as improve toxicological and pharmacokinetic properties. (Prakash, TP. An overview of sugar-modified oligonucleotides for antisense therapeutics. ChemBiodivers. Sep 2011; 8(9):1616-41.) In a more preferred embodiment, the synthetic scaffold domain containing RNA comprises about 5 to about 10 ribonucleotides, about 10 to about 20 ribonucleotides, about 20 to about 30 ribonucleotides, about 30 to about 40 ribonucleotides, about 40-50 ribonucleotides, about 50 to about 100 ribonucleotides, about 100 to about 500 ribonucleotides, about 500 to about 5,000 ribonucleotides or more.

[0147] Therefore, the present invention provides an isolated glycoligand composition comprising a nucleic acid molecule and variants thereof conjugated to one or more desired glycans. Exemplary nucleic acid sequences are non-coding sequences. Modifying sequences may be selected from nucleic acid sequences having greater than 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, 99.9%, or even higher identity to wild-type non-coding sequences. In other embodiments, the nucleic acid molecules of the present invention are partially non-coding.

[0148] In some embodiments, the nucleic acid polymer is siRNA. In some embodiments, the nucleic acid polymer is siRNA containing modifications to one or more nucleotides, including but not limited to 2-OMe modification, fluorine modification (such as 2-fluororibose modification), and phosphate thioester modification. In some embodiments, the nucleic acid is siRNA containing a modified backbone.

[0149] In some implementations, the nucleic acid is a circular RNA, wherein the circular RNA is modified by self-ligation compared to naturally occurring RNA, thus lacking a cap or tail. In some implementations, the nucleic acid is a circular RNA containing an IRES sequence selected from the following IRES: Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, simian virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, reticuloendotheliosis virus, human poliovirus 1, Plautia stall enterovirus, Kashmir bee virus, human rhinovirus 2, Homalodisca coagulata virus-1, human immunodeficiency virus type 1, Homalodisca coagulata virus-1, and Himetobi virus-1. P) virus, Hepatitis C virus, Hepatitis A virus, GB hepatitis virus, Foot-and-mouth disease virus, Human enterovirus 71, Equine rhinitis virus, Ectropis obliqua picoma-like virus, Encephalocarditis virus, Drosophila C virus, Human coxsackievirus B3, Crucifera tobamovirus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black Queen Cell virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscus chlorotic ringspot virus, Classical swine fever virus, Human FGF2, Human SFTPA1, Human AML1 / RUNX1, Drosophila antennae and legs Antennapedia), Human AQP4, Human AT1R, Human BAG-1, Human BCL2, Human BiP, Human c-IAPl, Human c-myc, Human eIF4G, Mouse NDST4L, Human LEF1, Mouse HIF1α, Human n.myc, Mouse Gtx, Human p27kipl, Human PDGF2 / c-sis, Human p53, Human Pim-1, Mouse Rbm3, Drosophila reaper, Canine Scamper, Drosophila Ubx, Human UNR, Mouse UtrA, Human VEGF-A, Human XIAP, Hairless Drosophila, Saccharomyces cerevisiae TFIID, Saccharomyces cerevisiae YAP1, Tobacco etch virus, Turnip wrinkle viruscrinkle virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, Picobirnavirus, HCVQC64, Human Cosavirus E / D, Human Cosavirus F, Human Cosavirus JMY, Rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21, Salivary Virus A SHI, Salivary Virus FHB, Salivary Virus NG-J1, Human Paraenteritis Virus 1, Crohivirus B, Yc-3, Rosavirus M-7, Shanbavirus A, Pasivirus A, Pasivirus A 2, Echovirus E14, Human Paraenteritis Virus 5, Aichi Virus, Hepatitis A Virus HA 16. Phopivirus, CVA10, Enterovirus C, Enterovirus D, Enterovirus J, Human Pegivirus 2. GBV-C GT110, GBV-C K1737, GBV-CIowa, Pegivirus A 1220, Pasi Virus A 3. Sapelovirus, Rose Virus B, Bakunsa Virus, Tremovirus A, Porcine Pasi Virus 1, PLV-CHN, Pasi Virus A, Sicinivirus, Hepatitis Virus K, Hepatitis Virus A, BVDV1, Border Disease Virus, BVDV2, CSFV-PK15C, SF573 Bicistronic Virus, Hubei Picoma-like Virus, CRPV, Salivary Virus A BN5, Salivary Virus A BN2, Salivary Virus A aptamers of 02394, salivary virus A GUT, salivary virus A CH, salivary virus A SZ1, salivary virus FHB, CVB3, CVB1, echovirus 7, CVB5, EVA71, CVA3, CVA12, EV24, or eIF4G (see PCT applications WO2020237227A1 and WO2021113777A2, both of which are incorporated herein by reference in their entirety). In some embodiments, the circular RNA comprises, in the following order: a) a spliced ​​intron of a 3' class I intron, b) IRES, c) an expression sequence, and d) a spliced ​​intron of a 5' class I intron. In some embodiments, the circular RNA polynucleotide is produced by circulylating an RNA polynucleotide comprising, in the following order: a) a 3' class I intron, b)IRES, c) the expression sequence, and d) the 5' class I intron fragment. In some embodiments, the circular RNA comprises a first spacer preceding the spliced ​​intron fragment of the 3' class I intron fragment and a second spacer following the spliced ​​intron fragment of the 5' class I intron fragment. In some embodiments, the length of each of the first and second spacers is about 10 to about 60 nucleotides. In some embodiments, the circular RNA polynucleotide is produced by circulylating an RNA polynucleotide comprising, in the following order: a) a 5' outer double-strand forming region, b) a 3' class I intron fragment, c) a 5' inner spacer (optionally including a 5' inner double-strand forming region), d) IRES, e) the expression sequence, f) a 3' inner spacer (optionally including a 3' inner double-strand forming region), g) a 5' class I intron fragment, and h) a 3' outer double-strand forming region.

[0150] In some embodiments, the circular RNA polynucleotide is produced by cyclizing an RNA polynucleotide comprising, in the following order: a) a 5' outer double-stranded region, b) a 5' outer spacer, c) a 3' class I intron fragment, d) a 5' inner spacer (optionally including a 5' inner double-stranded region), e) an IRES, f) an expression sequence, g) a 3' inner spacer (optionally including a 3' inner double-stranded region), h) a 5' class I intron fragment, i) a 3' outer spacer, and j) a 3' outer double-stranded region. In some embodiments, the circular RNA polynucleotide is produced by cyclizing an RNA polynucleotide comprising, in the following order: a) a 3' class I intron fragment, b) a 5' inner spacer (including a 5' inner double-stranded region), c) an IRES, d) an expression sequence, e) a 3' inner spacer (including a 3' inner double-stranded region), and f) a 5' class I intron fragment. In some embodiments, the circular RNA polynucleotide is produced by cyclizing an RNA polynucleotide comprising, in the following order: a) a 5' outer double-stranded region, b) a 5' outer spacer, c) a 3' class I intron fragment, d) a 5' inner spacer (containing a 5' inner double-stranded region), e) an IRES, f) an expression sequence, g) a 3' inner spacer (containing a 3' inner double-stranded region), h) a 5' class I intron fragment, i) a 3' outer spacer, and j) a 3' outer double-stranded region. In some embodiments, the circular RNA polynucleotide is produced by circulylating an RNA polynucleotide comprising, in the following order: a) a first polyA sequence, b) a 5' outer double-stranded region, c) a 5' outer spacer, d) a 3' class I intron fragment, e) a 5' inner spacer (containing a 5' inner double-stranded region), f) IRES, g) an expression sequence, h) a 3' inner spacer (containing a 3' inner double-stranded region), i) a 5' class I intron fragment, j) a 3' outer spacer, k) a 3' outer double-stranded region, and l) a second polyA sequence. In some embodiments, the circular RNA polynucleotide is produced by circulylating an RNA polynucleotide comprising, in the following order: a) a first polyA sequence, b) a 5' outer spacer, c) a 3' class I intron fragment, d) a 5' inner spacer (containing a 5' inner double-stranded region), e) an IRES, f) an expression sequence, g) a 3' inner spacer (containing a 3' inner double-stranded region), h) a 5' class I intron fragment, i) a 3' outer spacer, and j) a second polyA sequence.

[0151] In some embodiments, the circular RNA polynucleotide is produced by circulylating an RNA polynucleotide comprising, in the following order: a) a first polyA sequence, b) a 5' outer spacer, c) a 3' class I intron fragment, d) a 5' inner spacer (containing a 5' inner double-stranded region), e) an IRES, f) an expression sequence, g) a stop codon box, h) a 3' inner spacer (containing a 3' inner double-stranded region), i) a 5' class I intron fragment, j) a 3' outer spacer, and k) a second polyA sequence.

[0152] In some embodiments, at least one of the 3' or 5' inner or outer spacers is about 8 to about 60 nucleotides in length. In some embodiments, the lengths of the 3' and 5' outer double-stranded regions are each about 10-50 nucleotides. In some embodiments, the lengths of the 3' and 5' inner double-stranded regions are each about 6-30 nucleotides.

[0153] In some implementations, the modified nucleic acid is a capped RNA, wherein the 5' and / or 3' ends are capped by chemical alteration.

[0154] In a more preferred embodiment, the synthetic scaffold domain contains at least two desired modification sites for multiplexing. For example, a second glycan is paired with a second Y RNA to modify a second target region of the nucleic acid sequence. Thus, multiple glycans are paired with corresponding nucleic acid sequences (e.g., RNA) to modify multiple target regions.

[0155] This invention also provides nucleic acid molecules that hybridize with the aforementioned nucleic acid molecules under stringent conditions. As defined above and as is well known in the art, stringent hybridization is, under a specific set of conditions, hybridization at a temperature higher than the thermal melting point (T0) of a specific DNA hybrid. m The experiment was conducted at a temperature of approximately 25°C, where T... m This is the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe. Rigorous washing is performed under specific condition sets, at a temperature higher than that of a specific DNA heterozygote at T... m It was carried out at a temperature approximately 5°C lower.

[0156] Nucleic acid molecules containing fragments of any one of the aforementioned nucleic acid sequences are also provided. These fragments preferably contain at least 20 consecutive nucleotides. More preferably, the fragments of the nucleic acid sequences contain at least 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or even more consecutive nucleotides.

[0157] The nucleic acid sequence fragments of the present invention have demonstrated utility in various systems and methods. For example, the fragments can be used as probes for various hybridization techniques. Depending on the method, the target nucleic acid sequence can be DNA or RNA. Prior to hybridization, the target nucleic acid sequence can be graded (e.g., by gel electrophoresis) or hybridization can be performed in situ on the sample. Those skilled in the art will appreciate that nucleic acid probes with known sequences can be used to determine chromosome structure (e.g., by Southern blotting) and measure gene expression (e.g., by Northern blotting). In such experiments, it is preferable to detectably label the sequence fragments so that their specific hybridization with the target sequence can be detected and optionally quantified. Those skilled in the art will appreciate that the nucleic acid fragments of the present invention can be used in various blotting techniques not specifically described herein.

[0158] It should also be understood that the nucleic acid sequence fragments disclosed herein (optionally conjugated with glycans) can also be used as probes when immobilized on microarrays. Methods for generating microarrays by depositing and immobilizing nucleic acids onto a supporting substrate are well known in the art. Related reviews can be found in DNA Microarrays: A Practical Approach (Practical Approach Series), Schena (ed.), Oxford University Press (1999) (ISBN: 0199637768); Nature Genet. 21(1)(Supplement):1-60 (1999); Microarray Biochip: Tools and Technology, Schena (ed.), Eaton Publishing Company / BioTechniques Books Division (2000) (ISBN: 1881299376), the contents of which are incorporated herein by reference in their entirety. For example, the analysis of gene expression using microarrays containing nucleic acid sequence fragments, such as those disclosed herein, is a well-established application of sequence fragments in cell and molecular biology. Other uses of sequence fragments immobilized on microarrays are described in Gerhold et al., Trends Biochem. Sci. 24:168-173 (1999) and Zweiger, Trends Biotechnol. 17:429-436 (1999); DNA Microarrays: A Practical Approach (Practical Approach Series), Schena (ed.), Oxford University Press (1999) (ISBN: 0199637768); Nature Genet. 21(1)(Supplement): 1-60 (1999); Microarray Biochip: Tools and Technology, Schena (ed.), Eaton Publishing Company / BioTechniques Books Division (2000) (ISBN: 1881299376), the contents of each of these publications are incorporated herein by reference in their entirety.

[0159] As is well known in the art, enzyme activity can be measured in a variety of ways. For example, the pyrophosphate hydrolysis of OMP can be tracked by spectroscopic methods (Grubmeyer et al., (1993) J. Biol. Chem. 268:20299-20304). Enzyme activity can be tracked using chromatographic techniques, such as high performance liquid chromatography (Chung and Sloan, (1986) J. Chromatogr. 371:71-81). As an alternative, activity can be measured indirectly by determining the level of products produced by the enzyme activity. These levels can be measured using techniques including aqueous chloroform / methanol extraction, as known and described in the art (Cf. M. Kates (1986) Techniques of Lipidology; Isolation, analysis and identification of Lipids. Elsevier Science Publishers, New York (ISBN: 0444807322)). More modern techniques include the use of gas chromatography coupled with mass spectrometry (Niessen, WMA (2001). Current practice of gas chromatography-mass spectrometry. New York, NY: Marcel Dekker. (ISBN: 0824704738)). Additional modern techniques for identifying the activity and products of recombinant proteins include liquid chromatography-mass spectrometry (LCMS), high-performance liquid chromatography (HPLC), capillary electrophoresis, matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS), nuclear magnetic resonance (NMR), near-infrared (NIR) spectroscopy, viscosity determination (Knothe, G (1997) Am. Chem. Soc. Symp. Series, 666: 172–208), titration for determining free fatty acids (Komers (1997) Fett / Lipid, 99(2): 52–54), enzymatic methods (Bailer (1991) Fresenius J. Anal. Chem. 340(3): 186), physical property-based methods, wet chemical methods, etc., all of which can be used to analyze the level and identity of the products produced by the organisms of this invention. As is known to those skilled in the art, other methods and techniques may also be applicable to measuring enzyme activity.

[0160] isolated peptides

[0161] According to another aspect of the invention, isolated polypeptides (including mutant proteins, allele variants, fragments, derivatives, and analogs) encoded by the nucleic acid molecules of the invention are provided. In an alternative embodiment of the invention, the isolated polypeptide comprises a polypeptide sequence that is at least 85% identical to one or more coding polypeptide sequences. Preferably, the isolated polypeptide of the invention has at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or even higher identity with one or more coding polypeptide sequences.

[0162] According to other embodiments of the invention, isolated polypeptides comprising fragments of the above-described polypeptide sequences are provided. These fragments preferably comprise at least 20 consecutive amino acids, more preferably at least 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or even more consecutive amino acids.

[0163] The polypeptides of the present invention also comprise fusions between the aforementioned polypeptide sequences and heterologous polypeptides. For example, the heterologous sequence may include a sequence designed to facilitate purification (e.g., histidine tagging) and / or visualization of the recombinant expressed protein. Other non-limiting examples of protein fusions include fusions that allow the display of protein-coding fusions on the surface of bacteriophages or cells, fusions with intrinsic fluorescent proteins (such as green fluorescent protein (GFP)), and fusions with the Fc region of IgG.

[0164] Glycan synthesis and selection

[0165] This document provides methods and compositions for selecting or synthesizing one or more glycan components for glycoligand compositions. Preferably, the glycoligand composition is produced by synthesizing or selecting a desired glycan (based on its assumed association with cell signaling) and conjugating the glycan onto a synthetic scaffold. One or more glycans are selected or synthesized as cell signaling molecules to contact one or more cell surface proteins of target cells, thereby modulating desired biological effects.

[0166] For example, libraries of naturally occurring N-glycans can be generated by chemical enzymatic synthesis or other suitable methods. See, for example, Gao et al., Cell Chem Biology, Vol. 26, No. 4, 2019. Based on Gao et al., UDP-glycan substrates were chemically enzymatically transferred to glycan acceptor substrates using known glycosyltransferases. See, for example, Example 1.

[0167] As an alternative to chemically enzymatically synthesized glycans, any suitable method can be used to synthesize, purify, and / or isolate glycans to produce the desired type of glycan.

[0168] Other existing methods exist for producing glycans, which can be generated through recombinant methods, such as by overexpressing or heterologously expressing one or more glycosyltransferases, glycosidases, or glyconucleotide donors (e.g., UDP-N-acetylglucosamine, UDP-N-acetylglucosamine, CMP-N-acetylneuraminic acid, UDP-galactose, GDP-fucose, etc.), which are synthesized in the cytosol and transported to the Golgi apparatus, where they are attached to the core oligosaccharide by glycosyltransferases. See, for example (Sommers and Hirschberg, 1981 J. Cell Biol. 91 (2): A406-A406; Sommers and Hirschberg 1982 J. Biol. Chem. 257(18): 811-817; Perez and Hirschberg 1987 Methods in Enzymology 138: 709-715), epimerases (UDP-GlcNAc and UDP-Gal), UDP-N-acetylglucosamine transporter, GDP-fucose transporter, UDP-galactose transporter, and CMP-N-acetylneuraminic acid (CMP-sialic acid) transporter, for example, for catalyzing the assembly of desired glycans and their subsequent isolation from host cells (including CHO cells, yeast cells, insect cells, and plant cells).

[0169] In various aspects of the invention, the carbohydrate portion (e.g., a polysaccharide conjugated to a glycoligand) comprises one or more sugar residues or combinations thereof, including but not limited to D-glucose (“Glc”), galactose (“Gal”), mannose (“Man”), fucose (“Fuc”), N-acetylgalactosamine (“GalNAc”), N-acetylglucosamine (“GlcNAc”), N-acetyllactosamine (“LacNAc”), sialic acid (e.g., N-acetylneuraminic acid (“NANA” or “NeuAc”, where “Neu” is neuraminic acid and “Ac” refers to “acetyl”)), D-glucosamine (“GlcN”), D-glucuronic acid (“GlcA”), β-muramic acid (“Mur”), mannuronic acid (“ManA”), N-acetyl-muramic acid (“MurNAc”), Legionaminic acid. acid (“Leg”), acetaminophen (“Aci”), D-xylose (“Xyl”), N-acetyl-L-fucosamine (“FucNAc”), pseudoamine (“Pse”) and L-iduronic acid (“IdoA”).

[0170] As used in this article, the chemical modification "9-N-biphenylcarbamate" or "BPC" refers to the part with the following structure: .

[0171] Oligosaccharide structures attached to nucleic acid molecules (such as those found in naturally occurring RNA), while not fully characterized, can be classified into two categories (similar to glycoproteins): "N-linked glycans" or "N-linked oligosaccharides" and "O-linked glycans" or "O-linked oligosaccharides." Glycans can contain monosaccharides, disaccharides, and oligosaccharides. Without being theoretically constrained, the processing of carbohydrate portions on non-amino acid molecules (such as RNA) can occur via co-translation in the ER cavity and continue in the Golgi apparatus, similar to N-linked glycoproteins.

[0172] Various glycans can be selected for conjugation to the desired scaffold, including N-linked glycans (such as mixed or complex, branched, oligomannose-type glycans) or O-linked glycans. In some embodiments, the glycan is a complex N-glycan. In some embodiments, the glycan is a multi-tentacle complex N-glycan. In some embodiments, the glycan is a mixed N-glycan. In some embodiments, the glycan is an oligomannose-type N-glycan. In some embodiments, the glycan is an O-linked glycan.

[0173] In some embodiments, the glycan ligands of the present invention comprise glycans selected from those glycans listed in Tables 1A-1F below.

[0174] In some embodiments, the glycan targeting portion of this disclosure comprises a glycan selected from those listed in Table 1A below:

[0175] Table 1A: Exemplary polysaccharides

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183] In some embodiments, the glycan targeting portion of this disclosure comprises a glycan selected from those listed in Table 1B below:

[0184] Table 1B: Exemplary polysaccharides

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195] In some embodiments, the glycan targeting portion of this disclosure comprises a glycan selected from those listed in Table 1C below:

[0196] Table 1C: Exemplary polysaccharides

[0197]

[0198]

[0199]

[0200]

[0201]

[0202] In some embodiments, the glycan targeting portion of this disclosure comprises a glycan selected from those listed in Table 1D below:

[0203] Table 1D: Exemplary Glycans

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212] In some embodiments, the glycan targeting portion of this disclosure comprises a glycan selected from those listed in Table 1E below:

[0213] Table 1E: Exemplary polysaccharides

[0214]

[0215]

[0216] In some embodiments, the glycan-targeting portion of this disclosure comprises a glycan of a ganglioside compound. Gangliosides are a class of complex lipids naturally occurring in the gray matter of the brain, the central nervous system, and / or the peripheral nervous system. Typically, gangliosides comprise one or more sialic acid moieties as part of a glycosphingolipid. In some embodiments, the glycan-targeting portion of this disclosure comprises a glycan selected from those glycans described in Table 1F:

[0217] Table 1F: Exemplary Gangliosides

[0218]

[0219]

[0220] In some embodiments, the glycan portion is or contains a glycan that differs from the glycans in Tables 1A-1F in that a single monosaccharide is replaced. In some embodiments, the glycan portion is or contains a glycan that differs from the glycans in Tables 1A-1F in that two monosaccharides are replaced. As a non-limiting example, the glycan portion may contain the glycans in Tables 1A-1F, wherein mannose is replaced by galactose (or vice versa), but the rest of the glycan portion remains unchanged.

[0221] In some embodiments, the glycan moiety comprises glucose, N-acetylglucosamine, mannose, galactose, N-acetylglucosamine, sialic acid, glucuronic acid, iduronic acid, glucosamine, galactosamine, xylose, and fucose. In some embodiments, the glycan moiety comprises glucose, GlcNAc, mannose, galactose, sialic acid, N-acetylneuraminic acid (NANA), and fucose, or subsets or combinations thereof. In some embodiments, the glycan moiety comprises sialic acid and fucose, or combinations thereof. In some embodiments, the glycan moiety comprises sialic acid. In some embodiments, the glycan moiety comprises glucose. In some embodiments, the glycan moiety comprises fucose. In some embodiments, the glycan moiety comprises mannose. In some embodiments, the glycan moiety comprises GlcNAc (N-acetylglucosamine). In some embodiments, the glycan moiety comprises galactose. In some embodiments, the glycan moiety comprises fucose linked to a GlcNAc residue. In some embodiments, the glycan moiety comprises fucose linked to a galactose residue. In some embodiments, the glycan moiety comprises fucose linked to glucose residues. In some embodiments, the glycan moiety comprises GalNAc. In some embodiments, the glycan moiety does not comprise GalNAc.

[0222] In some embodiments, the glycan moiety comprises one or more hexuronic acid sugars. In some embodiments, the glycan moiety comprises IdoA. In some embodiments, the glycan moiety comprises GlcA.

[0223] In some embodiments, the glycan portion comprises glucose. In some embodiments, the glycan portion consists of a multi-tentacle glycan composed solely of glucose monosaccharides. In some embodiments, the glycan portion consists of a multi-tentacle glycan composed solely of galactosyl monosaccharides.

[0224] In some implementations, the polysaccharide portion contains β-muramic acid.

[0225] In some embodiments, the glycan moiety comprises one or more non-sugar components or modifications. For example, exemplary glycan H-33 comprises 9-N-biphenylcarbamate (BPC) modification. In some embodiments, the glycan moiety comprises BPC modification. In some embodiments, the glycan moiety comprises one or more non-sugar components or modifications selected from:

[0226] , , , , , , , , , , , , , , , , , , and .

[0227] In some embodiments, the glycan moiety comprises compounds disclosed by Büll et al. (Trends in Biochemical Sciences;41:6, P519-531, 2016), which are incorporated herein by reference in their entirety. In some embodiments, the glycan moiety comprises one or more sialic acid-mimicking chemical modifications or substituents disclosed by Büll et al.

[0228] In some embodiments, the glycan moiety comprises two or more repeating chains of HexNAc-hexuronic acid units. In some embodiments, the glycan moiety comprises two or more repeating chains of GlcN-GlcA units. In some embodiments, the glycan moiety comprises two or more repeating chains of GlcN-IdoA units. In some embodiments, the glycan moiety comprises two or more repeating chains of GalNAc-GlcA units.

[0229] In some implementations, the glycan moiety comprises chains of repeating galactose-GlcNAc- units.

[0230] In some embodiments, the glycan portion comprises a monotendril containing at least two monosaccharides. In some embodiments, the glycan portion comprises a monotendril containing at least three monosaccharides. In some embodiments, the glycan portion comprises a monotendril containing at least four monosaccharides. In some embodiments, the glycan portion comprises a monotendril containing at least five monosaccharides. In some embodiments, the glycan portion comprises a monotendril containing at least six monosaccharides. In some embodiments, the glycan portion comprises a monotendril containing at least seven monosaccharides. In some embodiments, the glycan portion comprises a monotendril containing at least eight monosaccharides. In some embodiments, the glycan portion comprises a monotendril containing at least nine monosaccharides. In some embodiments, the glycan portion comprises a monotendril containing at least ten monosaccharides. In some embodiments, the glycan portion comprises a monotendril containing at least eleven monosaccharides. In some embodiments, the glycan portion comprises a monotendril containing at least twelve monosaccharides.

[0231] In some embodiments, the glycan moiety includes a multi-tentacle glycan containing one or more mannoses at one or more locations on the glycan branch. In some embodiments, the multi-tentacle glycan contains at least three mannose moieties, one of which is located at a glycan branch and bonded to two other mannose moieties, each mannose moieties located on each branch of the multi-tentacle glycan. In some embodiments, the glycan moiety comprises a branched oligosaccharide consisting solely of mannose.

[0232] In some embodiments, the glycan moiety includes a multi-tentacle glycan containing one or more mannoses at one or more locations on the glycan branch. In some embodiments, the multi-tentacle glycan contains at least three mannose moieties, one of which is located at a glycan branch and bonded to two other mannose moieties, each mannose moiety located on each branch of the multi-tentacle glycan.

[0233] In some embodiments, the glycan portion comprises a biantennary glycan, wherein the biantennary glycan includes a first terminal residue and a second terminal residue. In some embodiments, at least one of the first or second terminal residues of the biantennary glycan comprises sialic acid. In some embodiments, at least one of the first or second terminal residues of the biantennary glycan comprises mannose. In some embodiments, at least one of the first or second terminal residues of the biantennary glycan comprises GlcNAc. In some embodiments, at least one of the first or second terminal residues of the biantennary glycan comprises NANA. In some embodiments, at least one of the first or second terminal residues of the biantennary glycan comprises GalNAc. In some embodiments, at least one of the first or second terminal residues of the biantennary glycan comprises a sialic acid residue, said sialic acid residue comprising one or more polysialic acid end modifications. In some embodiments, at least one of the first or second terminal residues of the biantennary glycan comprises fucose. In some embodiments, one of the first or second terminal residues of the biantennary glycan comprises fucose and the other comprises sialic acid. In some embodiments, both the first and second terminal residues of the biantennary glycan contain sialic acid. In some embodiments, both the first and second terminal residues of the biantennary glycan contain mannose. In some embodiments, both the first and second terminal residues of the biantennary glycan contain GlcNAc. In some embodiments, both the first and second terminal residues of the biantennary glycan contain NANA. In some embodiments, both the first and second terminal residues of the biantennary glycan contain GalNAc.

[0234] In some embodiments, the glycan portion comprises a tritendinous glycan, wherein the tritendinous glycan includes a first terminal residue, a second terminal residue, and a third terminal residue. In some embodiments, at least one of the first, second, or third terminal residues of the tritendinous glycan comprises sialic acid. In some embodiments, at least one of the first, second, or third terminal residues of the tritendinous glycan comprises a sialic acid residue, said sialic acid residue comprising one or more polysialic acid end modifications. In some embodiments, at least one of the first or second terminal residues of the tritendinous glycan comprises fucose. In some embodiments, at least one of the first, second, or third terminal residues of the tritendinous glycan comprises sialic acid, and at least one of the remaining terminal residues comprises fucose. In some embodiments, at least one of the first, second, and third terminal residues of the tritendinous glycan comprises sialic acid. In some embodiments, at least one of the first, second, and third terminal residues of the tritendinous glycan comprises mannose. In some embodiments, at least one of the first, second, and third terminal residues of the tritendolent glycan comprises GlcNAc. In some embodiments, at least one of the first, second, and third terminal residues of the tritendolent glycan comprises NANA. In some embodiments, at least one of the first, second, and third terminal residues of the tritendolent glycan comprises GalNAc. In some embodiments, all of the first, second, and third terminal residues of the tritendolent glycan comprises sialic acid. In some embodiments, all of the first, second, and third terminal residues of the tritendolent glycan comprises mannose. In some embodiments, all of the first, second, and third terminal residues of the tritendolent glycan comprises GlcNAc. In some embodiments, all of the first, second, and third terminal residues of the tritendolent glycan comprises NANA. In some embodiments, all of the first, second, and third terminal residues of the tritendolent glycan comprises GalNAc.

[0235] In some embodiments, the glycan portion comprises a tetraanthind glycan, wherein the tetraanthind glycan includes a first terminal residue, a second terminal residue, a third terminal residue, and a fourth terminal residue. In some embodiments, at least one of the first, second, third, or fourth terminal residues of the tetraanthind glycan comprises sialic acid. In some embodiments, at least one of the first, second, third, or fourth terminal residues of the tetraanthind glycan comprises a sialic acid residue, said sialic acid residue comprising one or more polysialic acid end modifications. In some embodiments, at least one of the first, second, third, or fourth terminal residues of the tetraanthind glycan comprises fucose. In some embodiments, at least one of the first, second, third, or fourth terminal residues of the tetraanthind glycan comprises sialic acid, and at least one of the remaining terminal residues comprises fucose. In some embodiments, at least one of the first, second, third, and fourth terminal residues of the tetraanthind glycan comprises sialic acid. In some embodiments, at least one of the first, second, third, and fourth terminal residues of the tetraanthinyl glycan comprises mannose. In some embodiments, at least one of the first, second, third, and fourth terminal residues of the tetraanthinyl glycan comprises GlcNAc. In some embodiments, at least one of the first, second, third, and fourth terminal residues of the tetraanthinyl glycan comprises NANA. In some embodiments, at least one of the first, second, third, and fourth terminal residues of the tetraanthinyl glycan comprises GalNAc. In some embodiments, all of the first, second, third, and fourth terminal residues of the tetraanthinyl glycan comprises sialic acid. In some embodiments, all of the first, second, third, and fourth terminal residues of the tetraanthinyl glycan comprises mannose. In some embodiments, all of the first, second, third, and fourth terminal residues of the tetraanthinyl glycan comprises GlcNAc. In some embodiments, all of the first, second, third, and fourth terminal residues of the tetraanthinyl glycan contain NANA. In some embodiments, all of the first, second, third, and fourth terminal residues of the tetraanthinyl glycan contain GalNAc.

[0236] In some embodiments, the glycan portion comprises fucose attached to the core or basal region of the glycan. In some embodiments, the glycan portion comprises fucose attached to non-terminal regions of the glycan. In some embodiments where the glycan portion comprises a bitendril, tritendril, or tetratendril glycan, the glycan comprises fucose attached to GlcNAc residues in the core or basal region of the glycan. In some embodiments where the glycan portion comprises a bitendril, tritendril, or tetratendril glycan, the glycan comprises fucose attached to GlcNAc residues in the dendritic, branching, or arm regions of the glycan.

[0237] In some embodiments, the glycan portion comprises a diglycosylcan. In some embodiments, the glycan portion comprises a biantennary glycan containing a GlcNAc moiety bound to a monosaccharide connecting two branches of the biantennary glycan, thereby forming a diglycosylcan. In some embodiments, the glycan portion comprises a triantennary glycan, wherein one of the three branches of the triantennary glycan is formed by a diglycosylcan bond between the other two branches. In some embodiments, the glycan portion comprises a tetraantennary glycan, wherein at least one of the branches of the tetraantennary glycan is formed by a diglycosylcan bond between the other two branches.

[0238] In some embodiments, the glycan moiety comprises a biantennary, triantennary, or tetraantennary glycan having at least two different terminal residue monosaccharides. For example, in some embodiments, the glycan moiety is a biantennary glycan, wherein the first and second terminal residues do not contain the same monosaccharide. In some embodiments, the glycan moiety is a triantennary glycan, wherein the first and second terminal residues contain the same monosaccharide, and the third terminal residue contains a different monosaccharide. In some embodiments, the glycan moiety is a triantennary glycan, wherein the first, second, and third terminal residues contain different monosaccharides. In some embodiments, the glycan moiety is a tetraantennary glycan, wherein the first and second terminal residues contain the same monosaccharide, and the third and fourth terminal residues contain monosaccharides different from the first and second terminal residues, wherein the third and fourth terminal residues optionally contain the same monosaccharide as each other. In some embodiments, the glycan moiety is a tetraantennae glycan, wherein the first, second, and third terminal residues contain the same monosaccharide, and the fourth terminal residue contains a monosaccharide different from the first, second, and third terminal residues. In some embodiments, the glycan moiety is a tetraantennae glycan, wherein the first, second, third, and fourth terminal residues contain different monosaccharides.

[0239] In some implementations, the glycan moiety is an N-linked glycan, which allows the glycan to be conjugated to the modified nucleic acid via a nitrogen atom.

[0240] In some embodiments, the glycan portion comprises a glycan containing a monosaccharide at a non-reducing end, and further comprises a conjugated handle covalently bonded to the non-reducing terminal monosaccharide. In some embodiments, the glycan portion comprises a glycan containing N-acetylglucosamine (GlcNAc) at a non-reducing end, and further comprises a conjugated handle covalently bonded to the non-reducing terminal GlcNAc. As used herein, the terms "non-reducing terminal monosaccharide" and "non-reducing terminal monosaccharide" refer to a monosaccharide residue that is part of the glycan and forms the end of the glycan at a non-reducing end. As an illustrative example, in exemplary glycan H-7, the "GlcNAc" at the end of the IUPAC name is a non-reducing terminal GlcNAc:

[0241] Neu5Ac(a2-8)Neu5Ac(a2-3)Gal(b1-4)GlcNAc

[0242] In some embodiments, the glycan portion includes a glycan, the glycan further comprising a conjugated handle covalently bonded to a non-reducing terminal GlcNAc. In some embodiments, the glycan portion includes a glycan, the glycan further comprising a conjugated handle covalently bonded to a non-reducing terminal GalNAc. In some embodiments, the glycan portion includes a glycan, the glycan further comprising a conjugated handle covalently bonded to a non-reducing terminal mannose. In some embodiments, the glycan portion includes a glycan, the glycan further comprising a conjugated handle covalently bonded to a non-reducing terminal IdoA. In some embodiments, the glycan portion includes a glycan, the glycan further comprising a conjugated handle covalently bonded to a non-reducing terminal glucose. In some embodiments, the glycan portion includes a glycan, the glycan further comprising a conjugated handle covalently bonded to a non-reducing terminal GlcN.

[0243] In some embodiments, the glycoligand comprises a glycan, said glycan further comprising an asparagine residue covalently bound to a non-reducing terminal monosaccharide. In some embodiments, the glycoligand comprises a glycan described in any of the glycans in Tables 1A-1F, said glycan further comprising an asparagine residue covalently bound to a non-reducing terminal monosaccharide, as follows:

[0244]

[0245] The asterisk (*) indicates the attachment site to the non-reducing terminal monosaccharide of the glycan, and the ** indicates the attachment site to the modified RNA or the adapter group attached to the modified RNA.

[0246] In some embodiments, the sugar ligand comprises a glycan, which further comprises asparagine residues covalently bound to a non-reducing terminal monosaccharide, as follows:

[0247]

[0248] The asterisk (*) indicates the attachment point to the non-reducing terminal monosaccharide of the glycan.

[0249] In some embodiments, the glycoligand comprises a glycan, said glycan further comprising an arginine residue covalently bound to a non-reducing terminal monosaccharide. In some embodiments, the glycoligand comprises a glycan, said glycan further comprising an azide click chemical handle covalently bound to the non-reducing terminal monosaccharide directly or via a linker group. In some embodiments, the linker group bridging the non-reducing terminal monosaccharide and the azide comprises one or more peptide residues. In some embodiments, the linker group bridging the non-reducing terminal monosaccharide and the azide comprises one or more polyethylene glycol (PEG) units. In some embodiments, the linker group bridging the non-reducing terminal monosaccharide and the azide comprises 1-10 PEG units. In some embodiments, the linker group bridging the non-reducing terminal monosaccharide and the azide comprises one PEG unit. In some embodiments, the linker group bridging the non-reducing terminal monosaccharide and the azide comprises two PEG units. In some embodiments, the linker group bridging the non-reducing terminal monosaccharide and the azide comprises three PEG units. In some embodiments, the linker group bridging the non-reducing terminal monosaccharide and the azide comprises four PEG units. In some embodiments, the linker group bridging the non-reducing terminal monosaccharide and the azide comprises five PEG units. In some embodiments, the linker group bridging the non-reducing terminal monosaccharide and the azide comprises an optionally substituted aliphatic chain. In some embodiments, the optionally substituted aliphatic chain is C1-C. 12 Alkylene chain. In some embodiments, the optionally substituted aliphatic chain is C2-C. 12 Alkenyl chain.

[0250] In some embodiments, the sugar ligand comprises a polysaccharide, said polysaccharide further comprising a conjugated handle covalently bonded to a non-reducing terminal monosaccharide, wherein the conjugated handle comprises an aminooxy-PEG3-azide.

[0251] Or, in terms of the entire glycoligand, the conjugation handle is the product of a click chemistry between an aminooxy-PEG3-azide and an alkyne portion of the ligand (e.g., nucleic acid) attached to the glycoligand.

[0252] In some embodiments, the sugar ligand comprises a polysaccharide, said polysaccharide further comprising an aminooxy-PEG3-azide covalently bound to a non-reducing terminal monosaccharide, as shown below:

[0253]

[0254] The asterisk (*) indicates the attachment point to the non-reducing terminal monosaccharide of the glycan.

[0255] In some embodiments, the glycan portion comprises a glycan, which further includes a linker covalently bound to a non-reducing terminal monosaccharide, as shown below:

[0256]

[0257] The asterisk (*) indicates the attachment site to the non-reducing terminal monosaccharide of the glycan, and the ** indicates the attachment site to the ligand component of the glycan ligand (e.g., RNA or modified RNA or a linker group attached to modified RNA).

[0258] In some embodiments, the sugar ligand comprises a polysaccharide, said polysaccharide further comprising a conjugated handle covalently bonded to a non-reducing terminal monosaccharide, wherein the conjugated handle comprises O-(3-azidopropyl)-N-methylhydroxylamine.

[0259] Or, in terms of the entire sugar ligand, the conjugation handle is the product of a click chemistry between O-(3-azidopropyl)-N-methylhydroxylamine and the alkyne portion of the ligand (e.g., nucleic acid) attached to the sugar ligand.

[0260] In some embodiments, the sugar ligand comprises a polysaccharide, said polysaccharide further comprising O-(3-azidopropyl)-N-methylhydroxylamine covalently bound to a non-reducing terminal monosaccharide, as shown below:

[0261]

[0262] The asterisk (*) indicates the attachment point to the non-reducing terminal monosaccharide of the glycan.

[0263] In some embodiments, the glycan portion comprises a glycan, which further includes a linker covalently bound to a non-reducing terminal monosaccharide, as shown below:

[0264]

[0265] The asterisk (*) indicates the attachment site to the non-reducing terminal monosaccharide of the glycan, and the ** indicates the attachment site to the ligand component of the glycan ligand (e.g., RNA or modified RNA or a linker group attached to modified RNA).

[0266] In some embodiments, the sugar ligand comprises a polysaccharide, said polysaccharide further comprising a conjugation handle covalently bonded to a non-reducing terminal monosaccharide, wherein the conjugation handle comprises O-(3-azidoethyl)-N-methylhydroxylamine.

[0267] Or, in terms of the entire sugar ligand, the conjugation handle is the product of a click chemistry between O-(3-azidoethyl)-N-methylhydroxylamine and the alkyne portion of the ligand (e.g., nucleic acid) attached to the sugar ligand.

[0268] In some embodiments, the sugar ligand comprises a polysaccharide, said polysaccharide further comprising O-(3-azidoethyl)-N-methylhydroxylamine covalently bound to a non-reducing terminal monosaccharide, as shown below:

[0269]

[0270] The asterisk (*) indicates the attachment point to the non-reducing terminal monosaccharide of the glycan.

[0271] In some embodiments, the glycan portion comprises a glycan, which further includes a linker covalently bound to a non-reducing terminal monosaccharide, as shown below:

[0272]

[0273] The asterisk (*) indicates the attachment site to the non-reducing terminal monosaccharide of the glycan, and the ** indicates the attachment site to the ligand component of the glycan ligand (e.g., RNA or modified RNA or a linker group attached to modified RNA).

[0274] polysaccharide conjugates

[0275] In one aspect, this disclosure provides a multivalent polynucleotide conjugate comprising a glycan conjugated with a polynucleotide. In some embodiments, the glycan-polynucleotide conjugate is a compound of formula (A-1):

[0276] (KX A -V 1 -X B ) m -X 1 -V 2 -X 2 -W A-1,

[0277] Or its pharmaceutically acceptable salt, wherein:

[0278] Each K is independently a glycan or glycan moiety disclosed or described herein;

[0279] Each X A C1-C independently as a bond or optionally substituted 24 Bivalent linear aliphatic chain, where X AOne or more methylene bonds may optionally be replaced by C3-C8 cycloalkylene, phenylene, -S(O2)-, -O-, -NH-, -N(C1-C6 alkyl)-, -C(=O)-, -C(=O)O- or -C(=O)NH-;

[0280] Each V 1 Independently linked by bonds, amides, divalent amino acids, divalent peptides, or heterobifunctional groups;

[0281] Each X B C1-C independently as a bond or optionally substituted 24 Bivalent linear aliphatic chain, where X B One or more methylene bonds may optionally be replaced by C3-C8 cycloalkylene, phenylene, -S(O2)-, -O-, -NH-, -N(C1-C6 alkyl)-, -C(=O)-, -C(=O)O- or -C(=O)NH-;

[0282] X 1 It is the optional substitution of C1-C 30 Divalent or multivalent straight or branched aliphatic chains, where X 1 One or more methylene bonds may optionally be replaced by C3-C8 cycloalkylene, phenylene, -S(O2)-, -O-, -NH-, -N(C1-C6 alkyl)-, -C(=O)-, -C(=O)O-, -C(=O)NH-, or divalent polypeptide bonds or multivalent polypeptide bonds; or X 1 It is a key;

[0283] V 2 It is a bond, amide, divalent amino acid bond, divalent peptide bond, or heterobifunctional group;

[0284] X 2 It is the optional substitution of C1-C 30 Divalent straight-chain or branched aliphatic chains, where X 2 One or more methylene bonds are optionally replaced by proline bonds, –OP(=O)(OH)O-, C3-C8 cycloalkylene, phenylene, -S(O2)-, -O-, -NH-, -N(C1-C6 alkyl)-, -C(=O)-, -C(=O)O- or -C(=O)NH-; or X 1 It is a key;

[0285] m is an integer greater than 2; and

[0286] W stands for polynucleotide.

[0287] K

[0288] In some embodiments, each K is independently any glycan or glycan moiety disclosed or described herein. In some embodiments, each K is covalently bound to X via any chemically available attachment site of K. A or V 1 This will be obvious to those skilled in the art. In some embodiments, each K is covalently bonded to X by replacing any covalent bond with a hydrogen atom in the polysaccharide disclosed herein. A or V 1 In some embodiments, each K is or contains any glycan independently selected from those glycans disclosed in Tables 1A-1F. In some embodiments, K contains a divalent connector disclosed elsewhere herein.

[0289] X A

[0290] In some implementation schemes, X A It is a key. In some implementations, X A It is the optional substitution of C1-C 24 Bivalent linear aliphatic chain, where X A One or more methylene bonds are optionally replaced by C3-C8 cycloalkylene, phenylene, -S(O2)-, -O-, -NH-, -N(C1-C6 alkyl)-, -C(=O)-, -C(=O)O-, or -C(=O)NH-. In some embodiments, X A It is the optional substitution of C1-C 16 Bivalent linear aliphatic chain, where X A One or more methylene bonds are optionally replaced by C3-C8 cycloalkylene, phenylene, -S(O2)-, -O-, -NH-, -N(C1-C6 alkyl)-, -C(=O)-, -C(=O)O- or -C(=O)NH-.

[0291] X B

[0292] In some implementation schemes, X B It is a key. In some implementations, X B It is the optional substitution of C1-C 24 Bivalent linear aliphatic chain, where X B One or more methylene bonds are optionally replaced by C3-C8 cycloalkylene, phenylene, -S(O2)-, -O-, -NH-, -N(C1-C6 alkyl)-, -C(=O)-, -C(=O)O-, or -C(=O)NH-. In some embodiments, X B It is the optional substitution of C1-C 16 Bivalent linear aliphatic chain, where X BOne or more methylene bonds are optionally replaced by C3-C8 cycloalkylene, phenylene, -S(O2)-, -O-, -NH-, -N(C1-C6 alkyl)-, -C(=O)-, -C(=O)O- or -C(=O)NH-.

[0293] X 1

[0294] In some implementation schemes, X 1 It is the optional substitution of C1-C 30 Divalent or multivalent straight or branched aliphatic chains, where X 1 One or more methylene bonds are optionally replaced by C3-C8 cycloalkylene, phenylene, -S(O2)-, -O-, -NH-, -N(C1-C6 alkyl)-, -C(=O)-, -C(=O)O-, -C(=O)NH-, or divalent or multivalent polypeptide bonds. In some embodiments, X 1 It is the optional substitution of C1-C 16 Divalent or multivalent straight or branched aliphatic chains, where X 1 One or more methylene bonds are optionally replaced by C3-C8 cycloalkylene, phenylene, -S(O2)-, -O-, -NH-, -N(C1-C6 alkyl)-, -C(=O)-, -C(=O)O-, -C(=O)NH-, or divalent or multivalent polypeptide bonds. In some embodiments, X 1 It is a key. In some implementations, X 1 It is the optional substitution of C1-C 16 Divalent straight-chain or branched aliphatic chains, where X 1 One or more methylene bonds are optionally replaced by C3-C8 cycloalkylene, phenylene, -S(O2)-, -O-, -NH-, -N(C1-C6 alkyl)-, -C(=O)-, -C(=O)O-, or -C(=O)NH-. In some embodiments, X 1 It is the optional substitution of C1-C 30 Bivalent linear aliphatic chain, where X 1 One or more methylene bonds are optionally replaced by C3-C8 cycloalkylene, phenylene, -S(O2)-, -O-, -NH-, -N(C1-C6 alkyl)-, -C(=O)-, -C(=O)O-, or -C(=O)NH-. In some embodiments, X 1 It is the optional substitution of C1-C 16 Bivalent linear aliphatic chain, where X 1One or more methylene bonds are optionally replaced by C3-C8 cycloalkylene, phenylene, -S(O2)-, -O-, -NH-, -N(C1-C6 alkyl)-, -C(=O)-, -C(=O)O-, or -C(=O)NH-. In some embodiments, X 1 It is the optional substitution of C1-C 16 Alkylene chain, wherein X 1 One or more methylene bonds are optionally replaced by C3-C8 cycloalkylene, phenylene, -S(O2)-, -O-, -NH-, -N(C1-C6 alkyl)-, -C(=O)-, -C(=O)O-, or -C(=O)NH-. In some embodiments, X 1 It is an optional C1-C6 divalent straight or branched aliphatic chain, wherein X 1 One or more methylene bonds are optionally replaced by C3-C8 cycloalkylene, phenylene, -S(O2)-, -O-, -NH-, -N(C1-C6 alkyl)-, -C(=O)-, -C(=O)O-, or -C(=O)NH-. In some embodiments, X 1 It is an optionally substituted C1-C6 alkylene chain, wherein X 1 One methylene bond is replaced by a C3-C8 cycloalkylene group, a phenylene group, -S(O2)-, -O-, -NH-, -N(C1-C6 alkyl)-, -C(=O)-, -C(=O)O-, or -C(=O)NH-. In some embodiments, X 1 It is an optionally substituted C1-C6 alkylene chain, wherein X 1 One methylene bond is replaced by -O-, -NH-, -N(C1-C6 alkyl)-, -C(=O)-, -C(=O)O-, or -C(=O)NH-. In some embodiments, X 1 It is an optionally substituted C1-C6 alkylene chain, wherein X 1 The two methylene bonds are replaced by C3-C8 cycloalkylene, phenylene, -S(O2)-, -O-, -NH-, -N(C1-C6 alkyl)-, -C(=O)-, -C(=O)O-, or -C(=O)NH-. In some embodiments, X 1 It is the optional substitution of C1-C 16 Multivalent straight or branched aliphatic chains, where X 1 One or more methylene bonds are optionally replaced by C3-C8 cycloalkylene, phenylene, -S(O2)-, -O-, -NH-, -N(C1-C6 alkyl)-, -C(=O)-, -C(=O)O-, -C(=O)NH-, or divalent or multivalent polypeptide bonds. In some embodiments, X 1 It is the optional substitution of C1-C16 Multivalent branched aliphatic chains, where X 1 One or more methylene bonds may optionally be replaced by C3-C8 cycloalkylene, phenylene, -S(O2)-, -O-, -NH-, -N(C1-C6 alkyl)-, -C(=O)-, -C(=O)O-, -C(=O)NH- or divalent polypeptide bonds or multivalent polypeptide bonds.

[0295] In some implementation schemes, X 1 One or more methylene bonds are replaced by divalent amino acid bonds. In some embodiments, X 1 One or more methylene bonds are replaced by divalent groups of naturally occurring amino acids. In some embodiments, X 1 One or more methylene bonds are replaced by divalent groups of non-naturally occurring amino acids. In some embodiments, X 1 One or more methylene bonds are replaced by divalent polypeptide bonds. In some embodiments, X 1 One or more methylene bonds are replaced by multivalent groups of non-naturally occurring amino acids. In some embodiments, X 1 One or more methylene bonds are replaced by polyvalent polypeptide bonds. In some embodiments, the divalent amino acid bonds and / or polyvalent amino acid bonds independently comprise at least 2, at least 3, at least 4, at least 5, at least 6, or more than 6 amino acids.

[0296] In some implementation schemes, X 1 It is a multivalent bond, where X 1 An open valence bond to V 2 And X 1 The remaining open-price bond is bonded to (KX) A -V 1 -X B X represents m independently selected instances. In some implementations, X... 1 One or more methylene bonds are replaced by multivalent polypeptide bonds, thereby allowing one open valence bond of the polypeptide to X. 1 And the remaining open valence bonds are bonded to m independently selected instances of K.

[0297] X 2

[0298] In some implementation schemes, X 2 It is a key. In some implementations, X 2 It is the optional substitution of C1-C 30 Divalent straight-chain or branched aliphatic chains, where X 2One or more methylene bonds are optionally replaced by –OP(=O)(OH)O-, C3-C8 cycloalkylene, phenylene, -S(O2)-, -O-, -NH-, -N(C1-C6 alkyl)-, -C(=O)-, -C(=O)O-, or -C(=O)NH-. In some embodiments, X 2 It is a key. In some implementations, X 2 It is the optional substitution of C1-C 16 Divalent straight-chain or branched aliphatic chains, where X 2 One or more methylene bonds are optionally replaced by –OP(=O)(OH)O-, C3-C8 cycloalkylene, phenylene, -S(O2)-, -O-, -NH-, -N(C1-C6 alkyl)-, -C(=O)-, -C(=O)O-, or -C(=O)NH-. In some embodiments, X 2 It is the optional substitution of C1-C 16 Bivalent linear aliphatic chain, where X 2 One or more methylene bonds are optionally replaced by –OP(=O)(OH)O-, C3-C8 cycloalkylene, phenylene, -S(O2)-, -O-, -NH-, -N(C1-C6 alkyl)-, -C(=O)-, -C(=O)O-, or -C(=O)NH-. In some embodiments, X 2 It is the optional substitution of C1-C 16 Alkylene chain, wherein X 2 One or more methylene bonds are optionally replaced by –OP(=O)(OH)O-, C3-C8 cycloalkylene, phenylene, -S(O2)-, -O-, -NH-, -N(C1-C6 alkyl)-, -C(=O)-, -C(=O)O-, or -C(=O)NH-. In some embodiments, X 2 It is an optional C1-C6 divalent straight or branched aliphatic chain, wherein X 2 One or more methylene bonds are optionally replaced by –OP(=O)(OH)O-, C3-C8 cycloalkylene, phenylene, -S(O2)-, -O-, -NH-, -N(C1-C6 alkyl)-, -C(=O)-, -C(=O)O-, or -C(=O)NH-. In some embodiments, X 2 It is an optionally substituted C1-C6 alkylene chain, wherein X 2 One methylene bond is replaced by –OP(=O)(OH)O-, C3-C8 cycloalkylene, phenylene, -S(O2)-, -O-, -NH-, -N(C1-C6 alkyl)-, -C(=O)-, -C(=O)O-, or -C(=O)NH-. In some embodiments, X 2It is an optionally substituted C1-C6 alkylene chain, wherein X 2 The two methylene bonds are replaced by –OP(=O)(OH)O-, C3-C8 cycloalkylene, phenylene, -S(O2)-, -O-, -NH-, -N(C1-C6 alkyl)-, -C(=O)-, -C(=O)O- or -C(=O)NH-.

[0299] m

[0300] In some implementations, m is an integer selected from 2, 3, 4, 5, 6, 7, 8, 9, and 10. In some implementations, m is 3. In some implementations, m is 4.

[0301] V 1

[0302] In some implementation schemes, V 1 It is a key. In some implementations, V 1 It is an amide. In some implementations, V 1 It is a divalent amino acid bond. In some implementations, V 1 It is a divalent group of a naturally occurring amino acid. In some implementations, V 1 It is a divalent group of an amino acid that does not exist naturally. In some implementations, V 1 It is a bivalent polypeptide bond. In some implementations, V 1 It contains at least 2, at least 3, at least 4, at least 5, at least 6, or more than 6 amino acids. In some embodiments, V 1 It contains polylysine chains.

[0303] V 2

[0304] In some implementation schemes, V 2 It is a key. In some implementations, V 2 It is an amide. In some implementations, V 2 It is a divalent amino acid bond. In some implementations, V 2 It is a divalent group of a naturally occurring amino acid. In some implementations, V 2 It is a divalent group of an amino acid that does not exist naturally. In some implementations, V 2 It is a bivalent polypeptide bond. In some implementations, V 2 It contains at least 2, at least 3, at least 4, at least 5, at least 6 or more amino acids.

[0305] In some implementation schemes, V 1 It is a heterodifunctional group. In some implementations, V 2It is a heterobifunctional group. In some embodiments, each heterobifunctional group is independently a divalent linker formed by a click chemistry reaction between two click chemistry handles. A click chemistry handle can be a reactant or reactive group that can participate in a click chemistry reaction. For example, a strained alkyne (e.g., cyclooctyne) is a click chemistry handle because it can participate in strain-promoted cycloaddition. Typically, a click chemistry reaction requires at least two molecules containing click chemistry handles that can react with each other. Such pairs of click chemistry handles that react with each other are sometimes referred to herein as partner click chemistry handles. For example, an azide is a partner click chemistry handle for cyclooctyne or any other alkyne. Exemplary click chemistry handles (click chemistry handle 1 and click chemistry handle 2) suitable for some aspects of this disclosure are described herein, for example, in Tables D and E. Other suitable click chemistry handles are known to those skilled in the art. For two molecules linked via click chemistry, the click chemistry handles of the molecules react with each other, for example, because the reactive portion of one click chemistry handle can react with the reactive portion of the second click chemistry handle to form a covalent bond. Those skilled in the art are familiar with such reactive click chemical handle pairs, including but not limited to the reactive click chemical handle pairs described in Table 2A:

[0306] Table 2A – Exemplary Click Chemical Handle and Reaction

[0307]

[0308] Table 2A provides examples of click chemical handles and reactions. When V 1 and / or V 2 When it is a heterodifunctional group, R, R 1 and R 2 It can represent the elements on either side of V1 and / or V2 in formula A-1, depending on the situation.

[0309] In some embodiments, a click chemistry handle is used that can react to form covalent bonds in the absence of a metal catalyst. Such click chemistry handles are well known to those skilled in the art and include those described in Becer, Hoogenboom, and Schubert, Click Chemistry beyond Metal-Catalyzed Cycloaddition, Angewandte Chemie International Edition (2009) 48: 4900–4908. See Table 2B below.

[0310] Table 2B – Exemplary Click Chemical Handle and Reaction

[0311]

[0312] RT = room temperature, DMF = N,N-dimethylformamide, NMP = N-methylpyrrolidone, THF = tetrahydrofuran, CH3CN = acetonitrile

[0313] In some implementation schemes, V 1 Selected from:

[0314] .

[0315] In some implementations, V 1 Selected from: , , , , , , , , , , or The * indicates the same as X. A The attachment point, and # indicates the connection to X. 1 The attachment point. In some implementations, V 1 yes: or The * indicates the same as X. A The attachment point, and # indicates the connection to X. 1 Attachment point.

[0316] In some implementation schemes, V 2 Selected from

[0317] .

[0318] In some implementations, V 2 Selected from: , , , , , , , , , , or The * indicates the same as X. 2 The attachment point, and # indicates the attachment point with W. In some implementations, V 2 yes: or The * indicates the same as X. 2The attachment point, and # indicates the attachment point with W.

[0319] In some embodiments, W is any polynucleotide disclosed or described herein. In some embodiments, W is siRNA or a portion thereof. In some embodiments, W is the siRNA positive strand (or guest strand, the two terms are used interchangeably herein and refer to the same thing). In some embodiments, W is the siRNA antisense strand (or guide strand, the two terms are used interchangeably herein and refer to the same thing). In some embodiments, W is attached to the V at the 3' end of the RNA. 2 or X 2 In some implementations, W is attached to the V at the 5' end of the RNA. 2 or X 2 In some implementations, W is attached to the non-terminal V of the polynucleotide. 2 or X 2 In some embodiments, the attachment site is a handle between two nucleotides inserted into the polynucleotide. In some embodiments, the attachment site is a chemically modified nucleotide of the polynucleotide. In some embodiments, W is covalently bound to V via the terminal phosphate ester or modified phosphate ester of the polynucleotide. 2 or X 2 .

[0320] In some embodiments, W is mRNA. In some embodiments, W is ASO. In some embodiments, W is a polynucleotide selected from snRNA, snoRNA, dsRNA, miRNA, lncRNA, circular RNA, Y RNA, ribosomal RNA, and small RNA fragments.

[0321] In some embodiments, the conjugate of formula A-1 is or comprises a structure selected from those structures shown in Table G below. The structure is shown below: This represents a single-stranded RNA. In some embodiments, the structure represents the sense (or guest) strand of the siRNA. In some embodiments, the structure represents the antisense (or guide) strand of the siRNA of this disclosure. In some embodiments, X' represents the 5' end of the single-stranded RNA, and Y' represents the 3' end of the single-stranded RNA. In other embodiments, X' represents the 3' end of the single-stranded RNA, and Y' represents the 5' end of the single-stranded RNA.

[0322] In some embodiments, this disclosure provides a polysaccharide-polynucleotide as shown in Table G.

[0323] Table 1. Non-limiting examples of G-polysaccharide-polynucleotide conjugates

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330] Regulation of target cell surface proteins and receptor-mediated signal transduction

[0331] This article provides methods and compositions for contacting glycoligands on cell surface proteins of target cells. Various cell surface proteins of target cells can contact glycoligands, thereby modulating biological effects.

[0332] This document provides methods and compositions for modulating cell surface proteins on target cells. Various embodiments are provided for modulating target cells by contacting a glycoligand composition with cell surface proteins, wherein the glycoligand composition comprises one or more glycans operatively linked to one or more sites on a synthetic scaffold domain. Additional targets are modulated by one or more glycoligand compositions of the present invention. Some embodiments induce agonistic effects on cell surface proteins or protein complexes on the surface of target cells or cell populations by contacting one or more glycans on the glycoligand. Other embodiments induce antagonistic effects on cell surface proteins or protein complexes on the surface of target cells or cell populations. Thus, the glycoligand compositions of the present invention induce signal transduction or signal transduction cascades in target cells or cell populations.

[0333] In various embodiments, methods and compositions for modulating cell surface proteins on target cells include synthesizing or selecting one or more desired glycans, conjugating the glycans to a synthetic scaffold structural domain, wherein the scaffold is modified to accept the glycans and contact one or more cell surface proteins comprising receptors, receptor complexes, or glycan-binding proteins.

[0334] The delivery of one or more glycoligand compositions of the present invention can address many of the drawbacks of protein therapeutics, such as changes in protein folding, solubility, protein hydrolysis and degradation, transport, translocation, compartmentalization, secretion, recognition by other proteins or factors, antigenicity or sensitization, or even many of the drawbacks of RNA therapeutics, such as targeted delivery, specificity, stability, immunogenicity and off-target toxicity.

[0335] Various cell surface proteins of target cells can be modulated by glycoligands to induce biological effects. Cell surface proteins include receptors, glycan-binding proteins, lectins, or other proteins containing carbohydrate recognition domains. Glycoligands can bind to cell surface proteins to produce the desired biological effects. In some preferred aspects of the invention, one or more lectins targeted by the glycoligand are selected from Table 3 below [Raposo CD, Canelas AB, Barros MT. Human Lectins, Their Carbohydrate Affinities and Where to Find Them. Biomolecules. Jan 29, 2021; 11(2):188]. In some embodiments, the glycan component of the glycoligand is a glycan of a lectin that binds to those lectins disclosed in Table 3. In some embodiments, the glycan component of the glycoligand is a glycan of a lectin that selectively binds to those lectins disclosed in Table 3.

[0336] Table 3: List of selected lectins

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365] In some embodiments, the glycan component of the glycoligand is a glycan that binds to plasma membrane lectins. In some embodiments, the glycan component of the glycoligand is a glycan that selectively binds to plasma membrane lectins.

[0366] In some embodiments, the glycan component of the glycoligand is a glycan that binds to MRC1 (macrophage Man-type receptor). In some embodiments, the glycan component of the glycoligand is a glycan that selectively binds to MRC1 (macrophage Man-type receptor). In some embodiments, the MRC1-binding glycan is H-1 or H-2.

[0367] In some embodiments, the glycan component of the glycoligand is a glycan bound to DC-SIGN. In some embodiments, the glycan component of the glycoligand is a glycan selectively bound to DC-SIGN. In some embodiments, the glycan bound to DC-SIGN is H-3.

[0368] In some embodiments, the glycan component of the glycoligand is a glycan that binds to MGL (macrophage galactose lectin). In some embodiments, the glycan component of the glycoligand is a glycan that selectively binds to MGL (macrophage galactose lectin). In some embodiments, the MGL-binding glycan is H-4 or H-5.

[0369] In some embodiments, the glycan component of the glycoligand is a glycan bound to Siglec 3. In some embodiments, the glycan component of the glycoligand is a glycan selectively bound to Siglec 3.

[0370] In some embodiments, the glycan component of the glycoligand is a glycan bound to Siglec 8. In some embodiments, the glycan component of the glycoligand is a glycan selectively bound to Siglec 8.

[0371] In some embodiments, the glycan component of the glycoligand is a glycan bound to Siglec 3 and Siglec 8. In some embodiments, the glycan bound to Siglec 3 and Siglec 8 is H-6.

[0372] In some embodiments, the glycan component of the glycoligand is a glycan bound to Siglec 9. In some embodiments, the glycan component of the glycoligand is a glycan selectively bound to Siglec 9. In some embodiments, the glycan bound to Siglec 9 is H-9.

[0373] In some embodiments, the glycan component of the glycoligand is a glycan bound to Siglec 2. In some embodiments, the glycan component of the glycoligand is a glycan selectively bound to Siglec 2. In some embodiments, the Siglec 2-bound glycan is H-33.

[0374] In some embodiments, the glycan component of the glycoligand is a glycan bound to Siglec 4a. In some embodiments, the glycan component of the glycoligand is a glycan selectively bound to Siglec 4a. In some embodiments, the glycan bound to Siglec 4a is H-17.

[0375] In some embodiments, the glycan component of the glycoligand is a glycan bound to Langerhansin. In some embodiments, the glycan component of the glycoligand is a glycan selectively bound to Langerhansin. In some embodiments, the glycan bound to Langerhansin is H-14, H-15, or H-16.

[0376] In some embodiments, the glycan component of the glycoligand is a glycan bound to Dectin-1. In some embodiments, the glycan component of the glycoligand is a glycan selectively bound to Dectin-1. In some embodiments, the glycan bound to Dectin-1 is H-18.

[0377] In some embodiments, the glycan component of the glycoligand is a glycan bound to Dectin-2. In some embodiments, the glycan component of the glycoligand is a glycan selectively bound to Dectin-2. In some embodiments, the glycan bound to Dectin-2 is H-10.

[0378] In some embodiments, the glycan component of the glycoligand is a glycan bound to CLEC4E. In some embodiments, the glycan component of the glycoligand is a glycan selectively bound to CLEC4E. In some embodiments, the glycan bound to CLEC4E is H-47, H-48, H-49, H-50, or H-51.

[0379] In some embodiments, the glycan component of the glycoligand is a glycan bound to CLEC12A. In some embodiments, the glycan component of the glycoligand is a glycan selectively bound to CLEC12A. In some embodiments, the glycan bound to CLEC12A is H-45 or H-46.

[0380] In some embodiments, the glycan component of the glycoligand is a glycan bound to CLEC14A. In some embodiments, the glycan component of the glycoligand is a glycan selectively bound to CLEC14A. In some embodiments, the glycan bound to CLEC14A is H-19 or H-20.

[0381] In some embodiments, the glycan component of the glycoligand is a glycan bound to CLEC4A. In some embodiments, the glycan component of the glycoligand is a glycan selectively bound to CLEC4A. In some embodiments, the glycan bound to CLEC4A is H-26.

[0382] In some embodiments, the glycan component of the glycoligand is a glycan bound to CLEC4C. In some embodiments, the glycan component of the glycoligand is a glycan selectively bound to CLEC4C. In some embodiments, the glycan bound to CLEC4C is H-27.

[0383] In some embodiments, the glycan component of the glycoligand is a glycan bound to CLEC5A. In some embodiments, the glycan component of the glycoligand is a glycan selectively bound to CLEC5A. In some embodiments, the glycan bound to CLEC5A is H-25 or H-32.

[0384] In some embodiments, the glycan component of the glycoligand is a glycan bound to CLEC2D. In some embodiments, the glycan component of the glycoligand is a glycan selectively bound to CLEC2D. In some embodiments, the glycan bound to CLEC2D is H-30.

[0385] In some embodiments, the glycan component of the glycoligand is a CD2-bound glycan. In some embodiments, the glycan component of the glycoligand is a CD2-selectively bound glycan. In some embodiments, the CD2-bound glycan is H-24.

[0386] In some embodiments, the glycan component of the glycoligand is a glycan that binds to E-selectin. In some embodiments, the glycan component of the glycoligand is a glycan that selectively binds to E-selectin. In some embodiments, the E-selectin-binding glycan is H-23.

[0387] In some embodiments, the glycan component of the glycoligand is a glycan that binds to P-selectin. In some embodiments, the glycan component of the glycoligand is a glycan that selectively binds to P-selectin. In some embodiments, the P-selectin-binding glycan is H-29.

[0388] In some embodiments, the glycan component of the glycoligand is a glycan that binds to L-selectin. In some embodiments, the glycan component of the glycoligand is a glycan that selectively binds to L-selectin. In some embodiments, the L-selectin-binding glycan is H-9 or H-34.

[0389] In some embodiments, the glycan component of the glycoligand is a glycan that binds to thrombomodulin. In some embodiments, the glycan component of the glycoligand is a glycan that selectively binds to thrombomodulin. In some embodiments, the glycan that binds to thrombomodulin is H-22 or H-31.

[0390] In some embodiments, the glycan component of the glycoligand is a glycan bound to SRCL. In some embodiments, the glycan component of the glycoligand is a glycan selectively bound to SRCL. In some embodiments, the SRCL-bound glycan is H-24.

[0391] In some embodiments, the glycan component of the glycoligand is a glycan that binds to serum lectins. In some embodiments, the glycan component of the glycoligand is a glycan that selectively binds to serum lectins.

[0392] In some embodiments, the glycan component of the glycoligand is a glycan bound to MBL (Man-binding lectin). In some embodiments, the glycan component of the glycoligand is a glycan selectively bound to MBL (Man-binding lectin). In some embodiments, the MBL-bound glycan is H-1o.

[0393] In some embodiments, the glycan component of the glycoligand is a glycan bound to galactoglobulin-2 (LGACS2). In some embodiments, the glycan component of the glycoligand is a glycan selectively bound to galactoglobulin-2 (LGACS2). In some embodiments, the glycan bound to galactoglobulin-2 is H-11.

[0394] In some embodiments, the glycan component of the glycoligand is a glycan that binds to the anti-α-Gal antibody. In some embodiments, the glycan component of the glycoligand is a glycan that selectively binds to the anti-α-Gal antibody. In some embodiments, the glycan that binds to the anti-α-Gal antibody is H-12.

[0395] In some embodiments, the glycan component of the glycoligand is a glycan bound to galactoglobulin-3. In some embodiments, the glycan component of the glycoligand is a glycan selectively bound to galactoglobulin-3.

[0396] In some embodiments, the glycan component of the glycoligand is a glycan bound to galactoglobulin-8. In some embodiments, the glycan component of the glycoligand is a glycan selectively bound to galactoglobulin-8.

[0397] In some embodiments, the glycan component of the glycoligand is a glycan bound to galactoglobulin-3 and galactoglobulin-8. In some embodiments, the glycan bound to galactoglobulin-3 and galactoglobulin-8 is H-13.

[0398] In some embodiments, the glycan component of the glycoligand is a glycan bound to Siglec-11. In some embodiments, the glycan component of the glycoligand is a glycan selectively bound to Siglec-11. In some embodiments, the Siglec-11-bound glycan is selected from H-35, H-36, H-37, H-38, H-39, H-40, H-41, H-42, H-43, and H-44.

[0399] In some embodiments, the glycan component of the glycoligand is a glycan bound to CD161. In some embodiments, the glycan component of the glycoligand is a glycan selectively bound to CD161. In some embodiments, the CD161-bound glycan is H-17, H-48, H-52, H-53, H-54, or H-55.

[0400] In some embodiments, the glycan component of the glycoligand is a Siglec-1-bound glycan. In some embodiments, the glycan component of the glycoligand is a Siglec-1-selectively bound glycan. In some embodiments, the Siglec-1-bound glycan is H-56.

[0401] In some embodiments, the glycan component of the glycoligand is a Siglec-2-bound glycan. In some embodiments, the glycan component of the glycoligand is a Siglec-2-selectively bound glycan. In some embodiments, the Siglec-2-bound glycan is H-57.

[0402] In some embodiments, the glycan component of the glycoligand is a Siglec-3-bound glycan. In some embodiments, the glycan component of the glycoligand is a Siglec-3-selectively bound glycan. In some embodiments, the Siglec-3-bound glycan is H-57.

[0403] In some embodiments, the glycan component of the glycoligand is a Siglec-4-bound glycan. In some embodiments, the glycan component of the glycoligand is a Siglec-4-selectively bound glycan. In some embodiments, the Siglec-4-bound glycan is H-58.

[0404] In some embodiments, the glycan component of the glycoligand is a Siglec-5-bound glycan. In some embodiments, the glycan component of the glycoligand is a Siglec-5-selectively bound glycan. In some embodiments, the Siglec-5-bound glycan is H-59.

[0405] In some embodiments, the glycan component of the glycoligand is a Siglec-7-bound glycan. In some embodiments, the glycan component of the glycoligand is a Siglec-7-selectively bound glycan. In some embodiments, the Siglec-7-bound glycan is H-60.

[0406] In some embodiments, the glycan component of the glycoligand is a Siglec-9-bound glycan. In some embodiments, the glycan component of the glycoligand is a Siglec-9-selectively bound glycan. In some embodiments, the Siglec-9-bound glycan is H-61.

[0407] In some embodiments, the glycan component of the glycoligand is a Siglec-10-bound glycan. In some embodiments, the glycan component of the glycoligand is a glycan selectively bound to Siglec-10. In some embodiments, the Siglec-10-bound glycan is H-62.

[0408] In some embodiments, the glycan component of the glycoligand is a CD28-bound glycan. In some embodiments, the glycan component of the glycoligand is a CD28-selectively bound glycan. In some embodiments, the CD28-bound glycan is one of K-1, K-2, K-3, K-4, K-5, K-6, K-7, K-8, K-9, K-10, K-11, K-12, and K-13.

[0409] In some embodiments, the glycan component of the glycoligand is a CD22-binding glycan. In some embodiments, the glycan component of the glycoligand is a CD22-selectively bound glycan. In some embodiments, the CD22-binding glycan is one of K-33, K-34, K-35, K-36, K-37, K-38, K-39, K-40, K-41, K-42, K-43, K-44, K-45, K-46, K-47, K-48, K-49, and K-50.

[0410] In some embodiments, the glycan component of the glycoligand is a CD83-bound glycan. In some embodiments, the glycan component of the glycoligand is a CD83-selectively bound glycan. In some embodiments, the CD83-bound glycan is one of K-14, K-15, K-16, and K-17.

[0411] In some embodiments, the glycan component of the glycoligand is a glycan that binds to KLRF1. In some embodiments, the glycan component of the glycoligand is a glycan that selectively binds to KLRF1. In some embodiments, the glycan that binds to KLRF1 is one of K-18, K-19, K-20, and K-21.

[0412] In some embodiments, the glycan component of the glycoligand is a CD93-binding glycan. In some embodiments, the glycan component of the glycoligand is a CD93-selectively bound glycan. In some embodiments, the CD93-binding glycan is one of K-31, H-37, H-35, and H-39.

[0413] In some embodiments, the glycan component of the glycoligand is a glycan that binds to DC-SignR. In some embodiments, the glycan component of the glycoligand is a glycan that selectively binds to DC-SignR. In some embodiments, the glycan that binds to DC-SignR is one of K-22, K-23, K-24, K-25, K-26, K-27, K-28, K-29, K-30, and K-31.

[0414] In some embodiments, the glycan component of the glycoligand is a glycan that binds to ASGR1. In some embodiments, the glycan component of the glycoligand is a glycan that selectively binds to ASGR1.

[0415] In some embodiments, the glycan component of the glycoligand is a glycan that binds to ASGR2. In some embodiments, the glycan component of the glycoligand is a glycan that selectively binds to ASGR2.

[0416] In some implementations, the glycan component contains one or more sialic acid moieties and facilitates penetration of the blood-brain barrier. Upregulation of sialyltransferases and excessive sialylation of the resulting tumor cell surface have been reported as established features of several cancers, including lung, breast, ovarian, pancreatic, and prostate cancer (Dobie et al., British Journal of Cancer, Vol. 124, pp. 76–90 (2021)). Excessive sialylation promotes tumor metastasis through several pathways, including enhancing immune evasion and tumor cell survival, and stimulating tumor invasion and migration. Bos et al., Nature, Vol. 459, pp. 1005–1009 (2009) reported that the epidermal growth factor receptor (EGFR) ligand HBEGF and α2,6-sialyltransferase ST6GALNAC5 are mediators for cancer cells crossing the blood-brain barrier. Sialinated glycans, including but not limited to sialic acid-Lewis x tetrasaccharide H-23, can facilitate the transport of payloads across the blood-brain barrier, thereby enabling the treatment of brain diseases and conditions. In some embodiments, the glycoligands of this disclosure can be used to treat brain diseases and conditions.

[0417] There are approximately 100 known glycan-binding receptors in the human body, indicating the type and selectivity of glycan receptor binding. [Taylor ME, Drickamer K, Schnaar RL, Etzler ME, and Varki A (2015) Discovery and classification of glycan-binding proteins. In Essentials of Glycobiology (AVarki, RD Cummings, JD Esko, P Stanley, GW Hart, M Aebi, AG Darvill, TKinoshita, NH Packer, JH Prestegard, RL Schnaar, and PH Seeberger, eds.), pp. 361–372. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.]

[0418] The four major classes of glycan-binding receptors contain different types of carbohydrate recognition domains (CRDs). These glycan-binding receptors include: Siglec, where the CRD is based on immunoglobulin folding; galactagglutinins, whose CRDs are formed by different β-sandwich folds; C-type lectins, where the sugar is directly linked to the calcium ion bound to the CRD; lectins containing R-type CRDs, whose structures are associated with the plant toxin ricin; and at least 10 additional structural classes of CRDs found in one or more mammalian glycan-binding receptors. [Taylor, ME and Drickamer, K. (2019) Mammalian sugar-binding receptors: known functions and unexplored roles. FEBS J, 286: 1800-1814].

[0419] Selectins are the best-characterized example to date of glycan-binding receptors that function in this role, mediating the initial transient interaction between leukocytes and endothelial cells, which causes leukocytes to roll along the endothelial surface [Taylor, ME and Drickamer, K. (2019) Mammalian sugar-binding receptors: known functions and unexplored roles. FEBS J, 286: 1800-1814].

[0420] Sialic acid-Lewis on endothelial cells at the site of inflammation x Tetrasaccharides serve as attachment sites for selectin-type C-type CRDs, mediating initial weak adhesion, which leads to leukocytes rolling along the endothelium [Taylor, ME and Drickamer, K. (2019) Mammalian sugar-binding receptors: known functions and unexplored roles. FEBSJ, 286: 1800-1814].

[0421] The molecular mechanism by which C-type CRDs interact with glycan ligands in the extracellular portion of each selectin involves sialic acid-Lewis. xThe fucose residues in the tetrasaccharide are directly linked to the conserved calcium ions specific to C-type CRDs, as well as having additional secondary interactions with other sugar residues in the tetrasaccharide [Taylor, ME and Drickamer, K. (2019) Mammalian sugar-binding receptors: known functions and unexplored roles. FEBS J, 286: 1800-1814].

[0422] The same phenotype was observed in mice lacking expression of the two GlcNAc-6-O-sulfotransferases, GlcNAc6ST-1 and GlcNAc6ST-2, which are sialic acid 6-sulfoLewis enzymes that produce L-selectin on high endothelial vein glycoproteins. x The glycan ligands required [Taylor, ME and Drickamer, K. (2019) Mammalian sugar-binding receptors: known functions and unexplored roles. FEBS J, 286: 1800-1814].

[0423] The transport of glycoproteins to the cell surface is facilitated by glycan-binding receptors in the endoplasmic reticulum-Golgi intermediate compartment, and the transport of hydrolases to lysosomes is directed by mannose-6-phosphate receptors [Taylor, ME and Drickamer, K. (2019) Mammalian sugar-binding receptors: known functions and unexplored roles. FEBS J, 286: 1800-1814].

[0424] Mannose receptors of oligomannose or high-mannose content. Patients with Gaucher disease (a lysosomal storage disorder) are now routinely and successfully treated with enzyme replacement therapy, in which the missing lysosomal hydrolases (with appropriate mannose-containing glycans) are injected into the bloodstream for uptake into macrophages via mannose [Taylor, ME and Drickamer, K. (2019) Mammalian sugar-binding receptors: known functions and unexplored roles. FEBS J, 286: 1800-1814].

[0425] ASGRs used to remove terminal sialic acid, such as galactose or GalNAc. Lewis x Trisaccharides are scavenger receptor C-type lectins found on glycoproteins released from secondary granules of neutrophils [Taylor, ME and Drickamer, K. (2019) Mammalian sugar-binding receptors: known functions and unexplored roles. FEBS J, 286: 1800-1814].

[0426] Glycoproteins bound to scavenger receptor C-type lectin (SRCL) are rapidly internalized into the cell and degraded. Therefore, SRCL appears to play a role similar to that of mannose receptors in clearing potentially dangerous glycoproteins released from sites of inflammation. [Taylor, ME and Drickamer, K. (2019) Mammalian sugar-binding receptors: known functions and unexplored roles. FEBS J, 286: 1800-1814].

[0427] Therapies targeting the desialyl glycoprotein receptor are also under development, which utilize the ability to control protein expression in hepatocytes by delivering interfering RNA molecules [Foster DJ, Brown CR, Shaikh S, Trapp C, Schlegel MK, Qian K, Sehgal A, Rajeev KG, Jadhav V, Manoharan M et al. (2018) Advanced siRNA designs further improve in vivo performance of GalNAc-siRNA conjugates. Mol Ther 26, 708–717.]. Understanding the glycoprotein turnover mechanism of the desialyl glycoprotein receptor also helps in the development of appropriate glycosylated therapeutic glycoproteins, such as erythropoietin, to ensure they have a suitable serum half-life [Taylor, ME and Drickamer, K. (2019) Mammalian sugar-binding receptors: known functions and unexplored roles. FEBS J, 286: 1800-1814.]. In addition to the C-type CRD that binds to mannose-containing oligosaccharides, the mannose receptor also contains the R-type CRD that selectively binds to the terminal 4-SO4-GalNAc [Taylor, ME and Drickamer, K. (2019) Mammalian sugar-binding receptors: known functions and unexplored roles. FEBS J, 286: 1800-1814].

[0428] Specific aspects of the glycan structure attached to glycoproteins can have a significant effect on their interaction with receptors. Glycoproteins formed by sialic acid linked to galactose or GalNAc residues in a 2-6 bond rather than a 2-3 bond can bind to receptors without removing sialic acid and are therefore constitutively cleared [Taylor, ME and Drickamer, K. (2019) Mammalian sugar-binding receptors: known functions and unexplored roles. FEBSJ, 286: 1800-1814].

[0429] The levels of these glycoproteins are increased in mice lacking the receptor. Highly branched triantennae and tetraantennae glycans have higher binding affinity to the receptor, which may result in a hierarchy of clearance rates [Taylor, ME and Drickamer, K. (2019) Mammalian sugar-binding receptors: known functions and unexplored roles. FEBS J, 286: 1800-1814].

[0430] The glycoligands of the present invention can interact with glycan-binding proteins, including signal transduction domains of T cells, B cells, NK cells, RBCs, macrophages, monocytes, platelets, granulocytes, γδT cells, other immune cells, and immunomodulatory cells.

[0431] Glycan-binding receptors

[0432] Glycan-binding receptors include immunotyrosine repressive motifs (ITIMs) in the cytoplasmic domains of many Siglec cells, such as CD22 on B lymphocytes [Taylor, ME, and Drickamer, K. (2019) Mammalian sugar-binding receptors: known functions and unexplored roles. FEBS J, 286: 1800-1814]. ITIMs interact with sialylated glycans (such as those on host cells) and then with SHP-1 phosphatase, thereby inhibiting B cell activation by modulating Ca2+-dependent signaling [Taylor, ME, and Drickamer, K. (2019) Mammalian sugar-binding receptors: known functions and unexplored roles. FEBS J, 286: 1800-1814]. This pathway may prevent targeting of widely sialylated self-antigens. Dendritic cell inhibitory receptors (DCIRs) function similarly and contain ITIM in their cytoplasmic domain, although in this case the extracellular sugar-binding domain is a C-type CRD and the bound ligand contains mannose [Taylor, ME and Drickamer, K. (2019) Mammalian sugar-binding receptors: known functions and unexplored roles. FEBS J, 286: 1800-1814].

[0433] C-type lectins mincle and dectin-2 on macrophages and blood dendritic cell antigen 2 (BDCA-2) on plasmacytoid dendritic cells lack signaling motifs but interact with common Fc receptor γ chains [Taylor, ME and Drickamer, K. (2019) Mammalian sugar-binding receptors: known functions and unexplored roles. FEBS J, 286: 1800-1814].

[0434] CRDs are typically rigid and their binding sites do not change upon ligand binding [Taylor, ME, and Drickamer, K. (2019) Mammalian sugar-binding receptors: known functions and unexplored roles. FEBS J, 286: 1800-1814]. Additionally, CRDs are usually spaced away from the cell surface by stem regions. Activation is more likely to involve induced interactions between multiple receptor peptides, whether dimers or larger clusters. One proposed way dimerization may initiate signaling is targeting dectin-1, as binding to β-glucan aggregates two receptor peptides together, thereby generating a fully functional ITAM from the semi-ITAM present in the cytoplasmic domain of each peptide [Taylor, ME, and Drickamer, K. (2019) Mammalian sugar-binding receptors: known functions and unexplored roles. FEBS J, 286: 1800-1814].

[0435] Galactolectins interact with glycosylated membrane receptors, providing an alternative model for how glycan-binding proteins can regulate signal transduction [Taylor, ME, and Drickamer, K. (2019) Mammalian sugar-binding receptors: known functions and unexplored roles. FEBS J, 286: 1800-1814]. Galactolectins are typically at least divalent, either because of the presence of tandem CRDs within a single polypeptide or because non-covalent oligomers are formed from individual CRDs [Taylor, ME, and Drickamer, K. (2019) Mammalian sugar-binding receptors: known functions and unexplored roles. FEBS J, 286: 1800-1814]. At the cell surface, polyvalent galactolectins can aggregate glycoproteins to form lattices, thereby stimulating or inhibiting signal transduction. For example, crosslinking of galactoglobulin-1 with CD45 leads to activation of the phosphatase domain in the receptor's cytoplasmic domain, thereby modulating T cell responses such as apoptosis [Taylor, ME and Drickamer, K. (2019) Mammalian sugar-binding receptors: known functions and unexplored roles. FEBS J, 286: 1800-1814]. Conversely, lattice formation between polygalactoglobulin and T cell receptors with multiple glycans prevents the tight aggregation of the receptor polypeptide's cytoplasmic domain, thereby increasing the threshold for antigen activation of the receptor [Taylor, ME or Drickamer, K. (2019) Mammalian sugar-binding receptors: known functions and unexplored roles. FEBS J, 286: 1800-1814].

[0436] CRDs in pathogen-binding receptors typically have extended binding sites that bind common disaccharide motifs, such as Manα1-2Man (a common terminal structure on mannans in yeast and other fungi) or GlcNAcβ1-2Man (exposed to unprocessed viral glycans) [Taylor, ME and Drickamer, K. (2019) Mammalian sugar-binding receptors: known functions and unexplored roles. FEBS J, 286: 1800-1814]. Some CRDs have even more extended sugar-binding sites, such as the cleft in the CRD of DC-SIGN, which binds several mannose residues in the high-mannose oligosaccharide present on the HIV surface [Taylor, ME and Drickamer, K. (2019) Mammalian sugar-binding receptors: known functions and unexplored roles. FEBS J, 286: 1800-1814].

[0437] In a preferred aspect, the present invention provides a glycoligand composition that acts as a direct ligand for the Siglec receptor and modulates immune regulation in a subject. In some embodiments, a negatively charged or enriched glycan comprising one or more sialic acid residues mediates the binding of the glycan receptor to a Siglec active site containing a conserved arginine residue.

[0438] In a further aspect, the glycoligands of the present invention are characterized by positive or negative regulatory factors on the target receptor. Elimination of N-glycosylation of CD28 expressed on T cells significantly increases the binding of CD28 to CD80 and amplifies downstream signal activation, indicating that N-linked glycosylation negatively regulates CD28 function. (Ma, Bruce Y, et al., “CD28 T cell costimulatory receptor function is negatively regulated by N-linked carbohydrates.” Biochemical and biophysical research communications, Vol. 317, 1(2004): 60-7.) Furthermore, the glycoligands of the present invention are characterized by exhibiting bidirectional regulation. (Nitschke L, Carsetti R, Ocker B, Köhler G, Lamers MC (February 1997). "CD22 is a negative regulator of B-cell receptor signalling.")

[0439] The binding of glycoligands can mediate or trigger intracellular signaling in Siglec cells by contacting desired glycans that are oriented in a specific way, leading to the aggregation of signaling proteins. Glycoligands can bind in a specific orientation or conformation, or bind to multiple receptors to mediate biological effects. Therefore, a preferred embodiment of the invention provides glycoligands that bind to target receptors via glycan-glycan interactions. For example, the glycan on the glycoligand interacts with agglutinin or a glycan on the receptor.

[0440] Siglec receptors are expressed in various cell types, including but not limited to macrophages, monocytes, B cells, Schwann cells, ODCs, DCs, osteoclasts, MyPro cells, monocytes, granulocytes, microglia, mast cells, neutrophils, trophoblasts, NK cells, T cells, eosinophils, basophils, platelets, and glycoligands. Preferably, the glycoligands of the present invention regulate one or more of the following siglec receptors: Siglec-2, Siglec-3, Siglec-4A, Siglec-5, Siglec-6, Siglec-7, Siglec-8, Siglec-9, Siglec-10, Siglec-11, Siglec-14, and Siglec-16. Similarly, the glycoligands of the present invention also regulate CD33 and conserved siglec receptors, including sialic acid adhesion, MAG, CD22, and Siglec-15.

[0441] The glycoligands of the present invention may also include a targeting group, such as a cell or tissue targeting agent or group, such as a lectin, glycoprotein, lipid, or protein, such as an antibody, which binds to a specific cell type (such as kidney cells). The targeting group may be thyroid-stimulating hormone, melanocyte-stimulating hormone, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polylactose, polygalactose, N-acetylgalactosamine, N-acetylglucosamine, polymannose, polyfucose, glycosylated polyamino acids, polygalactose, transferrin, bisphosphonates, polyglutamic acid, polyaspartic acid, lipids, cholesterol, steroids, bile acids, folic acid, vitamin B12, biotin, RGD peptide, RGD peptide mimic, or aptamer.

[0442] Targeting groups can be proteins, such as glycoproteins; or peptides, such as molecules with a specific affinity for a coligand; or antibodies, such as antibodies that bind to specific cell types (such as cancer cells, endothelial cells, or osteoblasts). Targeting groups can also include hormones and hormone receptors. They can also include non-peptide substances such as lipids, lectins, carbohydrates, vitamins, cofactors, polylactose, polygalactose, N-acetylgalactosamine, N-acetylglucosamine, polymannose, polyfucose, or aptamers.

[0443] The targeting group can be any ligand capable of targeting a specific receptor. Examples include, but are not limited to, folic acid, GalNAc, galactose, mannose, mannose-6P, aptamers, integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands. In a particular embodiment, the targeting group is an aptamer. The aptamer can be unmodified or can have any combination of modifications disclosed herein.

[0444] In other embodiments, the glycoligand is covalently conjugated to the cell-penetrating peptide. The cell-penetrating peptide may also contain a signaling sequence. The conjugates of the present invention can be designed to have increased stability; increased cell transfection rate; and / or altered biodistribution (e.g., targeting specific tissues or cell types).

[0445] Conjugation groups can be added to antibodies that interact with glycans, enabling the conjugation groups to tag targets for clearance. Such tagging molecules include, but are not limited to, ubiquitin, fluorescent molecules, human influenza hemagglutinin (HA), c-myc [a 10-amino acid segment of the human proto-oncogene myc, sequence EQKLISEEDL (SEQ ID NO: 10)], histidine (His), flag [a short peptide with the sequence DYKDDDDK (SEQ ID NO: 11)], glutathione S-transferase (GST), V5 (a paramyxoviral epitope of simian virus 5), biotin, avidin, streptavidin, horseradish peroxidase (HRP), and digoxigenin.

[0446] In some implementations, antibodies that interact with glycans can be combined with another molecule or other molecules to treat diseases or conditions.

[0447] In some embodiments, the glycoligand composition binds to the ligand-binding domain, α, and / or β subunits of a chimeric antigen receptor (CAR) or T-cell receptor (TCR). The CAR and TCR may comprise an antigen-binding domain, a transmembrane domain, and an intracellular domain. In some embodiments, the glycoligand composition binds to one or more antigen-binding proteins comprising an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the antigen-binding domain is linked to a transmembrane domain, which in turn links to an intracellular signaling domain, thereby creating a chimeric antigen receptor. In some embodiments, the antigen-binding domain binds to a tumor antigen, a tolerogen, or a pathogen antigen, or the antigen is a tumor antigen or a pathogen antigen. In some embodiments, the antigen-binding domain is an antibody or an antibody fragment thereof (e.g., scFv, Fv, Fab, dAb). In some embodiments, the antigen-binding domain is a bispecific antibody.

[0448] In some embodiments, the bispecific antibody has a first immunoglobulin variable domain that binds to a first epitope and a second immunoglobulin variable domain that binds to a second epitope. In some embodiments, the first and second epitopes are the same. In some embodiments, the first and second epitopes are different. In some embodiments, a transmembrane domain connects the binding domain and the intracellular signal transduction domain. In some embodiments, the transmembrane domain is a hinge protein (e.g., an immunoglobulin hinge), a peptide linker (e.g., a GS linker), a KIR2DS2 hinge, a CD8a hinge, or a spacer.

[0449] In some embodiments, the co-stimulatory intracellular signal transduction domain comprises one or more of the following: TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, signal transduction lymphocyte activation molecule, or activated NK cell receptor protein. In some embodiments, the co-stimulatory intracellular signal transduction domain comprises one or more of the following: CD27, CD28, 4-1BB, OX40, GITR, CD30, CD40, PD-1, ICOS, BAFFR, HVEM, ICAM-1, LFA-1, CD2, CDS, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, CD19, CD4, CD8α. CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6, VLA6, CD49f, ITGAD, CD103, ITGAL, ITGAM, ITGAX, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE / TRANKL, CD226, SLAMF4, CD84, CD96, CEACAM1, CRTAM, CD229, CD160, PSGL1, CD100, CD69, SLAMF6, SLAMF1, SLAMF8, CD162, LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, B7-H3, or ligands bound to CD83.

[0450] In some embodiments, the intracellular signaling domain comprises at least a portion of a T cell signaling molecule. In some embodiments, the intracellular signaling domain comprises an activation motif based on an immune receptor tyrosine residue. In some embodiments, the intracellular signaling domain comprises at least a portion of CD3ζ, the common FcRγ (FCER1G), FcγRlla, FcRβ (Fcε Rib), CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10, DAP12, or any combination thereof. In some embodiments, the intracellular signaling domain further comprises a co-stimulatory intracellular signaling domain.

[0451] Specific cell-targeting ligands that deliver other bioactive molecules to specific target cells

[0452] In other respects, the pharmaceutical composition further comprises a target or effector (e.g., a bioactive molecule) associated with or operatively linked to a nucleic acid molecule conjugated to a glycan. For example, a radioligand, toxin, enzyme, protein, or peptide may be conjugated to a glycan ligand.

[0453] In some embodiments, the glycoligand composition is modified 1-2 times by using stop codon inhibition or by incorporating non-natural amino acids to create a single conjugation site or to make each amino acid a conjugation site so as to enzymatically conjugate with one or more proteins.

[0454] In other embodiments, the glycoligand composition is conjugated to one or more proteins via chemical synthesis. These glycoligand compositions are programmable when conjugated to peptides having <12 amino acids, but producing correctly folded long peptides is a challenge. Preferably, the conjugated peptide should fold correctly.

[0455] In other embodiments, the glycoligand composition is enzymatically configured onto nucleic acids, which may contain modified nucleosides for transcription or linked to long RNA. In yet another embodiment, the glycoligand composition is conjugated (<120 nt) via programmable chemical synthesis, and the specific structure and orientation of the glycan for receptor binding can be defined. In one embodiment, the glycoligand is in a specific orientation or conformation to facilitate binding to one or more receptors.

[0456] In a preferred embodiment, the glycoligand is operatively linked to one or more bioactive molecules to bind to target cells. In some embodiments, the bioactive molecules comprise toxins such as azaribine, anastrozole, azacitidine, bleomycin, bortezomib, bryostatin-1, busulfan, camptothecin, 10-hydroxycamptothecin, carmustine, celecoxib, chlorambucil, cisplatin, irinotecan, carboplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, docetaxel, dactinomycin, dactinomycin gluconate, daunorubicin, dexamethasone, diethylstilbestrol, and doxorubicin. (doxorubicin), doxorubicin glucoside, epirubicin, ethinyl estradiol, estramustine, etoposide, etoposide glucoside, fluorouracil, fludarabine, flutamide, fluorouracil, fluorometholone, gemcitabine, progesterone caproate, hydroxyurea, idarubicin, ifosfamide, folate, lomustine, nitrogen mustard, medroxyprogesterone acetate Acetate), medroxyprogesterone acetate, melphalan, mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, phenyl butyrate, prednisone, procarbazine, paclitaxel, pentostatin, semustine, streptozocin, tamoxifen, taxane, paclitaxel, testosterone propionate, thalidomide, thioguanine, thiotepa, teniposide, topotecan, uracil mustard, vinblastine, vinorelbine, and vincristine.In other embodiments, the bioactive molecules include enzymes such as glycosidases, including sialidase, galactosidase, hexosamine diase, fucosidase, mannosidase, PNGase, etc. In some embodiments, the bioactive molecules include proteins and peptides.

[0457] Glycoligand analysis

[0458] Glycoligands can be analyzed using MALDI-TOF-MS, NMR spectroscopy, glycosidase degradation, and other known methods in glycobiology.

[0459] Glycans are typically purified from culture media by chromatography, and then released using glycosidases such as peptidyl-N-glycosidase F (PNGase F). The glycans are detected by MALDI-TOF-MS, as described in Example 4. Generally, the quality of a particular glycan is related to its structure (+ / - ionization).

[0460] Since MS measurements of glycans only provide the mass of ionized glycans, the structure of specific hexosylglycans cannot be identified without glycoside analysis. Therefore, NMR is used to detect glycoside linkages, i.e., specific glycan linkages (α, β) between glycan structures. The NMR scheme and analytical methods are adapted from Gao et al.

[0461] 1 H and 13 C10 NMR spectra were recorded on a Bruker Avance II 600 MHz and an Agilent 700 MHz NMR magnet system. The compounds were exchanged three times with heavy water before analysis following redissolution with heavy water. The resulting glycan / glycan conjugates were characterized as follows: chemical shift (parts per million (ppm), with water as an internal standard), multiplicity (s = singlet, d = doublet, t = triplet, dd = doublet, m = multiplet and / or multiple resonances), coupling constant (Hertz (Hz)), and integral. All NMR signals were based on... 1 H NMR, 1 H- 1 H COSY、 1 H- 1 H TCOSY and 1 H- 13 The C HSQC experiment was assigned.

[0462] Cell-based assays

[0463] This article also provides methods for detecting the bioactivity of glycoligands and the interaction between glycoligands and cell surface proteins of target cells.

[0464] In some embodiments, the glycoligands provided herein are characterized by enzyme-linked lectin assays, fluorescence-based solid-phase assays, or cell-based assays. Cell-based assays can be performed in vitro (using cultured cells) or in vivo. For example, the cells used in cell-based assays may express one or more target receptors recognized by one or more glycoligands of the present invention. The target receptors may be naturally expressed by such cells or may be induced to express one or more desired target receptors. Induced expression may be performed by upregulating the expression of genes of proteins that regulate receptors through one or more therapeutic methods. In some embodiments, induced expression may include transfection, transduction, or other forms of introduction of one or more genes or transcripts to endogenously overexpress cell surface proteins involved in receptor regulation.

[0465] In some implementations, cell-based assays may include the use of cancer cells, macrophages, microglia, neutrophils, monocytes, B cells, T cells, NK cells, and eosinophils.

[0466] In some embodiments, cell-based assays may include the use of cancer cells that express or are induced to express the target receptor. Alternatively, cancer cell lines, representing cancer stem cells (CSCs), may be used to test the glycoligands of the present invention.

[0467] In some implementations, ovarian cancer cell lines may be used. Such cell lines may include, but are not limited to, SKOV3, OVCAR3, OV90, and A2870 cell lines. In some cases, CSC cells can be isolated from these cell lines by isolating cells expressing the cellular markers CD44 and / or CD133.

[0468] OVCAR3 cells were initially established using malignant ascites obtained from patients with progressive ovarian adenocarcinoma (Hamilton, TC et al., 1983. Cancer Res. 43: 5379-89). Cancer stem cell populations can be isolated from OVCAR3 cell cultures by selection based on specific cell surface markers such as CD44 (involved in cell adhesion and migration), CD133, and CD117 (Liang, D. et al., 2012. BMC Cancer. 12: 201, the contents of which are incorporated herein by reference in their entirety). OV90 cells are epithelial ovarian cancer cells, similarly derived from human ascites (see U.S. Patent No. 5,710,038). OV-90 cells also express CD44 upon activation (Meunier, L. et al., 2010. Transl Oncol. 3(4): 230-8).

[0469] In some implementations, cell lines derived from gastric cancer may be used. Such cell lines may include, but are not limited to, SNU-16 cells (see the description in Park JG et al., 1990. Cancer Res. 50: 2773-80, the contents of which are incorporated herein by reference in their entirety). SNU-16 cells naturally express STn, but at low levels.

[0470] Treatment

[0471] Methods for treating diseases or disorders are also provided, comprising administering to a subject in need a therapeutically effective amount of a pharmaceutical composition comprising the glycoligands described herein. In some embodiments, wherein the synthetic scaffold domain is or comprises a therapeutic polynucleotide, such as mRNA or siRNA, the present disclosure contemplates administering to a subject a therapeutically effective amount of glycoRNA such that one or more glycan moieties enable and facilitate the delivery of the therapeutic polynucleotide to an organ or cell of interest. In some embodiments, one or more glycan moieties result in increased delivery efficiency of the therapeutic polynucleotide (and thus a greater therapeutic effect) compared to a nonfunctionalized analogue. In some embodiments, the disease or disorder is any disease or disorder treatable by a therapeutic polynucleotide. Example diseases and disorders treatable by the methods of the present disclosure include, but are not limited to, cancer, metabolic diseases, coagulation disorders, anticoagulation disorders, autoimmune diseases, and infections (e.g., viral infections, bacterial infections).

[0472] Also provided are glycoligands of this disclosure for use in the manufacture of pharmaceutical agents for treating diseases or disorders. Further provided are methods for treating a disease or disorder in a subject in need using the pharmaceutical compositions disclosed herein.

[0473] carriers and delivery media

[0474] Vectors are also provided, including expression vectors containing the nucleic acid molecules of the present invention, as further described herein. In a first embodiment, the vector contains the isolated nucleic acid molecules described above. In an alternative embodiment, the vector of the present invention contains the nucleic acid molecules described above operably linked to one or more expression control sequences. Thus, the vectors of the present invention can be used to express polypeptides. Vectors for expressing nucleic acids are well known in the art.

[0475] In another aspect of the invention, delivery of the glycoligand comprises a non-viral composition. In some embodiments, the delivery medium comprises nanoparticles, lipids, lipid-based nanoparticles, and polymers, said delivery medium containing the nucleic acid molecule of the invention, wherein one or more mediators carry the glycan-conjugated nucleic acid sequence of the invention.

[0476] Delivery media are selected based on lower toxicity and immunogenicity, improved half-life, increased stability, and efficiency.

[0477] Combination with other drugs

[0478] In one embodiment, the present invention relates to a method for killing cancer cells in a subject by administering a therapeutically effective amount of glyconucleic acid (such as glycoRNA and glycoDNA) to the subject. In one aspect of this embodiment, the glyconucleic acid (such as glycoRNA and glycoDNA) is administered intravenously to the subject. In another aspect of this embodiment, the glyconucleic acid (such as glycoRNA and glycoDNA) is administered into the tumor of the subject. In yet another aspect of this embodiment, the glyconucleic acid (such as glycoRNA and glycoDNA) is applied near the tumor or administered systemically via a medium that allows delivery to the tumor.

[0479] In another embodiment, the present invention relates to a method for treating a subject's cancer by administering a therapeutically effective amount of glyconucleotides (such as glycoRNAs and glycoDNAs) to the subject. In one aspect of this embodiment, the glycoRNA is administered intravenously to the subject. In another aspect of this embodiment, the glycoRNA is administered into the subject's tumor. In yet another aspect of this embodiment, the glycoRNA is applied near the tumor or administered systemically via a medium that allows delivery to the tumor.

[0480] Cancer (and cancer cells) is any cancer that invades a subject's body. Such cancers include liver cancer, colon cancer, pancreatic cancer, lung cancer, and bladder cancer. Liver cancer can be primary liver cancer or cancer that has metastasized to the liver from another tissue. Primary liver cancer includes hepatocellular carcinoma and hepatoblastoma. Metastatic liver cancer includes colon cancer and pancreatic cancer.

[0481] In one embodiment, the present invention relates to a method for killing cancer cells in a subject by administering a therapeutically effective amount of an immune checkpoint inhibitor and a therapeutically effective amount of a glyconucleic acid (such as glycoRNA and glycoDNA). In one aspect of this embodiment, administering the immune checkpoint inhibitor together with the glyconucleic acid (e.g., glycoRNA) increases the efficacy of the glyconucleic acid (e.g., glycoRNA).

[0482] In another embodiment, the present invention relates to a method of treating a subject's cancer by administering to a subject a therapeutically effective amount of an immune checkpoint inhibitor and a therapeutically effective amount of a glyconucleic acid (such as glycoRNA and glycoDNA). In one aspect of this embodiment, administering the immune checkpoint inhibitor together with the glyconucleic acid (e.g., glycoRNA) increases the efficacy of the glyconucleic acid (e.g., glycoRNA).

[0483] As described above, immune checkpoint inhibitors and glyconucleotides (such as glycoRNAs and glycoDNAs) are administered intravenously to a subject, administered into a subject's tumor, said immune checkpoint inhibitors and glyconucleotides are applied near the tumor, or administered systemically via a medium that allows delivery to the tumor. In one aspect of this embodiment, the immune checkpoint inhibitor is a monoclonal antibody that blocks the interaction between receptors (such as PD-1, PD-L1, CTLA4, Lag3, and Tim3) and ligands of those receptors on mammalian cells (such as human cells). In a particular aspect, the monoclonal antibody is a monoclonal antibody targeting PD1 or PDL1. Examples of monoclonal antibodies include atezolizumab, durvalumab, pembrolizumab, and ipilimumab.

[0484] In another aspect of this embodiment, immune checkpoint inhibitors are small molecules that block the interaction between receptors (such as PD-1, PD-L1, CTLA4, Lag3, and Tim3) and ligands of those receptors on mammalian cells (such as human cells). In a specific aspect, the small molecule blocks the binding between PD1 and PDL1. BMS202 and similar ligands are examples of such small molecules. Immune checkpoint inhibitors administered together with glyconucleotide molecules (such as glycoRNAs and glycoDNAs) are monoclonal antibodies or small molecules as described above. They can be administered before, after, or simultaneously with the glyconucleotide molecule combination.

[0485] In another embodiment, this pharmaceutical composition is used in combination with an immune checkpoint inhibitor as described herein. Therefore, this embodiment of the invention relates to a combination of therapeutic agents comprising an immune checkpoint inhibitor and a pharmaceutical composition comprising glyconucleic acid (such as glycoRNA and glycoDNA) in a pharmaceutically acceptable carrier as described herein.

[0486] In another embodiment, the pharmaceutical composition comprising glyconucleotides (such as glycoRNA and glycoDNA) is used in combination with a chemotherapeutic agent. Illustrative examples of chemotherapeutic agents that can be administered with the pharmaceutical composition and have cytotoxic effects include: azaribine, anastrozole, azithromycin, bleomycin, bortezomib, lichenin-1, busulfan, camptothecin, 10-hydroxycamptothecin, carmustine, celecoxib, chlorambucil, cisplatin, irinotecan, carboplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, docetaxel, dextrin, dextrin gluconate, daunorubicin, dexamethasone, diethylstilbestrol, doxorubicin, doxorubicin gluconate, epirubicin, ethinyl estradiol, estradiol, etoposide, etoposide gluconate, and fluorine. Urate, fludarabine, flutamide, fluorouracil, fluorometholone, gemcitabine, progesterone caproate, hydroxyurea, idarubicin, ifosfamide, leucovorin, lomustine, nitrogen mustard, degarelix acetate, megestrol acetate, melphalan, mercaptopurine, methotrexate, mitoxantrone, styromycin, mitomycin, mitotane, phenylbutyrate, prednisone, procarbazine, paclitaxel, pentostatin, semustine, streptozotocin, tamoxifen, taxane, paclitaxel, testosterone propionate, thalidomide, thioguanine, thiotepa, teniposide, topotecan, uracil nitrogen mustard, vinblastine, vinorelbine, and vincristine.

[0487] In some implementation schemes, the chemotherapeutic agents are selected from the group consisting of: panobinostat, actinomycin, all-trans retinoic acid, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel, 5-fluorouracil, deoxyfluorouridine, doxorubicin, epirubicin, adriamycin, and epothilone. The following are listed: othilone, etoposide, fluorouracil, gemcitabine, hydroxyurea, idarubicin, imatinib, irinotecan, nitrogen mustard, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, teniposide, thioguanine, topotecan, valrubicin, vemurafenib, vinblastine, vincristine, vindesine, vinorelbine, and hydroxycamptothecin.

[0488] In some implementation schemes, the chemotherapy agents are selected from the group consisting of: docetaxel, pabistat, 5-fluorouracil, paclitaxel, cisplatin, irinotecan, topotecan, and etoposide.

[0489] If desired, the therapeutic component (such as a radioisotope, chemotherapy agent, or any therapeutic agent disclosed herein) may be conjugated to a glyconucleotide (such as glycoRNA and glycoDNA). If desired, the glyconucleotide (such as glycoRNA and glycoDNA) may be conjugated to a targeting antibody or antibody fragment. This enhances the targeting of the glyconucleotide to the desired cell or organ and may further stabilize (e.g., increase serum half-life) the glyconucleotide.

[0490] "Chemotherapy agents" are biological (macromolecule) or chemical (small molecule) compounds that can be used to treat cancer. Types of chemotherapeutic drugs include, but are not limited to, histone deacetylase inhibitors (HDACIs), alkylating agents, antimetabolites, alkaloids, cytotoxic / anticancer antibiotics, topoisomerase inhibitors, tubulin inhibitors, proteins, antibodies, and kinase inhibitors.

[0491] Chemotherapy agents include compounds used in targeted therapies and non-targeted compounds used in conventional chemotherapy. Non-limiting examples of chemotherapeutic agents include: erlotinib, afatinib, docetaxel, doxorubicin, 5-FU (5-fluorouracil), pabistat, gemcitabine, cisplatin, carboplatin, paclitaxel, bevacizumab, trastuzumab, pertuzumab, metformin, temozolomide, tamoxifen, doxorubicin, rapamycin, lapatinib, hydroxycamptothecin, and trimetinib. Further examples of chemotherapy drugs include: oxaliplatin, bortezomib, sunitinib, letrozole, imatinib, PI3K inhibitors, fulvestrant, leucovorin, lonafarnib, sorafenib, gefitinib, crizotinib, irinotecan, topotecan, valrubicin, vemurafenib, telbivinib, capecitabine, vandetanib, chlorambucil, panitumumab, cetuximab, rituximab, and tosimomab. temsirolimus, everolimus, pazopanib, canfosfamide, thiotepa, cyclophosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; ethyleneimine, phenyldopa, carboquinone, meturedopa, uredopa, methylmelamine (including atratamine), triethylenemelamine, triethylphosphamide, triethylthiophosphamide, and trimethylenemelamine; boletine, bolete; lichenin; callystatin, CC-1065 (including its synthetic analogues adozelesin, carzelesin, and bizelesin)); cryptophycin (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogues KW-2189 and CB1-TM1);Eleutherobin; pancratistatin, sarcodictyin, spongistatin; nitrogen mustards, such as chlorambucil, naphazoline, cyclophosphamide, estradiol, ifosfamide, bis(chloroethyl)methylamine, nitric oxide (melphalan, novembizine, phenethylamine, prednimustine, trophosphamide, uramustine); nitrosoureas, such as carmustine, chlorzotocin, fotemustine, lomustine, nimustine. tine), ranimnustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, calicheamicin γ1I, calicheamicin ωI1, dynemicin, dynemicin A; bisphosphonates, such as clophosphonate, esperamicin and neocarcinogens; chromophores and related chromogens of enediyne antibiotics), aclacinomysins, actinomycins, all-trans retinoic acid, autramycin, azaserine, bleomycin, actinomycin C, kalabi Carabicin, carminomycin, carzinophilin, chromomycinis, actinomycin D, daunorubicin, deoxyfluorouracil, detorubicin, 6-diazo-5-oxo-L-leucine, morpholino-doxorubicin, cyano-morpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogamycin (nogalamycin), olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercindin, ubenimex, zinostatin, zorubicin; antimetabolites, such as methotrexate;Folic acid analogs include dimethylfolate, methotrexate, pteropterin, and trimetrexate; purine analogs include fludarabine, 6-mercaptopurine, methotrexate, thiamiprine, and thioguanine; pyrimidine analogs include ancitabine, azacitidine, azathioprine, bleomycin, 6-nitrouridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and fluorouridine; and androgens include calusterone and dromostanolone. Propionate, epitiostanol, mepitiostane, testolactone; anti-adrenergic agents, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as folinic acid; aceglatone; aldehyde phosphoramide glycoside; aminolevulinic acid; eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elfornithine, elliptinium acetate), epothilone, etoglucid; gallium nitrate; hydroxyurea; lentinan, lonidainine, maytansinoid, maytansine, ansamitocin, mitoguazone, mitoxantrone, mopidamol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, podophyllinic; 2-ethylhydrazine;Procarbazine, PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.), razoxane, rhizoxin, sizofiran, spirogermanium, tenuazonic acid, triadimefon; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin). A and anguidine; ethyl carbamate, vinorelbine, dacarbazine, mannomustine; dibromomannitol; dibromoeusol; piperobromo, cytosine, arabinoside (“Ara-C”); cyclophosphamide; thiotepa; guanine; 6-mercaptopurine; methotrexate; vinblastine; etoposide, ifosfamide, mitoxantrone, vincristine, vinorelbine, novanone; emetrexate; teniposide, edaraxazole, daunorubicin; aminopterin; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; DMFO, retinoids, such as retinoic acid; and pharmaceutically acceptable salts or derivatives thereof.

[0492] Target biology

[0493] In various aspects, the pharmaceutical compositions produced by the methods of the present invention are used as therapies for treating diseases or health conditions. Such diseases or health conditions include, but are not limited to, autoimmune diseases, autoantibody-mediated diseases, complement dysregulation-related diseases, immune complex-related diseases, amyloidosis, diseases associated with infectious pathogens or pathogens (e.g., bacterial, fungal, viral, parasitic infections), diseases associated with toxic proteins, diseases associated with lipid accumulation, diseases associated with apoptosis, necrosis, abnormal or carcinogenic mammalian cells, metabolic diseases, and rare congenital disorders.

[0494] In some respects, the glycoligand binds to at least one of the following receptors: lectin, galactose, DC-SIGN, GLUT transporter, Gp120, and SIGN-R-1. In other respects, targets include macrophages, liver, glioma, inflammation, and antitumor immune responses.

[0495] An additional aspect of this invention considers glycan matrices as signaling molecules to modulate one or more desired receptors in target host cells, thereby mediating biological effects. Such glycoligands directly contact or bind to receptors on target cells. In some cases, the glycoligands are internalized within the target cells. Defined matrices that produce a variety of specific glycoligand structures can be deployed for studying one or more targets to determine receptor binding affinity, specificity, pharmacokinetic properties (half-life), and subsequent biological effects.

[0496] Glycoligand structures can also bind to receptors and then be internalized to express the payload. For example, as previously demonstrated with GalNAc-conjugated siRNA molecules (e.g., Givacsilan), targeted delivery of GalNAc-conjugated siRNAs includes hepatocytes. In such cases, tri-GalNAc-conjugated siRNA binds to the desialylate glycoprotein receptor (ASGPR) and then undergoes endocytosis. GalNAc residues are released or dissociated from the ASGPR, where the glycan is degraded in lysosomes, and the ASGPR is recovered to the cell surface. Similarly, in some embodiments, methods and compositions target various glycoligands to one or more receptors.

[0497] In some implementations, the mRNA is translated once the synthetic scaffold domain (e.g., mRNA) dissociates in the cytoplasm. See Aaron D. Springer and Steven F. Dowdy. Nucleic Acid Therapeutics. June 2018. 109-118.

[0498] In a preferred embodiment, the glycoligand is specific to the receptor and exhibits a nanomolar or picomolar binding affinity constant (e.g., 10) for the target antigen. 9 M, 10 10 M, 10 11 M, 10 12 M, 10 13 M or more compact). Use typical conventional analytical techniques, such as surface plasmon resonance (SPR) BIAcore™ instruments.

[0499] Therefore, the products of this invention can be used directly or with minimal processing for research, diagnostic, and therapeutic purposes. The glycoligands of this invention can be used as reagents in immunoassays, radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), or protein arrays.

[0500] Preferred applications include cell-cell interactions and / or cell-cell communication mediated by glycoligands.

[0501] The present invention provides, in various aspects, conserved small non-coding RNAs operably linked to sialylated and / or fucoidan glycans, glycans enriched with sialic acid and / or fucose residues, and synthetic glycans displaying terminal sialic acid and / or fucose residues. Additional embodiments include small non-coding RNAs operably linked to at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher amounts of sialylated glycans. Further embodiments include small non-coding RNAs operably linked to at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher amounts of fucoidan glycans. Still other embodiments include small non-coding RNAs operably linked to at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or higher amounts of both sialylated and fucoidan glycans.

[0502] Certain aspects of the present invention provide glycoligands that mediate cell-cell interactions or cell-cell communication. In some preferred embodiments, the glycoligands of the present invention mediate the binding of glycosylated RNA, glycosylated lipids, and / or glycoproteins to the cell surface of target cells.

[0503] target cell surface proteins

[0504] In some respects, the glycoligand composition is applied to one or more target cells, including but not limited to macrophages, monocytes, B cells, Schwann cells, ODCs, DCs, osteoclasts, MyPro cells, monocytes, granulocytes, microglia, mast cells, neutrophils, trophoblast cells, NK cells, T cells, eosinophils, basophils, and platelets.

[0505] Siglec is a class of receptor molecules expressed on various cell types and can be drug-treated via glycoligands. Flynn et al. (2021) showed that glycoRNAs can attach to two specific sialic acid-binding immunoglobulin-type lectins (Siglecs) associated with a family of immune receptors linked to several diseases, including systemic lupus erythematosus (SLE), suggesting that glycoRNAs may be involved in immune signaling. Flynn et al., (2021), Cell 184(12): 3109-3124.

[0506] Therefore, glycoligand compositions are applied to bind to one or more Siglec receptors. Certain glycoligand compositions are considered to have specificity and bioactivity for modulating one or more Siglec receptors.

[0507] In various aspects, glycoligand compositions are used as delivery mediators to deliver payloads. For example, a glycoligand composition binds to a receptor (e.g., CD22), thereby leading to the internalization and function of nucleic acids. In one embodiment, the glycoligand composition binds to a receptor to form a dimer complex, wherein the complex is internalized, the glycoligand is released and activated, and then the receptor is recovered. In such embodiments, the glycoligand includes, but is not limited to, mRNA, siRNA, ASO, and circRNA. In related embodiments, the glycoligand composition further comprises a conjugation to a toxin or radioactive nucleotide and binds to a receptor on a target cell, killing the target cell. In other embodiments, the glycoligand composition comprises one or more sequences encoding peptides delivered to the target cell.

[0508] Glycoligand preparations

[0509] The pharmaceutical composition can be formulated based on the desired route of administration.

[0510] Several factors are considered when determining the amounts of each component in the glycoligand formulation of the present invention. The glycoligand composition may contain single-stranded or double-stranded RNA, which may be linear or cyclic. For example, the synthetic scaffold containing RNA may comprise 50% of the pharmaceutical composition of the final product. In some cases, approximately 100% of the RNA is operatively linked to one or more glycans. Such glycans may be a single glycoform or a mixture of one or more glycoforms. Furthermore, the glycoligand composition may contain excipients, lyophilized using mannitol, as preservatives and stabilizers, to minimize degradation and precipitation, and to readily reconstitute in liquid for subcutaneous or intradermal administration via injectable microneedles.

[0511] In addition to the many advantages of this invention in alleviating diseases such as cancer, inflammatory diseases, and autoimmune diseases, other advantages of the pharmaceutical compositions disclosed herein include: improved stability, improved PK / PD, resistance to protease degradation, increased half-life, cold chain storage and distribution capability, configurability and programmability, specific orientation leading to signal transduction protein aggregation, altered nucleic acid aggregation ability to change biophysical properties, and the ability to functionalize DNA origami structures. [Jiang Q, Song C, Nangreave J, Liu X, Lin L, Qiu D, Wang ZG, Zou G, Liang X, Yan H, Ding B. DNA origami as a carrier for circumvention of drug resistance. J Am Chem Soc. 2012 Aug 15;134(32):13396-403]. In a preferred embodiment, functionalizing DNA origami structures improves drug efficacy.

[0512] Example

[0513] Example 1: Polysaccharide Synthesis

[0514] The chemical enzymatic polysaccharide synthesis and purification protocol was as described by Gao et al., 2019. Sialylglycopeptide (SGP) was prepared from egg yolks according to a pre-established protocol (Bingyang Sun, Wenzheng Bao, Xiaobo Tian, ​​Mingjing Li, Hong Liu, Jinhua Dong, Wei Huang, A simplified procedure for gram-scale production of sialylglycopeptide (SGP) from egg yolks and subsequent semi-synthesis of Man3GlcNAc oxazoline. Carbohydrate Research, Vol. 396, 2014, 62-69; Zou, Yang and Wu, Zhigang and Chen, Leilei and Liu, Xianwei and Gu, Guofeng and Xue, Mengyang and Wang, Peng and Chen, Min. (2012). An Efficient Approach for Large-Scale Production of Sialyglycopeptides from Egg Yolks. J Carbohyd. Chem. 31. 436-446) with slight modifications. In short, egg yolk powder (Magic Flavors, purchased directly from Amazon) was weighed and suspended in 3 volumes of diethyl ether, and washed twice. After filtration, the residue was resuspended in 3 volumes of 70% acetone and washed. SGP was then extracted using 1.5 volumes of 40% acetone. The crude extract containing SGP was dried on a rotary evaporator and purified using an activated carbon column (activated carbon:diatomaceous earth = 2:1). The column was pretreated with 3 bed volumes of acetonitrile, followed by 3 bed volumes (BV) of water containing 0.1% TFA. After sample loading, the column was washed sequentially with H₂O containing 0.1% TFA, 5% acetonitrile containing 0.1% TFA, and 10% acetonitrile containing 0.1% TFA, for 3 BV washes each. SGP was eluted with 3 BV of 25% acetonitrile, and the resulting fractions were combined, concentrated on a rotary evaporator, and lyophilized to dryness. This SGP-containing powder can be used directly in the following reactions without further purification. In the embodiment, the powder can be desalted by size exclusion chromatography on a BioGel P2, and the product SGP can be structurally analyzed by NMR or MS.

[0515] Preparation of Fmoc-labeled desialyl-lactose-biantennae-type N-glycan

[0516] SGP was treated to produce the substrate for enzymatic synthesis (GO-Fmoc). SGP powder was reconstituted with water, and HCl was added. The final concentrations of SGP and HCl were adjusted to 20 mg / ml and 0.1 M, respectively. After incubation at 80 °C for 2 h, the solution was neutralized with NaOH, yielding desialylated SGP (Man5-AEAB). (Song et al., (2009). Novelfluorescent glycan microarray strategy reveals ligands for galectins. Chem. Biol. 16, 36–47.) The pH of this solution was adjusted to 5.2 by adding sodium acetate and concentrated acetic acid. Galactosidase was then added to a final concentration of 10 mg / ml, and the mixture was incubated at 37 °C for 4 h. Desialylated and degalactosylated SGP (G2-AEAB) was heated at 85 °C for 10 min, the pH was adjusted to 8.0 by adding 200 mM Tris base, and protease was added to a final concentration of 1 mg / mL for proteolytic digestion. The mixture was incubated at 55 °C, with the same amount of protease added every 12 h until the starting material was undetectable by MALDI-MS. After centrifugation, the supernatant was lyophilized to dryness, and the residue was reconstituted with water, passed through a Sep-Pak C18 SPE column, and purified by size exclusion chromatography on a Bio-Gel P2 column. Asn-linked desialylated, galactose-free, biantennary N-glycan (G0) was obtained. This compound was reacted overnight with Fmoc-OSu (3 eq.) in 1,4-dioxane:H2O = 1:2, labeled with Fmoc, and the product (G0-N-Fmoc) was finally purified on a pretreated Sep-Pak C18 column.

[0517] Six glycosyltransferases, namely FUT8, MGAT4a, MGAT5, B4GalT1, ST3Gal4, and ST6Gal1, were expressed using suitable expression plasmids (e.g., plasmids available from Professor Kelley Moremen at the ComplexCarbohydrate Research Center, University of Georgia). The constructs contained soluble domains of the glycosyltransferases, with a His tag at the N-terminus and a GFP tag following the secretion signal (pGEn2-DEST vector). Transient transfection of suspensions and serum-free adapted HEK293 cells (Freestyle 293-F cells, Invitrogen) was performed using polyethyleneimine. Five to seven days post-transfection, the proteins were purified from the culture supernatant using His-Pur Ni-NTA resin (Thermo Scientific) via nickel affinity chromatography. After elution with an imidazole-containing buffer (50 mM sodium phosphate, 300 mM sodium chloride, and 400 mM imidazole, pH 8.0), the enzymes were dialyzed into storage buffer (20 mM Tris, pH 7.5, containing 300 mM sodium chloride) and rapidly frozen. All glycosyltransferases were stored as chimeric GFP fusion proteins at -80°C until use.

[0518] Glycosyltransferase reaction

[0519] A) FUT8 (2 mg / ml)-catalyzed α1,6-core fucosylation

[0520] The reaction was carried out in 100 mM MES buffer (pH 7.0). The final concentrations of glycan and GDP-Fuc were 2.5 mM and 3.75 mM, respectively. The glycosyltransferase FUT8 was 1 mg / ml. The reaction was incubated overnight at 37 °C and then terminated by freezing at -80 °C. The mixture was lyophilized to dryness and purified by elution with 0% to 50% MeOH on a pretreated Sep-Pak C18. The lichenol-positive fractions were examined by MALDI-MS, and fractions containing the predicted m / z were combined and dried to harvest the targeted glycan.

[0521] B) β1,4-GlcNAc branching catalyzed by MGAT4a (1 mg / ml)

[0522] The reaction was carried out in 500 mM MOPS buffer (pH 7.3) containing 30 mM MnCl2. The final concentrations of glycan and UDP-GlcNAc were 5 mM and 10 mM, respectively. The glycosyltransferase MGAT4a was 0.25 mg / mL. The mixture also contained phosphatase. The reaction was incubated at 37 °C and monitored by MALDI-MS. Typically, the reaction was allowed to proceed overnight and then terminated by cooling to -80 °C, followed by lyophilization to dryness. The product was purified by elution with 0% to 50% MeOH on a pretreated Sep-Pak C18 plate. The lichenol-positive fractions were examined by MALDI-MS, and fractions containing the predicted m / z were combined and dried to harvest the targeted glycan.

[0523] C) β1,6-GlcNAc branching catalyzed by MGAT5 (1 mg / ml)

[0524] The reaction was carried out in 125 mM MES buffer (pH 6.25). The final concentrations of glycan and UDP-GlcNAc were 5 mM and 10 mM, respectively. The concentration of glycosyltransferase MGAT5 was 0.25 mg / ml. The mixture also contained phosphatase to digest the product UDP. The reaction was incubated overnight at 37 °C and then terminated by placing it at -80 °C. The mixture was lyophilized to dryness and purified by elution with 0% to 50% MeOH on a pretreated Sep-Pak C18. The lichenol-positive fractions were examined by MALDI-MS, and fractions containing the predicted m / z were combined and dried to harvest the targeted glycan.

[0525] D) B4GalT1 (2 mg / ml)-catalyzed β1,4-galactosylation

[0526] The reaction was carried out in 125 mM Tris buffer (pH 7.5) containing 100 mM NaCl, 50 mM MgCl2, and 50 mM MnCl2. The final concentration of the glycan was 5 mM. The concentration of UDP-Gal varied from 15 mM for trianthopteric N-glycans to 20 mM for tetraanthopteric N-glycans. B4GalT1 was added to bring the final concentration to 0.3 mg / ml. The products of MGAT4a and MGAT5 could also be directly extended by B4GalT1, in which case Tris base, NaCl, MgCl2, and MnCl2 were added to the reaction mixture to bring the final concentrations to 125, 100, 50, and 50 mM, respectively. Hydrochloric acid was added to adjust the pH to 7.5. The final concentrations of glycans, UDP-Gal, and B4GalT1 were 1.2, 7.3 mM (or 9.6 mM for tetraanthopteric N-glycans), and 0.47 mg / ml, respectively. In all cases, phosphatase was present. The reaction was incubated overnight at 37°C and terminated by placing at -80°C. The mixture was lyophilized to dryness and purified by elution with 0% to 50% MeOH on a pretreated Sep-Pak C18. The lichenol-positive fractions were examined by MALDI-MS, and fractions containing the predicted m / z were combined and dried to harvest the targeting glycan.

[0527] E) ST3Gal4 (1 mg / ml)-catalyzed 2,3-sialylation

[0528] The reaction was carried out in 100 mM arsenopyrate-Na buffer (pH 6.2) containing 50 mM MnCl2. The final concentration of the glycan was adjusted to 2.5 mM. For biantennary, triantennary, and tetraantennary N-glycans, the CMP-sialic acid concentrations were 15, 22.5, and 30 mM, respectively, while the ST3Gal4 concentrations were 0.3, 0.4, and 0.5 mg / ml, respectively. The mixture also contained phosphatase to digest the product CMP. The reaction was incubated overnight at 37°C and then terminated by placing it at -80°C. The mixture was lyophilized to dryness, and the product was purified by HPLC on a Zorbax NH2 column (250 × 10 mm) as described below. Fractions with predicted m / z were combined and dried to harvest the target glycan.

[0529] F) ST6Gal1 (2 mg / ml)-catalyzed 2,6-sialylation

[0530] The conditions for 2,6-sialylation were the same as those for 2,3-sialylation, except for the amount of glycosyltransferase added. For biantennary, triantennary, and tetraantennary N-glycans, ST6Gal1 was adjusted to 0.6, 0.8, and 1 mg / ml, respectively.

[0531] Example 2: RNA Synthesis

[0532] RNA was prepared using PCR products as templates after the transcription reaction was terminated by standard T7 RNAP and purified by urea-PAGE as described. The RNA yield of in vitro transcription was optimized for each individual DNA template in 25 μL test reactions by varying the concentrations of Mg2+, NTPs

[22] and incubation time. A typical 10 mL large-scale transcription reaction mixture contained 30 mM Tris (pH 8.1 at 37 °C), 15 mM Mg2+, 10 mM dithiothreitol (DTT), 2 mM spermidine, 0.01% (v / v) Triton X-100, 4 mM each NTP, 1 mL of PCR-generated DNA template and 0.1 mg / mL T7 RNAP [6, 8]. After incubation at 37 °C for 2.5 h, the pyrophosphate formed during the in vitro transcription reaction was precipitated by centrifugation, and additional Mg2+ was added to the reaction. The reaction was continued for 5 h. To concentrate the reaction mixture, a Millipore centrifuge with appropriate MWCO was used. Transcriptional screening involved only one variable component per test, while the remaining components remained constant. Mg2+ concentrations tested included 5 mM, 15 mM, 25 mM, 35 mM, 45 mM, 55 mM, 65 mM, 75 mM, 85 mM, and 95 mM, while NTP concentrations were 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, and 10 mM. Transcription incubation times at 37°C were 5 h, 6 h, 7 h, 8 h, 9 h, and 10 h. Results were evaluated by image quantification (BioRad) of target RNA bands on 12% TAE urea-PAGE. Target RNA yield reached a peak of 180% under storage conditions, approximately 45 mM Mg2+. Generally, RNA yield increased with increasing NTP from 1 mM to a maximum of 10 mM, and stabilized at approximately 8 mM for each NTP. Finally, the reaction time was extended, and a steady increase in product was observed up to 9 hours. [Lu C et al., CellPhysiol. Biochem., 2018;48:1915-1927].

[0533] In BL21(DE3) *E. coli* cells, in vivo RNA synthesis was performed using a plasmid containing DNA and a template corresponding to the mRNA of interest. Transcription was induced using IPTG when the cell culture had a UV absorbance of approximately 0.5 OD at 600 nm, and the solution was shaken at 37°C for 3 hours. Following induction, as described by Mao and Wang et al., 1 ml of bacterial culture was centrifuged in a 1.5 ml centrifuge tube, and the suspension was removed. The precipitate was resuspended in 100 μL of buffer L containing 10 mM Tris-HCl (pH 7.4) and 10 mM Mg(OAc)2. The bacterial membrane was disrupted by adding 100 μL of phenol solution (Sigma). The aqueous layer was pipetted and added directly to a native PAGE gel. To prepare denaturing PAGE gels or gel-purified samples, 10 μL NaOAc (3 M, pH 5.2) and 200 μL ethanol were added to a 100 μL aqueous layer, followed by ethanol on dry ice to remove salt. The cell pellet was resuspended in 15 mL of buffer L and placed in an ice-water bath. Cells were then sonicated (phenol-free) using a Branson digital sonicator (10% amplitude) for 5 s, followed by a 5 s pause; this was repeated for a total of 10 min. Cell debris could be removed by centrifugation at 16,000 × g for 30 min, and the supernatant could be diluted with TAE / Mg2+ buffer. [Li, M., Zheng, M., Wu, S. et al. In vivo production of RNA nanostructures via programmed folding of single-stranded RNAs. NatCommun 9, 2196 (2018)].

[0534] Circular RNAs were prepared as described by Wesselhoeft et al., Nat Commun 9, 2629 (2018). The first step was cloning and mutagenesis, in which the protein-coding, class I self-splicing introns, and IRES sequences were chemically synthesized (IntegratedDNA Technologies) and cloned into a PCR-linearized plasmid vector containing a T7 RNA polymerase promoter using the NEBuilder HiFi DNA Assembly Kit (New England Biolabs) via Gibson assembly. Spacer regions, homologous arms, and other minor alterations were introduced using the Q5 site-directed mutagenesis kit (New England Biolabs). Next was the design and purification of the circRNA. RNA structure was predicted using RNAFold18. Modified linear GLuc mRNAs were obtained from Trilink Biotechnologies and consisted of a codon-optimized GLuc coding region, a proprietary synthetic 5' untranslated region, an α-globin 3' untranslated region, a cap 1 structure, a 120-nucleotide polyA tail, and uridine and cytosine were completely replaced throughout the mRNA with pseudouridine and 5-methylcytosine, respectively. The modified hEpo mRNA was also obtained from Trilink Biotechnologies and had the same structure as the Trilink GLuc mRNA described above, except that it was modified with 5-methoxyuridine and its coding region encodes human erythropoietin. The unmodified linear RNA consisted of either the GLuc or hEpo coding region but did not contain the specific untranslated region. Unmodified linear mRNA or circRNA precursors were synthesized using the T7 High Yield RNA Synthesis Kit (New England Biolabs) via in vitro transcription from a linearized plasmid DNA template. After in vitro transcription, the reaction was treated with DNase I (New England Biolabs) for 20 min. Following DNase treatment, the unmodified linear mRNA was column purified using the MEGAclear Transcription Purification Kit (Ambion). The RNA was then heated to 70°C for 5 min and immediately placed on ice for 3 min, after which it was capped using mRNA cap-2'-O-methyltransferase (NEB) and vaccinia virus capping enzyme (NEB) according to the manufacturer's instructions. Using E. coli PolyA polymerase (NEB) according to the manufacturer's instructions, polyadenylated tails were added to capped linear transcripts, and the fully processed mRNA was purified by column. For circRNA, after DNase treatment, additional GTP was added to bring the final concentration to 2 mM, and the reaction was carried out at 55 °C for 15 min. The RNA was then purified by column.In some cases, purified RNA may be recirculated: The RNA is heated to 70°C for 5 min, then immediately placed on ice for 3 min. GTP is then added to a final concentration of 2 mM, along with a magnesium-containing buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT, pH 7.5; New England Biolabs). The RNA is then heated to 55°C for 8 min, followed by column purification. To enrich circRNA, 20 μg of RNA is diluted in water (final volume 86 μL), heated at 65°C for 3 min, and cooled on ice for 3 min. 20 U RNase R and 10 μL of 10×RNase R buffer (Epicenter) are added, and the reaction is incubated at 37°C for 15 min; an additional 10 U RNase R is added halfway through the reaction. The RNase R-digested RNA is then column purified. RNA was separated on an E-gel iBase (Invitrogen) using a pre-prepared 2% E-gel EX agarose gel (Invitrogen) with the E-gel EX 1%–2% program; ssRNA Ladder (NEB) was used as the standard. Bands were visualized using blue light transmission and quantified using ImageJ. For gel extraction, bands corresponding to circRNA were excised from the gel and extracted using the Zymoclean Gel RNA Extraction Kit (Zymogen). For high-performance liquid chromatography (HPLC), 30 μg of RNA was heated at 65 °C for 3 min and then placed on ice for 3 min. RNA was passed through a 4.6 × 300 mm size size exclusion column (5 μm particle size and 200 Å pore size (Sepax Technologies; part number: 215980P-4630)) on an Agilent 1100 series HPLC system (Agilent). RNA was run at a flow rate of 0.3 mL / min in RNase-free TE buffer (10 mM Tris, 1 mM EDTA, pH: 6). RNA was detected by UV absorbance at 260 nm, but collection was performed without UV detection. The resulting RNA fractions were precipitated with 5 M ammonium acetate, resuspended in water, and then, in some cases, treated with RNase R as described above. [Wesselhoeft, RA, Kowalski, PS, and Anderson, DG Engineering circularRNA for potent and stable translation in eukaryotic cells. Nat Commun 9, 2629 (2018)].

[0535] RNA modification can be introduced to reduce cellular response. As described by Kariko K et al., in vitro transcription reactions can be generated by replacing one (or two) basic NTPs with one or more triphosphate derivatives of the modified nucleotides 5-methylcytidine, 5-methyluridine, 2-thiouridine, N6-methyladenosine, or pseudouridine (TriLink, San Diego, CA) to produce modified RNA, thereby reducing cellular response. For this type of transcription reaction, all four nucleotides or their derivatives are present in equimolar (7.5 mM) concentrations. Additionally, a 6 mM m7GpppG cap analogue (New England BioLabs, Beverly, MA) can be included to obtain capped RNA. Kariko K et al. Immunity 23:16575 (2005). In particular, replacing uridine with pseudouridine is beneficial for inhibiting RNA immunogenicity in vitro and in vivo, and also enhances RNA translation ability. Kariko K et al. Mol Ther 16:1833-40 (2008). The reduced immunogenicity and enhanced translational capacity of pseudouridine-modified RNA are due to the activation of RNA-dependent protein kinase R (PKR), which then phosphorylates translation initiation factor 2-α (eIF-2α) and inhibits translation. When pseudouridine is incorporated into the transcript, PKR activation is lower and translation is not inhibited. Anderson BR et al. NAR (2010).

[0536] RNA can be purified via natural gel or column purification using the MEGAclear Transcription Purification Kit (Ambion). Mao and Wang et al., Nat Commun 9, 2196 (2018) and Wesselhoeft et al., Nat Commun 9, 2629 (2018).

[0537] RNA modifications used for glycoconjugation include the use of 5-substituted pyrimidines and 7-substituted 7-deazapurines with terminal triple bonds during RNA synthesis, such as 3',5-octadiynyl dU. Seela F, Sirivolu VR. DNA containing side chains with terminal triple bonds: Base-pair stability and functionalization of alkynylated pyrimidines and 7-deazapurines. ChemBiodivers. May 2006;3(5):509-14.

[0538] Example 3: Sugar ligand conjugation

[0539] As described by Meng, G., Guo, T., Ma, T. et al., diazotizing compounds such as fluorosulfonyl azide (FSO2N3) can be used in click chemistry reactions to generate azides from terminal amines of glycans. [Meng, G., Guo, T., Ma, T. et al. Modular click chemistry libraries for functional screens using a diazotizing reagent. Nature 574, 86–89 (2019)].

[0540] Recent research by Flynn et al. has shown that by labeling precursor glycosyl groups with azide groups (e.g., AC4ManNAz used in this study), they can be conjugated with biotin once they are integrated into glycoprotein (and lipid) groups in cells (Flynn et al., (2021), Cell 184(12): 3109-3124). Probes are cross-linked for enrichment and subsequent identification analysis. Using such a system, the authors enriched high-purity RNA samples in labeled cells, indicating that glycosylation may also be present on RNA.

[0541] As disclosed in U.S. Patent 10,550,385 B2, GalNAc-siRNA conjugates can be generated by introducing two or more 2'-modifications into RNA, wherein the RNA has a 2'-O substituent containing an alkyl ester functional group at the 2'-position of one or more ribosome rings on one strand and a 2'-O substituent containing an alkyne functional group at the 2'-position of one or more ribosome rings on the same strand. The method comprises: a) adding an amine compound to the RNA to form an amidation product having an alkyl ester functional group; b) dissolving the RNA modified in step (a) in a solvent to form a solution; and c) adding an organic azide and a copper or ruthenium catalyst to the solution obtained in step (b) to form a 2'-azido-alkyne cycloaddition product having an alkyne functional group. When the organic azide is a GalNAc azide, GalNAc-siRNA is generated.

[0542] Example 4: Validation of Glycoligands

[0543] Release of N-linked glycans

[0544] By modifying a previously reported method, glycans were released and isolated from glycoligands or glycoproteins (Papac et al. AJS (1998) Glycobiology 8, 445-454). The wells of a 96-well MultiScreen IP (Immobilon-P membrane) plate (Millipore) were moistened with 100 μL of methanol and washed with 3 × 150 μL of water and 50 μL of RCM buffer (8M urea, 360 mM Tris, 3.2 mM EDTA, pH 8.6), draining gently under vacuum after each addition. The dried protein sample was dissolved in 30 μL of RCM buffer and transferred to wells containing 10 μL of RCM buffer. The wells were drained and washed twice with RCM buffer. Protein reduction was achieved by adding 60 μL of 0.1 M DTT to the RCM buffer and reacting at 37°C for 1 h. The wells were washed three times with 300 μL of water, and carboxymethylation was performed for 30 min by adding 60 μL of 0.1 M iodoacetic acid at room temperature in the dark. The wells were washed three times again with water, and the membrane was sealed for 1 h by adding 100 μL of an aqueous solution of 1% PVP360 at room temperature. The wells were drained and washed three times with 300 μL of water, and deglycosylated by adding 30 μL of 10 mM NH4HCO3 containing 1 mIU of N-glycosidase (Glyko) at pH 8.3. After incubation at 37 °C for 16 hours, the solution containing glycans was removed by centrifugation and evaporated to dryness.

[0545] Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry

[0546] The molecular weight of the glycans was determined using a delayed extraction method with a Voyager DE PRO linear MALDI-TOF (Applied Biosciences) mass spectrometer. The dried glycans from each well were dissolved in 15 μL of water, and 0.5 μL was spotted onto a stainless steel sample plate and mixed with 0.5 μL of S-DHB matrix (9 mg / mL dihydroxybenzoic acid and 1 mg / mL 5-methoxysalicylic acid in a 1:1 water / acetonitrile (0.1% TFA) solution) and allowed to dry.

[0547] Ions were generated by irradiation with a pulsed nitrogen laser (337 nm) for 4 ns. The instrument operated in delayed extraction mode with a delay of 125 ns and an accelerating voltage of 20 kV. The gate voltage could be 93.00%, the lead voltage 0.10%, the internal pressure less than 5 × 10⁻⁷ Torr, and the low-mass gate 875 Da. Spectra were generated by superimposing 100–200 laser pulses and acquired using a 2 GHz digitizer. The sialylated N-glycan complex NeuNAc₂Gal₂GlcNAc₂Man₃GlcNAc₂Fuc was used as an external molecular weight standard. All spectra were generated in the instrument's positive ion mode. The spectral quality accuracy was estimated to be approximately 0.5%.

[0548] The mass of N-glycans eluted from the column is typically related to the cation adduct, which increases the mass due to the molecular weight of the cation. The most common adduct is H... + Na + and K + .

[0549] Polysaccharide preparation for NMR

[0550] As described in EP1910838B1, the experimental procedure for proton NMR analysis of glycan fractions is detailed below. Glycans are released from glycoligands via enzymatic or chemical means. The glycans are then fractionated into neutral and acidic fractions by graphitized carbon chromatography. A useful purification step prior to NMR analysis is gel filtration high-performance liquid chromatography (HPLC). For glycans derived from glycoproteins or glycolipids, a Superdex Peptide HR10 / 300 column (Amersham Pharmacia) can be used. For larger glycans, a Superdex 75 HR10 / 300 column can be used. The Superdex column is eluted with water or 50–200 mM ammonium bicarbonate at a flow rate of 1 ml / min for both neutral and acidic glycan fractions, and the absorbance at 205–214 nm is recorded. The fractions (typically 0.5–1 ml) are collected and dried. Repeated dissolution in water and evaporation may be necessary to remove residual ammonium bicarbonate salts from the fractions. The fractions can be subjected to MALDI-TOF-MS, and all fractions containing glycans can be pooled. The pooled fractions are then dissolved in heavy water and evaporated. For glycan formulations containing approximately 100 nmol or more, the final sample is dissolved in 600 μL of heavy water (99.9%–99.996%) and transferred to an NMR analysis tube. An approximately equimolar amount of an internal standard, such as acetone, is typically added to the solution. For glycan formulations derived from small tissue samples or small cell counts (5 million to 25 million cells), it is preferable to evaporate the sample two or more times with very high-quality heavy water (99.996%) to remove H₂O as efficiently as possible, before finally dissolving it in 99.996% heavy water. These low-content samples are preferably analyzed using more sensitive NMR techniques. For example, smaller NMR analysis tubes can be used to obtain higher concentrations of glycans. This type of tube includes, for example, nanotubes (Varian), in which the sample is typically dissolved in a volume of 37 μL. In this implementation, higher sensitivity is achieved by analyzing the sample in a low-temperature NMR instrument, which increases analytical sensitivity by reducing electronic noise. This latter technique enables the collection of high-quality proton NMR data from glycan samples containing approximately 1-5 nmol of glycan material.

[0551] Analysis of NMR data

[0552] It has been recognized that numerous studies have demonstrated the ability of proton NMR data to indicate the presence of several structural features in glycan samples. Furthermore, by carefully integrating the spectra, the relative abundance of these structural features in glycan samples can be obtained. For example, the proton bound to the carbon-1 of a monosaccharide, known as H-1, produces a distinctive signal at low fields, clearly separated from the protons of other sugar residues. Most monosaccharide residues, such as those in N-glycans, are identified by their H-1 signals. Additionally, the H-2 signals of mannose residues indicate their bonding.

[0553] Sialic acids lack an H-1 group, but their H-3 signals (H-3 axial and H-3 flattened) are clearly separated from the protons of other sugar residues. Furthermore, different binding sialic acids can be identified by their H-3 signals. For example, the Neu5Ac H-3 signal of the Neu5Acα2-3Gal structure is located at 1.797 ppm (axial) and 2.756 ppm (flattened). On the other hand, the Neu5Ac H-3 signal of the Neu5Acα2-6Gal structure is located at 1.719 ppm (axial) and 2.668 ppm (flattened). The molar ratios of these structural features are obtained by comparing the integrated areas of these signals.

[0554] Other structural reporting signals are generally known, and those skilled in the art will use a large body of literature as a reference for glycan NMR assignment. Fu D., Chen L., and O'Neill RA (1994) Carbohydr. Res. 261, 173-186. Hård K., Mekking A., Kamerling JP, Dacremont GAA, and Vliegenthart J.FG (1991) Glycoconjugate J. 8, 17-28. Hård K., Van Zadelhoff G., Moonen P., Kamerling JP, and Vliegenthart JFG (1992) Eur. J. Biochem. 209, 895-915. Helin J., Maaheimo H., Seppo A., Keane A., and Renkonen O. (1995) Carbohydr. Res. 266, 191-209.

[0555] Example 5: Determination of how carbohydrate-binding receptors interact with their glycan ligands

[0556] Surface plasmon resonance spectroscopy (SPR spectroscopy) can be used to determine the binding kinetics between a selected glycoligand (e.g., G2FS2 glycoligand) and a target receptor. To obtain these binding kinetics, receptors or proteins, such as Siglec11 and Siglec14, are immobilized on the surface of a sensor chip. The glycoligands flow through a microflow cell with a buffer solution. The binding of the glycoligands to the immobilized receptor or protein on the sensor chip surface causes a change in the refractive index of the surface layer, which is monitored by a detector such as a diode array. The change in refractive index over time is recorded as a sensing map. The sensing map provides information about binding or non-binding, as well as information about the kinetics and strength of the interaction.

[0557] Example 6A: A general procedure for the synthesis of azide polysaccharides

[0558] Materials and methods

[0559] Free reduced terminal glycans were obtained from Glycobia, Inc., Ithaca, NY or Chemily Glycoscience, Peachtree Corners, GA, and manufactured according to literature procedures known in the art.

[0560] General characterization procedure

[0561] Matrix-assisted laser desorption / ionization (MALDI) analysis of the glycan conjugate: A glycan conjugate (1 μL, 1 mM) in mini-Q water was mixed with a matrix of 2,5-dihydroxybenzoic acid (DHB, 1 μL, 20 mg / mL) in 50% (v / v) aqueous acetonitrile solution. The mixture was then loaded onto an MTP 384 target plate. After air drying and co-crystallization, the sample was analyzed using a Bruker MALDI-TOF system.

[0562] HPLC analysis of the polysaccharide conjugates: The polysaccharide conjugates in ethanol (0.2 mg / mL, 50 μL) were analyzed by an analytical reversed-phase HPLC column. Mobile phase A was methanol containing 0.1% TFA, and mobile phase B was water containing 0.1% TFA. The flow rate was 1 mL / min. Gradient: 75% to 100% of mobile phase A.

[0563] Functionalization of asparagine azide

[0564] Asparagine-linked N-glycans were prepared in a solution of mini-Q water with added Na₂CO₃ (20 eq.) and FSO₂N₃ (40 eq.). The mixture was rotated at rt for 1 h, and MALDI mass spectrometry analysis showed complete conversion. The reaction mixture was centrifuged under vacuum for 30 min and then lyophilized. The residue (white powder) was reconstituted with mini-Q water and loaded onto pretreated Carb SPE tubes. The tubes were washed with distilled water (10 × 1.2 mL) and then eluted with 50% acetonitrile (4 × 1.2 mL) containing 100 mM (NH₄)₂CO₃. The eluates were combined and lyophilized to obtain the desired azide polysaccharide.

[0565] Table 4A – Illustrative Examples of Asparagine Azide-Functionalized Polysaccharides

[0566]

[0567]

[0568]

[0569] aminooxy-PEG3-azide addition

[0570] The polysaccharide with free reducing ends was incubated with a 10-fold molar excess of aminooxy-PEG3-azidolinker O-(2-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)hydroxylamine; molecular weight 234.26 mol). The reaction was carried out in 1X PBS (pH 4.0) at 37 °C for 30 h. The reaction was desalted using a PGC SPE column (Thermo Fisher Scientific®). The column was pretreated with 3 × 1 mL acetonitrile, followed by 3 × 1 mL H2O. The reaction mixture was diluted with water to a maximum of 500 μL and passed through the column. After loading the reaction mixture, the column was washed with 3 × 1 mL H2O, followed by elution with 2 × 750 μL of 10 mM NH4HCO3 in 50 / 50 acetonitrile and H2O. The acetonitrile was removed under vacuum and dried by lyophilization.

[0571] Table 4B – Examples of aminooxy-PEG3-azidofunctionalized polysaccharides

[0572]

[0573]

[0574]

[0575]

[0576] N-methoxyamine-ethyl-azide linker addition

[0577] The polysaccharide with free reducing ends was incubated with a 100-fold molar excess of N-methoxyamine-ethylamine dihydrochloride linker 2-((methylamino)oxy)ethylamine dihydrochloride (molecular weight 163.05 mol) and a 100-fold molar excess of anhydrous sodium acetate. The reaction was carried out in DMSO / HOAc (7 / 3, volume / volume) at 65 °C for 2 h. The reaction was quenched by adding 15 volumes of acetonitrile, followed by centrifugation to remove the supernatant, and the precipitate was purified by envi-Carb SPE column (Sigma-Aldrich®). The column was pretreated with 3 × 1 mL acetonitrile, followed by 3 × 1 mL H2O. The precipitate was redissolved in 300 μL of water and passed through the column. After precipitate loading, the column was washed with 5 × 1 mL H2O, followed by elution with 2 × 750 μL of 10 mM NH4HCO3 in a 50 / 50 acetonitrile and H2O solution. Acetonitrile was removed under vacuum and then dried by freeze-drying.

[0578] Add Na₂CO₃ (20 eq.) and FSO₂N₃ (40 eq.) to a solution of the labeled polysaccharide in mini-Q water. Rotate the mixture at rt for 1 h, and MALDI mass spectrometry analysis showed complete conversion. Centrifuge the reaction mixture under vacuum for 30 min, then lyophilize. Reconstitute the residue (white powder) with mini-Q water and load it onto pretreated Carb SPE tubes. Wash the tubes with distilled water (3 × 1 mL) and elute with 50% acetonitrile (2 × 0.75 mL) containing 10 mM NH₄HCO₃. Combine the eluates and lyophilize to obtain the desired azide polysaccharide.

[0579] Table 4C – Examples of N-methoxyamine-propyl-azidofunctionalized polysaccharides

[0580]

[0581]

[0582]

[0583] O-ethyl-azide linker addition

[0584] Na₂CO₃ (20 eq.) and FSO₂N₃ (40 eq.) were added to a solution of O-ethylamine-labeled polysaccharide in mini-Q water. The mixture was stirred at rt for 1 h, and MALDI mass spectrometry analysis showed complete conversion. The reaction mixture was centrifuged under vacuum for 30 min and then lyophilized. The residue (white powder) was reconstituted with mini-Q water and then loaded onto pretreated Carb SPE tubes. The tubes were washed with distilled water (3 × 1 mL) and then eluted with 50% acetonitrile (2 × 0.75 mL) containing 10 mM NH₄HCO₃. The eluates were combined and lyophilized to obtain the desired azide polysaccharide.

[0585] Table 4D – Examples of O-ethyl-azidofunctionalized polysaccharides

[0586]

[0587] Table 4E – Examples of N-methoxyamine-PEG3-azidofunctionalized polysaccharides

[0588]

[0589]

[0590] Table 4F – Examples of O-phenyl-azidofunctionalized polysaccharides

[0591]

[0592] N-methoxyamine-butyric acid linker addition

[0593] The polysaccharide with free reducing ends was incubated with a 100-fold molar excess of N-methoxyamine-butyric acid and a 100-fold molar excess of anhydrous sodium acetate. The reaction was carried out in DMSO / HOAc (7 / 3, v / v) at 65 °C for 2 h. The reaction was quenched by adding 15 v / v of ethyl acetate, followed by centrifugation to remove the supernatant, and the precipitate was purified by an Envi-Carb SPE column (Sigma-Aldrich®). The column was pretreated with 3 × 1 mL acetonitrile, followed by 3 × 1 mL H2O. The precipitate was redissolved in 300 μL of water and passed through the column. After precipitate loading, the column was washed with 5 × 1 mL H2O, followed by elution with 2 × 750 μL of 10 mM NH4HCO3 in a 50 / 50 acetonitrile and H2O solution. The acetonitrile was removed under vacuum and the product was dried by lyophilization.

[0594] Table 4G – Examples of N-methoxyamine-ethylamine functionalized polysaccharides

[0595]

[0596]

[0597]

[0598] Example 6B: General Procedure for Joint and Support Assembly

[0599] The organic connector support of the present invention is manufactured according to procedures known in the art.

[0600] Synthesis of BCN-functionalized dilysine linkers

[0601]

[0602] Step 1: Add di-lysine 2HCl salt and NaHCO3 to a round-bottom flask, followed by DMF and TEA. Stir and sonicate the mixture to dissolve the reactants. Then add BCN-NHS and stir overnight at RT. DMF is removed by vacuum evaporation. The residue is redissolved in DCM and washed with water. The organic layer is dried over anhydrous Na2SO4, concentrated, and purified by rapid silica gel chromatography to provide BCN-dri-lysine.

[0603] Step 2: BCN-dilysine and FmocNH-PEG5-C2-NH2 HCl salt were added to DMF, followed by DIPEA and DIC. The mixture was stirred overnight at RT. DMF was removed by vacuum evaporation. The residue was reconstituted in DCM and washed with water. The organic layer was dried over anhydrous Na2SO4, concentrated, and purified by rapid silica gel chromatography to provide the BCN-functionalized trilysine linker (intermediate C).

[0604]

[0605] Step 3: The BCN-functionalized dilysine linker (intermediate C; 1 equivalent) and azide-functionalized glycans (4 equivalents) were added to DMF / water (50 / 50, volume / volume). The mixture was incubated overnight at 25°C. TLC and MALDI analysis showed complete conversion of the BCN-functionalized dilysine linker. The reaction mixture was lyophilized. The residue was reconstituted in water and then purified by solid-phase extraction (C18 SPE column) to obtain the trilysine linker conjugated to the four glycans (intermediate D).

[0606] Synthesis of BCN-functionalized trilysine linkers

[0607]

[0608] Step 1: Trilysine 3HCl salt and NaHCO3 were added to a round-bottom flask, followed by DMF and TEA. The mixture was stirred and sonicated to dissolve the reactants. BCN-NHS was then added, and the mixture was stirred overnight at RT. DMF was removed by vacuum evaporation. The residue was redissolved in DCM and washed with water. The organic layer was dried over anhydrous Na2SO4, concentrated, and purified by rapid silica gel chromatography to provide BCN-trilysine.

[0609] Step 2: BCN-trilysine and FmocNH-PEG5-C2-NH2 HCl salt were added to DMF, followed by DIPEA and DIC. The mixture was stirred overnight at RT. DMF was removed by vacuum evaporation. The residue was redissolved in DCM and washed with water. The organic layer was dried over anhydrous Na2SO4, concentrated, and purified by rapid silica gel chromatography to provide the BCN-functionalized trilysine linker (intermediate A).

[0610]

[0611] Step 3: The BCN-functionalized trilysine linker (intermediate A; 1 equivalent) and azide-functionalized glycans (5 equivalents) were added to water / dioxane (50 / 50, v / v) or water / DMF (50 / 50, v / v). The mixture was incubated overnight at 37°C or 25°C. TLC and MALDI analysis showed complete conversion of the BCN-functionalized trilysine linker. The reaction mixture was lyophilized. The residue was reconstituted in water and then purified by solid-phase extraction (C18 SPE column) to obtain the trilysine linker conjugated to the four glycans (intermediate B).

[0612] Synthesis of BCN-functionalized dilysine linkers

[0613]

[0614]

[0615] Step 1: Purchase azidotriamine starting material E from commercial suppliers (including WuXi AppTec) and prepare it using methods well-established in the art. A 50 mL round-bottom flask was fitted with a magnetic stir bar, azidotriamine E, MeCN, and Et3N. The flask was then sealed with a Suba-Seal® diaphragm and stirred at room temperature for 5 minutes. An air balloon attached to a fixed-needle syringe was inserted into the flask through the diaphragm. After evacuating the air under slightly reduced pressure, bubbles formed inside the flask, and the balloon became taut. This facilitated the introduction of SOF4 gas from the gas cylinder into the flask via a plastic tube. Under an excess SOF4 atmosphere, the reaction proceeded rapidly and completely. The resulting mixture was cooled to -20°C, sample loading was performed by adding silica gel to the column, and then the solvent was removed under reduced pressure. Pure iminosulfonyl difluoride (intermediate F) was obtained by rapid column chromatography (hexane / ethyl acetate).

[0616] Step 2: Add iminosulfonyl difluoride F, H-23-N-methoxyamine-ethylamine, buffer solution, and MeCN to a vial equipped with a magnetic stir bar. Stir the mixture vigorously at 25°C until homogeneous and F is completely converted. Concentrate the mixture under vacuum and purify the residue using a C-18 column to obtain the product.

[0617] BCN-functionalized Tris linker conjugation with glycans and siRNA

[0618]

[0619] Step 1: BCN-functionalized Tris-NHS esters were purchased from commercial suppliers (including WuXi AppTec) and manufactured using methods well-established in the art. Amine-functionalized siRNA was manufactured using methods well-established in the art. The siRNA conjugate (1.5 eq.) and Tris linker (1 eq.) were dissolved in DMF, followed by the addition of TEA (3 eq.). The mixture was stirred overnight at 25°C. TLC analysis showed complete conversion of the Tris linker. DMF was removed by vacuum evaporation. The residue was reconstituted in EtOAc and washed with water. The organic layer was dried over anhydrous Na2SO4, concentrated, and purified by rapid silica gel chromatography to provide intermediate E.

[0620] Step 2: Intermediate E (1 equivalent) and azide-functionalized polysaccharide (4 equivalents) were incubated overnight at 25°C in DMF / water (50 / 50, volume / volume). MALDI-TOF mass spectrometry analysis showed that intermediate C was fully conjugated. The conjugated glycoPEG lipid was purified by HPLC, lyophilized, and then reconstituted in ethanol for direct use in LNP formulations.

[0621] Typically, the Fmoc-functionalized polylysine linker system can be conjugated with siRNA as described above for the tri-BCN-functionalized NHS linker system.

[0622] Example 7: A general procedure for coupling glycan ligands with modified siRNA

[0623] siRNA

[0624] The modified nucleic acids described in Table 7A may include optional base modifications, optional sugar modifications, and / or optional phosphate ester modifications. In Table 7A, the term "pos." refers to the nucleic acid position.

[0625] Table 7A – Exemplary Nucleic Acids

[0626]

[0627]

[0628]

[0629]

[0630]

[0631]

[0632]

[0633]

[0634]

[0635] Example 8: In vitro knockdown of target mRNA in 293T cells using glycoRNA

[0636] Twenty-four hours prior to the experiment, 200,000 293T cells were seeded in 1 mL of growth medium in 12-well plates. 3 μl of lipofectamine was added to 50 μl of serum-free medium, and the glycosiRNA duplex was added separately to the serum-free medium to a final concentration of 200 nM. The mixture of lipofectamine and the diluted duplex was added together at room temperature and incubated for 10 minutes. The medium was aspirated from the seeded 293T cells and replaced with 1 mL of fresh medium. 100 μl of the lipofectamine-glycosiRNA mixture was added to each well and incubated overnight. RNA was purified from the cells using RNA lysis buffer (Zymo), followed by RNA preparation buffer, wash buffer, and elution buffer, with each cycle performed at 10,000 g for 2 min (Zymo). A BioRad thermal cycler was used with SuperScript according to the manufacturer's instructions. TM IV cDNA synthesis system (Life Technologies / Thermo Scientific) and oligo(dT) primers were used to synthesize cDNA from 200 ng of RNA per sample. The cDNA was diluted to 15 ng / μL, resulting in 60 ng per qPCR reaction. Samples were run repeatedly, with biological replicates for each sample. 1× TaqMan qPCR probes targeting β-catenin and β-actin (β-catenin probe group assay number: Hs00355045_m1, endogenous human β-actin control assay number: Hs01060665_g1, both purchased from Applied Bio / Thermo Scientific) and 1× TaqMan master mix were used to amplify cDNA. Samples were incubated at 50°C for 30 min, then at 95°C for 10 min, followed by 40 cycles, each consisting of 30 s at 95°C and 1 min at 60°C. β-catenin Ct values ​​were normalized to β-actin Ct values ​​to report relative abundance (%β-catenin mRNA).

[0637] Example 9: In vitro knockdown of target mRNA in primary human hepatocytes using glycoRNA

[0638] 1×10 obtained from Lonza Bioscience 5Primary human hepatocytes from healthy donors were used. Cells were thawed in INVITROGRO HT medium and cultured in INVITROGRO HI medium (BioIVT). Cells were seeded in 96-well plates and incubated with Cy5-labeled double-stranded RNA in serum-free INVITROGRO HT medium for 24 h. After incubation, the medium was removed by aspiration and the cells were washed once with PBS. The dried precipitate was frozen at -80°C until RNA extraction was performed. Total RNA was isolated from the cells using an RNeasy micro-spinning column (QIAGEN) according to the manufacturer's instructions. Total RNA was eluted in water (total volume 30 μL) and an aliquot was placed in a NanoDrop. TM Quantization was performed on a Thermo Scientific instrument. Following the manufacturer's instructions, a BioRad thermal cycler was used with SuperScript. TM IV cDNA synthesis system (LifeTechnologies / Thermo Scientific) and oligo(dT) primers were used to synthesize cDNA from 100 ng RNA per sample. Gene expression was assessed using multiplex TaqMan probes for β-catenin and β-actin (β-catenin probe assay number: Hs00355045_m1, endogenous human β-actin control assay number: Hs01060665_g1, both purchased from Applied Bio / Thermo Scientific). 10 ng of sample cDNA was seeded into each well of a 96-well optically clear PCR plate, with biological and technical replicates set up (Applied Bio / Thermo Scientific). 20× TaqMan probes and 2× TaqMan gene expression premix were added to each well according to the manufacturer's instructions, for a total reaction volume of 20 μL per well (Applied Bio / Thermo Scientific). Samples were amplified using the QuantStudio 6 Pro real-time PCR system with the following amplification parameters: Stage 1: 50°C for 2 min. Phase 2: 95℃ for 10 min. Phase 3: 95℃ for 15 s, then 60℃ for 1 min. Repeated 40 times. The expression of β-catenin relative to β-actin and gene expression in untreated control cells were calculated using the ΔΔCT method, where a value less than 1 indicated siRNA-mediated β-catenin knockdown.

[0639] Example 10: HepG2 Transfection Protocol

[0640] Twenty-four hours prior to the experiment, HepG2 cells were seeded at 200,000 cells / well in 1 mL of growth medium into 12-well plates. 3 μl of lipofectamine was added to 100 μl of serum-free medium, and the glycosiRNA duplex was added separately to the serum-free medium to a final concentration of 200 nM. The mixture of lipofectamine and the diluted duplex was added together at room temperature and incubated for 10 minutes. The medium was aspirated from the seeded 293 T cells and replaced with 1 mL of fresh medium. 100 μl of the lipofectamine-glycosiRNA mixture was added to each well and incubated overnight. RNA was purified from the cells using RNA lysis buffer (Zymo), followed by RNA preparation buffer, wash buffer, and elution buffer, with each cycle performed at 10,000 g for 2 min (Zymo). A BioRad thermal cycler was used with SuperScript according to the manufacturer's instructions. TM IV cDNA synthesis system (Life Technologies / Thermo Scientific) and oligo(dT) primers were used to synthesize cDNA from 200 ng of RNA per sample. The cDNA was diluted to 15 ng / μL, resulting in 60 ng per qPCR reaction. Samples were run repeatedly, with biological replicates for each sample. 1× TaqMan qPCR probes targeting β-catenin and β-actin (β-catenin probe group assay number: Hs00355045_m1, endogenous human β-actin control assay number: Hs01060665_g1, both purchased from Applied Bio / Thermo Scientific) and 1× TaqMan gene expression premix were used to amplify cDNA. Samples were incubated at 50°C for 30 min, then at 95°C for 10 min, followed by 40 cycles, each consisting of 30 s at 95°C and 1 min at 60°C. β-catenin Ct values ​​were normalized to β-actin Ct values ​​to report relative abundance (%β-catenin mRNA).

[0641] Example 11: Imaging assay of glycosiRNA internalization

[0642] On day 1, cells were washed with an excess of 10 mL of 1× PBS and then incubated with 10 mL of ACCUTASE® (Sigma) at 37°C for 10 min. Cells were collected, centrifuged at 300 g for 5 min, and resuspended in 4 mL of OptiMEM to a total of 400,000 cells per 200 wells. Cell Mask Green plasma membrane dye (ThermoFisher) at a dilution of 1:5,000 and Hoechst at a dilution of 1:20,000 were added to the diluted cells, and the cells were incubated at 37°C for 5 min. Cells were then washed with 6 mL of OptiMEM and centrifuged at 300 g for 5 min. The culture medium was discarded, and the cells were resuspended in complete culture medium (DMEM + 10% FBS + 1% PEN / STREP, Gibco / Life Technologies) to a concentration of 1×10⁻⁶ cells / well. 4 Cells / mL. Cells were seeded at 2000 cells / well in 20 μL of complete culture medium in 384-well imaging plates (CORNING®). Cells were incubated overnight in a standard tissue culture incubator at 37°C and 5% CO2. On day 2, cells were injected with 15 nM, 2 nM, and 0 nM Cy5-labeled double-stranded RNA, respectively. The plates were then incubated at 37°C and 5% CO2, and live-cell imaging was performed every 30 min in the DAPI, FITC, and Cy5 channels on an Opera Phenix high-content screening system using a 40x water immersion objective for 4 hours. The collected images were analyzed using Harmony high-content imaging and analysis software (Perkin Elmer). Typically, cell nuclei are identified and screened based on size, shape, and intensity in the DAPI channel; cells are then identified from the selected nuclei and screened based on size, shape, and intensity in the FITC channel; and the signal of the glycosiRNA is identified by Cy5 signaling within the selected cells, which may manifest as spots or intensity depending on the cell type. All relevant metrics (count, intensity, and area) for all three channels are calculated and analyzed using a custom R script. This procedure can be run on at least eight cell lines: HepG2, A549, SK-N-DZ, Huh7, THP-1, Raji, PANC-1, and Jurkat.

[0643] Example 12: In vivo biodistribution of injected glycosiRNA

[0644] BALB / c mice were administered each test item via tail vein injection or subcutaneous injection at a dose of approximately 0.1–10 mg / kg (each glycoRNA), with a total volume of approximately 5–10 mL / kg. In an exemplary experiment, each test item was administered to 6 mice, with an additional 3 mice given a PBS control. At a first time point (e.g., 4 h or 24 h post-injection), whole-body imaging was performed on the 3 animals administered each test item using the IVIS Spectrum in vivo imaging system (PerkinElmer) to detect Cy5 bioluminescent signals. Similarly, at a second time point (e.g., 48 h or 72 h post-injection), whole-body imaging was performed on the remaining 3 animals using the IVIS Spectrum in vivo imaging system (PerkinElmer) to detect Cy5 bioluminescent signals. Immediately after whole-body imaging at each time point, the animals were euthanized via CO2 inhalation, and organs, including but not limited to liver, spleen, lungs, heart, and kidneys, were harvested within 10 minutes of sacrifice for bioluminescence imaging (BLI) analysis. Organs were collected after imaging and flash-frozen using liquid nitrogen. BLI images were examined in automatic exposure mode. The BLI signal was quantified using Living Image 4.7 software (Perkin Elmer) according to the manufacturer's instructions. The weight of the collected organs was measured after BLI analysis.

[0645] The rapidly frozen tissue was cooled in liquid nitrogen and then treated with GENOMAX. ® Homogenize at 1500 RPM for 1 minute and 45 seconds using a SPEX EW-41019-48 homogenizer. Store the powdered tissue at -80°C until ready for test specimen extraction. Use AMPURE. ® XP (Beckman Coulter A63881) 1.8X bead cleaning, using KINGFISHER according to the manufacturer's instructions. TM Apex extracts and cleans the test items.

[0646] Single-stranded cDNA was prepared according to the manufacturer's instructions using Thermo's SuperScript IV Reverse Transcription Kit (oligo(dT)). The cDNA was diluted to 15 ng / μL, providing 60 ng per qPCR reaction. Samples were run repeatedly, with biological replicates for each sample. 1× TaqMan qPCR probes targeting β-catenin and β-actin (β-catenin probe assay number: Hs00355045_m1, endogenous human β-actin control assay number: Hs01060665_g1, both purchased from Applied Bio / Thermo Scientific) and 1× TaqMan gene expression premix were used to amplify the cDNA. Samples were incubated at 50°C for 30 min, then at 95°C for 10 min, followed by 40 cycles, each consisting of 30 s at 95°C and 1 min at 60°C. β-catenin Ct values ​​were normalized to β-actin Ct values ​​to report relative abundance (%β-catenin mRNA).

[0647] All in vivo experiments in this study were conducted in accordance with approved animal care guidelines. Time points, dosages, and organs / tissues to be harvested may be modified as needed.

[0648] Example 13: In vitro cytokine analysis of glycosiRNAs in PBMCs and other primary immune cells using Luminex

[0649] Primary immune cells (either total PBMCs or purified immune cells, such as purified CD19+ B cells or CD3+CD8+ T cells) were inoculated and administered glycosiRNA assay items continuously for 24 h under resting and stimulation conditions. The cell culture supernatant was harvested and analyzed using a Luminex FlexMAP 3D instrument with ProCartaPlex. TM Magnetic bead arrays are used to assess cytokine levels. Assessing cytokine levels determines whether glycan binding inhibits / increases expression. Increased / decreased cytokine levels and expression are used to determine whether certain signaling pathways are altered by exposing primary immune cells to glycan siRNA test items and can be used to predict the clinical relevance of glycan siRNA therapy.

[0650] While the invention has been specifically shown and described with reference to its preferred embodiments and various alternative embodiments, those skilled in the art will understand that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.

[0651] All references, authorized patents and patent applications cited in the main body of this specification are incorporated herein by reference in their entirety for all purposes.

Claims

1. A pharmaceutical composition comprising a glycopolynucleotide, wherein the glycopolynucleotide comprises a compound of formula (A-1): (K-X A -V 1 -X B ) m -X 1 -V 2 -X 2 -W A-1, Or its pharmaceutically acceptable salt, wherein: Each K is independently a glycan or glycan moiety disclosed or described herein; Each X A C1-C independently as a bond or optionally substituted 24 Bivalent linear aliphatic chain, where X A One or more methylene bonds may optionally be replaced by C3-C8 cycloalkylene, phenylene, -S(O2)-, -O-, -NH-, -N(C1-C6 alkyl)-, -C(=O)-, -C(=O)O- or -C(=O)NH-; Each V 1 Independently linked by bonds, amides, divalent amino acids, divalent peptides, or heterobifunctional groups; Each X B C1-C independently as a bond or optionally substituted 24 Bivalent linear aliphatic chain, where X B One or more methylene bonds may optionally be replaced by C3-C8 cycloalkylene, phenylene, -S(O2)-, -O-, -NH-, -N(C1-C6 alkyl)-, -C(=O)-, -C(=O)O- or -C(=O)NH-; X 1 It is the optional substitution of C1-C 30 Divalent or multivalent straight or branched aliphatic chains, where X 1 One or more methylene bonds may optionally be replaced by C3-C8 cycloalkylene, phenylene, -S(O2)-, -O-, -NH-, -N(C1-C6 alkyl)-, -C(=O)-, -C(=O)O-, -C(=O)NH-, or divalent polypeptide bonds or multivalent polypeptide bonds; or X 1 It is a key; V 2 It is a bond, amide, divalent amino acid bond, divalent peptide bond, or heterobifunctional group; X 2 It is the optional substitution of C1-C 30 Divalent straight-chain or branched aliphatic chains, where X 2 One or more methylene bonds are optionally replaced by proline bonds, –OP(=O)(OH)O-, C3-C8 cycloalkylene, phenylene, -S(O2)-, -O-, -NH-, -N(C1-C6 alkyl)-, -C(=O)-, -C(=O)O- or -C(=O)NH-; or X 1 It is a key; m is an integer greater than 2; and W stands for polynucleotide.

2. The pharmaceutical composition of claim 1, wherein W comprises one or more RNA polymers selected from mRNA, siRNA, snRNA, snoRNA, dsRNA, miRNA, lncRNA, circular RNA, γ RNA, ribosomal RNA, and small RNA fragments.

3. The pharmaceutical composition according to any one of claims 1 or 2, wherein W comprises siRNA.

4. The pharmaceutical composition of claim 3, wherein the siRNA comprises one or more modifications to one or more nucleotides, the one or more modifications being selected from 2-OMe modification, fluorine modification, thiophosphate modification, or any combination thereof.

5. The pharmaceutical composition of any one of the preceding claims, wherein the polysaccharide portion comprises a monosaccharide selected from D-glucuronic acid ("GlcA"), β-muramic acid ("Mur"), mannuronic acid ("ManA"), N-acetyl-muramic acid ("MurNAc"), leguminoic acid ("Leg"), acetaminophenic acid ("Aci"), D-xylose ("Xyl"), N-acetyl-L-fucosamine ("FucNAc"), pseudoamine ("Pse"), and L-iduronic acid ("IdoA").

6. The pharmaceutical composition of any of the preceding claims, wherein the polysaccharide portion comprises hexuronic acid sugar.

7. The pharmaceutical composition of any of the preceding claims, wherein the polysaccharide moiety comprises a monosaccharide, the monosaccharide comprising a non-sugar substituent or modification selected from: , , , , , , , , , , , , , , , , , , and .

8. The pharmaceutical composition of any of the preceding claims, wherein the polysaccharide moiety comprises two or more repeating chains of HexNAc-hexuronic acid units.

9. The pharmaceutical composition of any of the preceding claims, wherein the polysaccharide moiety comprises chains of two or more repeating GlcN-GlcA- units.

10. The pharmaceutical composition of any of the preceding claims, wherein the polysaccharide moiety comprises chains of two or more repeating GlcN-IdoA- units.

11. The pharmaceutical composition of any of the preceding claims, wherein the polysaccharide moiety comprises chains of two or more repeating GalNAc-GlcA- units.

12. The pharmaceutical composition of any of the preceding claims, wherein the polysaccharide moiety comprises a chain of two or more repeating galactose-GlcNAc- units.

13. The pharmaceutical composition of any of the preceding claims, wherein the polysaccharide portion comprises at least one fucose, the at least one fucose being bound to at least one monosaccharide selected from GlcNAc, galactose and glucose.

14. The pharmaceutical composition of any of the preceding claims, wherein the polysaccharide portion comprises a multi-tentacle polysaccharide consisting solely of mannose.

15. The pharmaceutical composition of any of the preceding claims, wherein the polysaccharide portion comprises polysaccharides selected from: a. The polysaccharides (G-1 to G-39) described in Table 1A; b. The polysaccharides (H-1 to H-62) depicted in Table 1B; c. The polysaccharides described in Table 1C (J-1 to J-19); d. The polysaccharides (K-1 to K-53) depicted in Table 1D; e. The polysaccharides (M-1 to M-14) depicted in Table 1E; and f. The polysaccharides described in Table 1F.

16. The pharmaceutical composition of any of the preceding claims, wherein the polysaccharide portion comprises a polysaccharide selected from those polysaccharides (H-1 to H-62) depicted in Table 1B.

17. The pharmaceutical composition of any of the preceding claims, wherein the polysaccharide portion comprises a polysaccharide selected from those polysaccharides (J-1 to J-19) depicted in Table 1C.

18. The pharmaceutical composition of any of the preceding claims, wherein the polysaccharide portion comprises a polysaccharide selected from those polysaccharides (K-1 to K-53) depicted in Table 1D.

19. The pharmaceutical composition of any of the preceding claims, wherein the polysaccharide portion comprises a polysaccharide selected from those polysaccharides (M-1 to M-14) depicted in Table 1E.

20. The pharmaceutical composition of any of the preceding claims, wherein the polysaccharide portion comprises a polysaccharide selected from those polysaccharides depicted in Table 1F.

21. The pharmaceutical composition of any of the preceding claims, wherein the glycan portion comprises a glycan bound to a plasma membrane lectin selected from MRC1, DC-SIGN, MGL, Siglec 3, Siglec 8, Siglec 9, Siglec 2, Siglec 4a, Langerhansin, Dectin-1, Dectin-2, CLEC14A, CLEC4A, CLEC4C, CLEC5A, CLEC2D, CD2, E-selectin, P-selectin, L-selectin, thrombomodulin, and SRCL.

22. The pharmaceutical composition of any of the preceding claims, wherein the polysaccharide portion comprises a polysaccharide bound to a serum agglutinin selected from MBL, galactoglobulin-2, anti-α-Gal antibody, galactoglobulin-3, and galactoglobulin-8.

23. The pharmaceutical composition of any of the preceding claims, wherein the polysaccharide portion comprises a polysaccharide bound to a protein selected from Siglec-1, Siglec-2, Siglec-3, Siglec-4, Siglec-5, Siglec-6, Siglec-7, Siglec-8, Siglec-9, Siglec-10, Siglec-11, CLEC12A, CLEC4E, and CD161.

24. The pharmaceutical composition of any of the preceding claims, wherein the polysaccharide portion comprises a polysaccharide bound to a protein selected from CD93, CD83, KLRF1, CD22, and CD28.

25. The pharmaceutical composition of any of the preceding claims, wherein the one or more polysaccharide portions are bound to one or more lectins selected from those disclosed in Table 3.

26. The pharmaceutical composition of any of the preceding claims, wherein the polynucleotide of formula A-1 comprises the linker scaffold disclosed in Table G.

27. A method of treating a disease or ailment, comprising administering to a subject in need a therapeutically effective amount of the pharmaceutical composition of any one of claims 1 to 26.

28. Use of the pharmaceutical composition of any one of claims 1 to 26 for manufacturing a medicament for treating a disease or ailment.

29. Use of the pharmaceutical composition according to any one of claims 1 to 26 for treating a disease or ailment of a subject in need.

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