Target pointing component

Abstract

The present invention provides conjugates for targeted delivery of therapeutic agents to PDGFR-expressing cells to treat diseases involving these cells (e.g., liver fibrosis, non-alcoholic steatohepatitis (NASH), clear cell renal cell carcinoma, renal fibrosis, or alcoholic steatohepatitis (ASH)). TIFF2025531872000113.tif30128

Description

[Technical Field]

[0001] Priority This application claims priority to U.S. Provisional Application No. 63 / 405,697, filed September 12, 2022. The entire contents of the above-referenced applications are incorporated herein by reference. [Background technology]

[0002] background Liver fibrosis is caused by excessive accumulation of extracellular matrix during chronic liver injury. The activation of hepatic stellate cells (HSCs) is a key step in liver fibrogenesis. For the successful treatment of liver disease and / or kidney disease (e.g., liver fibrosis, nonalcoholic steatohepatitis (NASH), renal fibrosis, or alcoholic steatohepatitis (ASH)), it may be important that therapeutic agents be targeted and delivered to HSCs, for example, activated HSCs. Therefore, a means of delivering therapeutic agents to HSCs is needed. Summary of the Invention

[0003] Quick Overview The present invention provides compounds, compositions, and methods that can be used to target oligonucleotides to platelet-derived growth factor receptor (PDGFR)-expressing cells.

[0004] In one aspect, the present invention provides a compound of formula (I): TIFF2025531872000002.tif13128 or a salt thereof, wherein x is 2, 3, 4, or 5; Each R 1 is independently a targeting ligand, and the targeting ligand is a) SEQ ID NO: 1: C * SRNLIDC * (SEQ ID NO: 1) A cyclic polypeptide represented by the formula: *represents a disulfide bond linking two C residues thereby forming a cyclic polypeptide, and b) C * Any cyclic polypeptide having at least 80% sequence identity (e.g., at least 85%; e.g., at least 87.5%; e.g., at least 90%; e.g., at least 95%; e.g., at least 99% sequence identity) with a polypeptide set forth in SEQ ID NO:1, provided that both residues are present, wherein: * exhibits a disulfide bond linking two C residues thereby forming a cyclic polypeptide. Selected from L is a linking group, y is 1, 2, 3, 4, or 5; Each R 2 are independently an oligonucleotide, a label (e.g., a label derived from fluorescein isothiocyanate (FITC) or Cy5), a phenyl group substituted with a formyl (-CHO) group, or a group of the formula: The source of TIFF2025531872000003.tif17128 is The present invention provides a compound or a salt thereof.

[0005] In another aspect, the present invention provides a compound of formula (X): 1. A process for preparing a diblock polymer of TIFF2025531872000004.tif8128, comprising: a) reacting a compound of structure Va, Vb, Vc, or Vd to obtain a first product: R 27 However, (C1-C 12 ) alkyl TIFF2025531872000005.tif16128; R 28 However, (C1-C 12 ) alkyl TIFF2025531872000006.tif16128; R 25 and R 26However, independently, H, (C1-C 12 ) alkyl, aryl, or heteroaryl TIFF2025531872000007.tif25128; TIFF2025531872000008.tif27128, as follows: polyethylene glycol methacrylate (PEGMA) having 2 to 20 ethylene glycol units; (C4-C 18 ) alkyl methacrylate, (C4-C 18 ) branched alkyl methacrylates, cholesteryl methacrylate, (C4-C 18 ) alkyl methacrylates, and (C4-C 18 ) Branched alkyl methacrylate and one or more A monomers selected from the group consisting of: contacting in the presence of free radicals; b) reacting the first product with Equations B1, B2, and B3: one or more B monomers of TIFF2025531872000009.tif75128; contacting in the presence of free radicals; and c) optionally contacting the second product with a free radical source (e.g., AIBN) to remove the chain transfer agent and obtain the diblock polymer of formula (X). Including, During the ceremony, block A comprises one or more residues of an A monomer and has a molecular weight of about 1 kDa to about 25 kDa; block B comprises one or more residues of monomers B1, B2, and B3 and has a molecular weight of about 1 kDa to about 25 kDa; L1 is a linking moiety, Z is optionally protected by a protecting group and has the formula (XII): T-L2-Y (XII) is a functional group capable of reacting with Y of a compound of formula T is a ligand (optionally a targeting ligand); Y is a functional group capable of reacting with Z to form a conjugate; L2 is absent or a linking moiety; Provide a process.

[0006] In another aspect, the present invention provides a compound of formula (I): TIFF2025531872000010.tif13128 or a salt thereof, wherein x is 1, 2, 3, 4, or 5; Each R 1 is independently a targeting ligand, and the targeting ligand is a) SEQ ID NO: 1: C * SRNLIDC * (SEQ ID NO: 1) A cyclic polypeptide represented by the formula: * exhibits a disulfide bond linking two C residues thereby forming a cyclic polypeptide, b) C * Any cyclic polypeptide having at least 80% sequence identity (e.g., at least 85%; e.g., at least 87.5%; e.g., at least 90%; e.g., at least 95%; e.g., at least 99% sequence identity) with a polypeptide set forth in SEQ ID NO:1, provided that both residues are present, wherein: * exhibits a disulfide bond linking two C residues thereby forming a cyclic polypeptide. Selected from L is a linking group, y is 1, 2, 3, 4, or 5; Each R 2 is independently an endosomally released polymer, The present invention provides a compound or a salt thereof.

[0007] The present invention also provides synthetic intermediates and methods disclosed herein that are useful for preparing compounds of Formula I and Formula XI.

[0008] Other objects, features, and advantages of the present invention will become apparent to those skilled in the art from the following detailed description and drawings. [Brief explanation of the drawings]

[0009] [Figure 1] Western blot detection of PDGFRB expression in HSC-T6 and NIH3T3 cells. [Figure 2] Uptake of monovalent or trivalent pPB ligand-biotin-AF488-streptavidin complexes by HSC-T6 and NIH3T3 cells analyzed by flow cytometry. [Figure 3] Uptake of bivalent or trivalent pPB ligand-biotin-AF488-streptavidin complexes by pHHSCs analyzed by flow cytometry. [Figure 4] Western blot detection of PDGFRB expression in LX-2, pHHSC, and NIH3T3 cells. [Figure 5] Uptake of trivalent pPB ligand-biotin-AF488-streptavidin complex by LX-2, NIH3T3, and primary human HSCs analyzed by flow cytometry. [Figure 6] In vitro gene silencing by TripPB siRNA conjugates and chloroquine in NIH3T3 cells. [Figure 7] In vitro gene silencing by TripPB siRNA conjugates and pPB-ERP in pHHSCs. [Figure 8] In vivo gene silencing by TripPB siRNA conjugates and pPB-ERP in a mouse liver fibrosis model. [Figure 9]Dose response of pPB-ERP upon co-treatment with TripPB-siRNA conjugates in a mouse liver fibrosis model. [Figure 10] In vivo gene silencing by DipPB siRNA conjugates and TripPB siRNA conjugates with pPB-ERP in a mouse liver fibrosis model. DETAILED DESCRIPTION OF THE INVENTION

[0010] It should be understood that in this application, including the figures, examples, and schemes, an oligonucleotide can be, but is not limited to, a double-stranded siRNA molecule.

[0011] Detailed Description GalNAc-linked small interfering RNA (siRNA) is a modality for mediating RNA interference (RNAi) in hepatocytes. GalNAc-linked small interfering RNA (siRNA) contains two key components: a GalNAc-targeting ligand that binds to the asialoglycoprotein receptor (ASGPr) found on the surface of hepatocytes to mediate uptake, and an siRNA oligonucleotide that, once delivered to the hepatocyte cytoplasm, can mediate the destruction of specific mRNA sequences (determined by the siRNA sequence) to reduce the expression of the associated protein product.

[0012] Although siRNA delivery to hepatocytes has been demonstrated using this class of therapeutic agent, successful application to other cell types has presented greater challenges. A suitable receptor must be identified that is expressed relatively selectively and in sufficient numbers on the target cell surface. Next, a suitable ligand must be identified that binds to the target receptor with high specificity and affinity. The third major hurdle is endosomal escape. Upon receptor binding, the ligand conjugate is internalized into the cell and trapped in an endosome. To reach the cytoplasm, the ligand conjugate must escape from the endosome. Despite the lack of an active endosomal escape mechanism, GalNAc conjugates still appear to mediate activity, likely because the receptor is highly abundant and rapidly recycled (approximately 15 minutes) after internalization. Active endosomal escape may not be necessary because the vast number of conjugate molecules are internalized into hepatocytes. This is not the case for other ligand-based siRNA conjugate systems.

[0013] Hepatic stellate cells (HSCs) play a key role in the progression of fibrosis. In response to chronic liver injury, HSCs are activated and are primarily responsible for collagen deposition and scarring in the liver. Various genes are involved and are validated targets for treatment, including RNAi strategies. As described herein, siRNA conjugates have been synthesized to target HSCs via the platelet-derived growth factor receptor (PDGFR) using a cyclic octapeptide motif. Notably, as described herein, trivalent presentation of pPB ligands outperforms monovalent presentation in terms of binding and uptake. Gene silencing experiments have also demonstrated that trivalent ligand structures are particularly effective, for example, when compared to monovalent and bivalent conjugates.

[0014] The present invention also provides a pPB conjugate of an endosomal releasing polymer (ERP), also referred to as pPB-ERP. The inventors have discovered that monovalency, i.e., one ERP moiety linked to one pPB moiety, optionally via a linker, is sufficient for pPB-ERP. The terms "endosomal releasing polymer" (ERT) and "diblock polymer" are used interchangeably herein. In certain embodiments, specific ERTs provided by the present invention, optionally characterized by the presence of a functional group Z or the presence of a moiety T-L2-Y'-X' as described herein, can be obtained by a process according to the present invention. In certain embodiments, the ERTs according to the present invention are conjugated to specific ligands T, such as specific targeting ligands, as described herein.

[0015] The present inventors have designed a new process for individually preparing conjugated ERPs (i.e., pPB-ERPs). Previously known ERP preparation processes using other ligands, including, for example, N-acetylglucosamine, were found to be neither user-friendly nor technically applicable for ligands such as pPB, necessitating the design of a new process. pPB, as well as certain other ligands described herein, such as other polypeptides, which may or may not be cyclic, do not tolerate or are not amenable to RAFT polymerization as used in the prior art. Furthermore, as a result of expensive ligands, such as polypeptides including pPB and its derivatives, being conjugated in the final process step, the process according to the present invention is significantly more cost-effective with respect to the ligand.

[0016] The present invention also provides methods for using one or more pPB conjugates of the present invention, e.g., pPB-siRNA according to the present invention, pPB-ERP according to the present invention, alone and / or in combination, in the treatment of one or more diseases in humans or animals, preferably humans.

[0017] In certain embodiments, the disease is a liver disease.

[0018] In certain embodiments, the disease is a kidney disease.

[0019] In certain embodiments, the disease is liver fibrosis.

[0020] In certain embodiments, the disease is non-alcoholic steatohepatitis (NASH).

[0021] In certain embodiments, the disease is renal fibrosis.

[0022] In certain embodiments, the disease is clear cell renal cell carcinoma.

[0023] In certain embodiments, the disease is alcoholic steatohepatitis (ASH).

[0024] Also provided are methods for delivering a therapeutic agent to hepatic stellate cells (HSCs) in vivo or in vitro, comprising contacting the HSCs with a conjugate described herein.

[0025] Liver fibrosis is caused by excessive accumulation of extracellular matrix during chronic liver injury. Activation of hepatic stellate cells (HSCs) is a critical step in liver fibrogenesis. Targeted delivery of therapeutic agents to HSCs, such as activated HSCs, may be important for successful treatment of liver fibrosis. Numerous protein markers have been found to be overexpressed in activated HSCs, and their ligands have been used to specifically deliver various anti-fibrotic agents (see, e.g., Chen et al., Journal of Pharmacology and Experimental Therapeutics, 2019, 370 (3) 695-702). However, other methods for delivering therapeutic agents to HSCs require the use of other systems.

[0026] Liver fibrosis is caused by the formation of an abnormal amount of scar tissue in the liver. Liver fibrosis occurs as the liver tries to repair and replace damaged cells. Various disorders and drugs can injure the liver and cause fibrosis.

[0027] Nonalcoholic fatty liver disease (NAFLD) is a condition characterized by the accumulation of triglycerides in the liver. Nonalcoholic steatohepatitis (NASH) is a type of NAFLD. NASH is associated with inflammatory changes and liver cell injury. NASH is one of the leading causes of liver disease and often progresses to liver fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Nonalcoholic steatohepatitis (NASH) and alcoholic steatohepatitis (ASH) share similar pathogenesis and histopathology, but differ in etiology and epidemiology. NASH and ASH are advanced stages of nonalcoholic fatty liver disease (NAFLD) and alcoholic fatty liver disease (AFLD). Alcoholic steatohepatitis (ASH) is a chronic, progressive liver disease caused by excessive, prolonged alcohol use, characterized by hepatic fibrosis and possible necrosis of liver tissue. Women are more susceptible to this disease because their alcohol metabolism is slower than men's.

[0028] Liver fibrosis is an important underlying cause of liver dysfunction and predicts many deaths. Progression to cirrhosis and HCC leads to eventual liver failure and thus the need for liver transplantation. The current prevalence of NASH-associated fibrosis (F2 or later) in the United States is approximately 3.8 million patients. Physicians typically recommend weight loss to treat NAFLD and NASH. While weight loss can reduce hepatic fat, inflammation, and fibrosis, no medications are approved for the treatment of NAFLD and NASH. Specifically, no medications are approved for the treatment of liver fibrosis (Clin Liver Dis. 2008 Nov;12(4):733-46, N Engl J Med. 2017 Nov 23;377(21):2063-2072, J Hepatol. 2017 Dec;67(6):1265-127). Thus, new therapeutic treatment options, including delivery options, are needed to treat liver fibrosis, for example, in the setting of NASH or ASH.

[0029] As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0030] As used herein, the term "conjugate" includes a compound of formula (I) comprising an oligonucleotide (e.g., an siRNA molecule) linked to a targeting ligand. Thus, the terms compound and conjugate may be used interchangeably herein.

[0031] As used herein, the term "small interfering RNA" or "siRNA" refers to a double-stranded RNA (i.e., duplex RNA) that can reduce or inhibit expression of a target gene or sequence (e.g., by mediating degradation of mRNA complementary to the siRNA sequence or by inhibiting translation of mRNA complementary to the siRNA sequence) when the siRNA is present in the same cell as the target gene or sequence. The siRNA may have substantial or complete identity to the target gene or sequence or may contain regions of mismatch (i.e., mismatch motifs). In certain embodiments, the siRNA may be about 19-25 (duplex) nucleotides in length, preferably about 20-24, 21-22, or 21-23 (duplex) nucleotides in length. The siRNA duplex may include a 3' overhang of about 1 to about 5 nucleotides or about 2 to about 3 nucleotides and a 5' phosphate terminus. Examples of siRNA include, but are not limited to, double-stranded polynucleotide molecules assembled from two separate stranded molecules, one strand being the sense strand and the other being the complementary antisense strand.

[0032] In certain embodiments, the 5' overhang and / or 3' overhang on one or both strands of the siRNA comprises 1 to 5 (e.g., 1, 2, 3, 4, or 5) modified and / or unmodified deoxythymidine (t or dT) nucleotides, 1 to 5 (e.g., 1, 2, 3, 4, or 5) modified (e.g., 2'OMe) and / or unmodified uridine (U) ribonucleotides, and / or 1 to 5 (e.g., 1, 2, 3, 4, or 5) modified (e.g., 2'OMe) and / or unmodified ribonucleotides or deoxyribonucleotides that are complementary to a target sequence (e.g., a 3' overhang in the antisense strand) or its complementary strand (e.g., a 3' overhang in the sense strand).

[0033] Preferably, siRNAs are chemically synthesized. Alternatively, siRNAs can be generated by cleaving longer dsRNAs (e.g., dsRNAs longer than about 25 nucleotides) with E. coli RNase III or Dicer. These enzymes process dsRNA into biologically active siRNA (see, e.g., Yang et al., Proc. Natl. Acad. Sci. USA, 99:9942-9947 (2002); Calegari et al., Proc. Natl. Acad. Sci. USA, 99:14236 (2002); Byrom et al., Ambion TechNotes, 10(1):4-6 (2003); Kawasaki et al., Nucleic Acids Res., 31:981-987 (2003); Knight et al., Science, 293:2269-2271 (2001); and Robertson et al., J. Biol. Chem., 243:82 (1968)). Preferably, dsRNA is at least 50 nucleotides to about 100, 200, 300, 400, or 500 nucleotides in length.DsRNA can be as long as 1000, 1500, 2000, 5000 nucleotides, or even longer.DsRNA can encode the entire gene transcript, or can encode a partial gene transcript.In some cases, siRNA can be encoded by a plasmid (for example, can be transcribed as a sequence that automatically folds into a double strand with a hairpin loop).

[0034] The phrase " inhibit the expression of target gene " refers to that the siRNA of the present invention can stop, reduce or inhibit the expression of target gene.To examine the degree of gene silencing, test sample (for example, the biological sample from the organism of interest that expresses target gene, or the sample of cells in culture that expresses target gene) is contacted with the siRNA that stops, reduces or inhibits the expression of target gene.The expression of target gene in test sample is compared with the expression of target gene in control sample (for example, the biological sample from the organism of interest that expresses target gene, or the sample of cells in culture that expresses target gene) that is not contacted with siRNA.Control sample (for example, the sample that expresses target gene) can be assigned a value of 100%. In certain embodiments, silencing, inhibition, or reduction of expression of a target gene is achieved when the value for the test sample is about 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% compared to a control sample (e.g., buffer only, an siRNA sequence targeting a different gene, a scrambled siRNA sequence, etc.). Suitable assays include, but are not limited to, examining protein or mRNA levels using techniques known to those skilled in the art, such as dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays.

[0035] The term "synthetic activating group" refers to a group that can be attached to an atom to activate the atom so that it can form a covalent bond with another reactive group. It is understood that the nature of a synthetic activating group can depend on the atom being activated. For example, when a synthetic activating group is attached to an oxygen atom, the synthetic activating group is a group that activates the oxygen atom to form a bond with another reactive group (e.g., an ester, carbamate, or ether bond). Such synthetic activating groups are known. Examples of synthetic activating groups that can be attached to an oxygen atom include, but are not limited to, acetate, succinate, triflate, and mesylate. When a synthetic activating group is attached to the oxygen atom of a carboxylic acid, the synthetic activating group can be a group derived from a known coupling reagent (e.g., a known amide coupling reagent). Such coupling reagents are known. Examples of such coupling reagents include, but are not limited to, N,N'-dicyclohexylcarbodimide (DCC), hydroxybenzotriazole (HOBt), N-(3-dimethylaminopropyl)-N'-ethyl carbonate (EDC), (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), or O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU).

[0036] An "effective amount" or "therapeutically effective amount" of a therapeutic nucleic acid, such as an siRNA, is an amount sufficient to produce a desired effect, e.g., inhibition of expression of a target sequence compared to normal expression levels detected in the absence of the siRNA. In certain embodiments, inhibition of expression of a target gene or target sequence is achieved when the value obtained with the siRNA is about 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% compared to a control (e.g., buffer only, an siRNA sequence targeting a different gene, a scrambled siRNA sequence, etc.). Suitable assays for measuring expression of a target gene or target sequence include, but are not limited to, examining protein or mRNA levels using techniques known to those skilled in the art, such as dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays.

[0037] As used herein, the term "nucleic acid" refers to a polymer containing at least two nucleotides (i.e., deoxyribonucleotides or ribonucleotides) in either single- or double-stranded form, including DNA and RNA. A "nucleotide" contains a sugar, deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together via the phosphate group. "Base" includes purines and pyrimidines, including the naturally occurring compounds adenine, thymine, guanine, cytosine, uracil, and inosine, as well as natural analogs and synthetic derivatives of purines and pyrimidines, including, but not limited to, modifications that place new reactive groups, such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides. Nucleic acids include synthetic, natural, and non-natural nucleic acids containing known nucleotide analogs or modified backbone residues or linkages that have similar linkages as the reference nucleic acid. Examples of such analogs and / or modified residues include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Additionally, nucleic acids may contain one or more UNA moieties.

[0038] The term "oligonucleotide" includes nucleotides containing up to about 60 nucleotides. Deoxyribooligonucleotides consist of a pentose sugar called deoxyribose, with phosphate covalently attached to the 5' and 3' carbons, forming an alternating, unbranched polymer. Ribooligonucleotides consist of a similar repeating structure in which the pentose sugar is ribose. RNA can take the form of, for example, small interfering RNA (siRNA), Dicer-substrate dsRNA, small hairpin RNA (shRNA), small activating RNA (saRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), tRNA, rRNA, tRNA, and viral RNA (vRNA). Thus, the term "oligonucleotide" refers to a polymer or oligomer of nucleotide or nucleoside monomers composed of natural bases, sugars, and intersugar (backbone) linkages. The term "oligonucleotide" also includes polymers or oligomers, or portions thereof, containing unnatural monomers. Such modified or substituted oligonucleotides may possess properties such as improved cellular uptake, reduced immunogenicity, and / or increased stability in the presence of nucleases.

[0039] The term "gene" refers to a nucleic acid (eg, DNA or RNA) sequence that comprises partial or full-length coding sequences necessary to produce a polypeptide or precursor polypeptide.

[0040] As used herein, "gene product" refers to the product of a gene, e.g., an RNA transcript or a polypeptide.

[0041] As used herein, the term "label" includes a group that allows for detection of a compound in vitro or in vivo. The term "labeling substance" is used synonymously with the term label. The label may be selected from the group including, but not limited to, a fluorophore, a chromophore, and a radionucleotide. In one embodiment, the label can be detected by spectroscopy. In one embodiment, the label can be detected by fluorescence spectroscopy. In one embodiment, the label is a group derived from fluorescein isothiocyanate (FITC) or Cy5. In one embodiment, the label can be detected by an instrument suitable for detecting any one or more of alpha particles, beta particles, gamma rays, and X-rays.

[0042] The term "alkyl," as used herein, by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain hydrocarbon radical having the specified number of carbon atoms (i.e., C 1-8 means 1 to 8 carbons). Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. The term "alkenyl" refers to unsaturated alkyl groups having one or more double bonds. Similarly, the term "alkynyl" refers to unsaturated alkyl groups having one or more triple bonds. Examples of such unsaturated alkyl groups include vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and higher homologs and isomers.

[0043] The term "alkylene," by itself or as part of another substituent, means a divalent group derived from an alkane (including straight-chain or branched-chain alkanes), as exemplified by -CH2CH2CH2CH2- and -CH(CH3)CH2CH2-.

[0044] The terms "cycloalkyl," "carbocyclic," or "carbocycle" refer to a hydrocarbon ring structure having a total number of ring atoms from 3 to 20 (e.g., a 3-20 membered cycloalkyl is a cycloalkyl having 3 to 20 ring atoms, or C 3-20 Cycloalkyl is a cycloalkyl having 3 to 20 carbon ring atoms; 3-5 membered cycloalkyls are fully saturated or have one or less double bonds between the ring vertices, and 6-membered or larger cycloalkyls are fully saturated or have two or less double bonds between the ring vertices. As used herein, "cycloalkyl," "carbocyclic," or "carbocycle" refers to, for example, bicyclo[2.2.1]heptane, pinane, bicyclo[2.2.2]octane, adamantane, norbornene, spirocyclic C 5-12 It is also intended to refer to bicyclic, polycyclic, and spirocyclic hydrocarbon ring structures such as alkanes. As used herein, the terms "alkenyl," "alkynyl," "cycloalkyl," "carbocycle," and "carbocyclic" are intended to include mono- and poly-halogenated versions thereof.

[0045] The terms "heterocycloalkyl," "heterocyclic," or "heterocycle" refer to a saturated or partially unsaturated ring structure radical having 3 to 20 ring atoms containing 1 to 10 heteroatoms selected from N, O, and S (e.g., a 3-20 membered heterocycloalkyl is a heterocycloalkyl group having 3 to 20 ring atoms, C 2-19Heterocycloalkyl refers to a heterocycloalkyl having 3 to 10 ring atoms, of which 2 to 19 ring atoms are carbon, wherein the nitrogen and sulfur ring atoms are optionally oxidized and the nitrogen atom is optionally quaternized. Unless otherwise specified, a "heterocycloalkyl," "heterocyclic," or "heterocyclic" ring can be a monocyclic, bicyclic, spirocyclic, or polycyclic ring structure. Non-limiting examples of "heterocycloalkyl," "heterocyclic," or "heterocyclic" rings include pyrrolidine, piperidine, N-methylpiperidine, imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide, piperidine, pyrimidine-2,4(1H,3H)-dione, 1,4-dioxane, morpholine, thiomorpholine, thiomorpholine-S-oxide, thiomo Examples of heterocyclic groups include methylpyrroline-S,S-oxide, piperazine, pyran, pyridone, 3-pyrroline, thiopyran, pyrone, tetrahydrofuran, tetrahydrothiophene, quinuclidine, tropane, 2-azaspiro[3.3]heptane, (1R,5S)-3-azabicyclo[3.2.1]octane, (1s,4s)-2-azabicyclo[2.2.2]octane, (1R,4R)-2-oxa-5-azabicyclo[2.2.2]octane, and the like. A "heterocycloalkyl," "heterocyclic," or "heterocycle" group can be attached to the remainder of the molecule through one or more ring carbons or heteroatoms. A "heterocycloalkyl," "heterocyclic," or "heterocycle" may include monohalogenated and polyhalogenated versions thereof.

[0046] The terms "alkoxy" and "alkylthio" are used in the conventional sense to refer to an alkyl group attached to the remainder of the molecule via an oxygen atom ("oxy") or a thio group, and further include mono- and poly-halogenated versions thereof.

[0047] The terms "halo" or "halogen," by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. The term "(halo)alkyl" is intended to include both "alkyl" and "haloalkyl" substituents. Furthermore, the term "haloalkyl" is intended to include monohaloalkyl and polyhaloalkyl. For example, "C 1-4 The term "haloalkyl" is meant to include trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, difluoromethyl and the like.

[0048] The term "aryl" means a carbocyclic aromatic group having 6 to 14 carbon atoms, whether fused to one or more groups. Examples of aryl groups, unless otherwise specified, include phenyl, naphthyl, biphenyl, and the like.

[0049] The term "heteroaryl" refers to an aryl ring containing 1 to 5 heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom is optionally quaternized. A heteroaryl group may be attached to the remainder of the molecule through a heteroatom. Examples of heteroaryl groups include pyridyl, pyridazinyl, pyrazinyl, pyrimidinyl, triazinyl, quinolinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, benzotriazinyl, purinyl, benzimidazolyl, benzopyrazolyl, benzotriazolyl, benzisoxazolyl, isobenzofuryl, isoindolyl, indolizinyl, benzotriazinyl, thienopyridinyl, thienopyrimidinyl, pyrazolopyrimidinyl, imidazopyridine, benzothiaxolyl, benzofuranyl, benzothienyl, indolyl, quinolyl, isoquinolyl, isothiazolyl, pyrazolyl, indazolyl, pteridinyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiadiazolyl, pyrrolyl, thiazolyl, furyl, thienyl, and the like.

[0050] The term "animal" includes mammalian species, such as humans, mice, rats, dogs, cats, hamsters, guinea pigs, rabbits, farm animals, and the like.

[0051] The term "alkylamino" includes groups of the formula: --N(H)R, where R is alkyl as defined herein.

[0052] The term "dialkylamino" includes groups of the formula: -NR2, where each R is independently alkyl as defined herein.

[0053] The term "salt" includes any anionic and cationic complexes, such as complexes formed between a cationic lipid and one or more anions. Non-limiting examples of anions include inorganic and organic anions, such as hydride, fluoride, chloride, bromide, iodide, oxalate (e.g., hemioxalate), phosphate, phosphonate, hydrogen phosphate, dihydrogen phosphate, oxide, carbonate, bicarbonate, nitrate, nitrite, nitride, bisulfate, sulfide, sulfite, bisulfate, sulfate, thiosulfate, hydrogen sulfate, borate, formate, acetate, benzoate, citrate, tartrate, lactate, acrylate, polyacrylate, fumarate. Cationic lipid salts include salts, maleate, itaconate, glycolate, gluconate, malate, mandelate, tiglate, ascorbate, salicylate, polymethacrylate, perchlorate, chlorate, chlorite, hypochlorite, bromate, hypobromite, iodate, alkylsulfonate, arylsulfonate, arsenate, arsenite, chromate, dichromate, cyanide, cyanate, thiocyanate, hydroxide, peroxide, permanganate, and mixtures thereof.In certain embodiments, the salt of cationic lipid disclosed herein is crystalline salt.

[0054] The term "acyl" includes any alkyl, alkenyl, or alkynyl in which the carbon at the point of attachment is substituted with an oxo group, as defined below. The following are non-limiting examples of acyl groups: -C(=O)alkyl, -C(=O)alkenyl, and -C(=O)alkynyl.

[0055] It is understood by those skilled in the art that compounds of the present invention having chiral centers can exist and be isolated in optically active and racemic forms. Some compounds may exhibit polymorphism. It should be understood that the present invention encompasses any racemic, optically active, polymorphic, or stereoisomeric form of the compounds of the present invention, or mixtures thereof, that have the useful properties described herein. Methods for preparing optically active forms (for example, by resolving racemic forms by recrystallization, synthesis from optically active starting materials, chiral synthesis, or chromatographic separation using chiral stationary phases) are well known in the art.

[0056] When a bond in the formulas of compounds herein is drawn in a non-stereochemical manner (e.g., flat), the atoms attached to the bond include all stereochemical possibilities. Unless otherwise specified, when a bond in the formulas of compounds herein is drawn in a defined stereochemical manner (e.g., bold, bold wedge, dashed line, or dashed wedge), it is to be understood that the atoms attached to the stereochemical bond are enriched in the absolute stereoisomer depicted. In one embodiment, the compound may be at least 51% of the absolute stereoisomer depicted. In another embodiment, the compound may be at least 60% of the absolute stereoisomer depicted. In another embodiment, the compound may be at least 80% of the absolute stereoisomer depicted. In another embodiment, the compound may be at least 90% of the absolute stereoisomer depicted. In another embodiment, the compound may be at least 95% of the absolute stereoisomer depicted. In another embodiment, the compound may be at least 99% of the absolute stereoisomer depicted.

[0057] As used herein, the term "peptide" includes oligopeptides and polypeptides and refers to a substance comprising two or more, preferably three or more, preferably four or more, preferably six or more, e.g., eight or more amino acids covalently linked by peptide bonds. Peptides may include natural and unnatural amino acids, e.g., D-amino acids. In one embodiment, peptides contain only natural amino acids. In one embodiment, all amino acids in a peptide are L-amino acids. Peptides consisting of amino acids may also be referred to as "polypeptides." For example, a polypeptide has 6 to 10 amino acid residues, e.g., 7 to 9 amino acid residues, preferably about 8 amino acid residues.

[0058] As used herein, a "cyclic peptide" or "cyclized peptide" refers to a peptide or polypeptide chain that forms a ring, preferably resulting in a peptide or polypeptide having an intramolecular bond between two non-adjacent amino acids within the peptide. The intramolecular bond may be selected from the group consisting of, but is not limited to, (i) backbone-to-backbone (i.e., peptide bond), (ii) side chain-to-backbone, and (iii) side chain-to-side chain cycle formation. Cyclic peptides of the invention may include a linking group resulting from any of the foregoing, such as, for example, a peptide bond in the case of backbone-to-backbone cyclization, or a disulfide bond resulting from two residues containing thiol groups, e.g., cysteines, that can form an intramolecular disulfide bridge to form a cyclic peptide.

[0059] As used herein, the term "percent identity" refers to the percentage of identical amino acid residues between two sequences to be compared, obtained after optimally aligning the two sequences over their entire length. Sequence comparison aims to determine the percentage of sequence identity between two amino acid sequences, and is typically performed by comparing these sequences after optimal alignment. The comparison is performed by segments, or "comparison windows," to identify and compare local regions of sequence similarity. Optimal alignment of sequences for comparison can be generated by the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, the local homology algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48, 443, the similarity search method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85, 2444, or computer programs using these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA, available from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). Alternatively, two sequences can be manually aligned. This is convenient, for example, for short polypeptide sequences, e.g., those of 10 amino acid residues or less. "Percent identity" is calculated as follows: Aligning and comparing the number of identical positions between the two sequences; Divide by the number of positions to compare, Multiply the result by 100 This gives the percent identity between these two sequences.

[0060] Unless otherwise specified herein, the term "about," when used in connection with a value or range of values, means plus or minus 5% of the stated value or range of values.

[0061] Creation of siRNA molecules siRNA can be provided in several forms, including, for example, one or more isolated small interfering RNA (siRNA) duplexes, longer double-stranded RNA (dsRNA), or siRNA or dsRNA transcribed from a transcription cassette in a DNA plasmid.In some embodiments, siRNA can be produced enzymatically or by partial / total organic synthesis, and modified ribonucleotides can be introduced by in vitro enzymatic synthesis or organic synthesis.In certain cases, each strand is chemically prepared.Methods for synthesizing RNA molecules, such as the chemical synthesis method described in Verma and Eckstein (1998) or the chemical synthesis method described herein, are known in the art.

[0062] Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids, creating and screening cDNA libraries, and performing PCR are well known in the art (see, for example, Gubler and Hoffman, Gene, 25:263-269 (1983); Sambrook et al., supra; Ausubel et al., supra), and PCR methods are also well known in the art (see U.S. Patent Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990)). Expression libraries are also well known to those skilled in the art. Further basic textbooks that disclose general methods for use in the present invention include Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994). The disclosures of these references are incorporated herein by reference in their entireties for all purposes.

[0063] Typically, siRNA is chemically synthesized.The oligonucleotides comprising the siRNA molecules of the present invention can be synthesized by any of various techniques known in the art, for example, Usman et al., J. Am. Chem. Soc., 109:7845 (1987); Scaringe et al., Nucl. Acids Res., 18:5433 (1990); Wincott et al., Nucl. Acids Res., 23:2677-2684 (1995); and Wincott et al., Methods Mol. Bio., 74:59 (1997).Synthesis of oligonucleotides utilizes common protecting groups and coupling groups, such as dimethoxytrityl at 5'-end and phosphoramidite at 3'-end.As a non-limiting example, small-scale synthesis can be carried out using a 0.2 μmol scale protocol on an Applied Biosystems synthesizer. Alternatively, synthesis can be performed on a 0.2 μmol scale in a Protogene (Palo Alto, CA) 96-well plate synthesizer. However, larger or smaller scale syntheses are within the scope of the present invention. Reagents suitable for oligonucleotide synthesis, methods for RNA deprotection, and methods for RNA purification are known to those of skill in the art.

[0064] An siRNA molecule can be assembled from two different oligonucleotides, one containing the sense strand of the siRNA and the other containing the antisense strand of the siRNA. For example, each strand can be synthesized separately and then joined together by hybridization or ligation after synthesis and / or deprotection.

[0065] Linking Group (L, L1, or L2) The linking group may be variable, provided that the targeting conjugate functions as described herein. The linking group may vary in length and atomic composition, for example, branched, unbranched, cyclic, or a combination thereof. The linking group may also adjust the properties of the targeting conjugate, including, but not limited to, solubility, stability, and aggregation.

[0066] In one embodiment, the linker comprises about 3 to 5,000 atoms. In one embodiment, the linker comprises about 3 to 4,000 atoms. In one embodiment, the linker comprises about 3 to 2,000 atoms. In one embodiment, the linker comprises about 3 to 1,000 atoms. In one embodiment, the linker comprises about 3 to 750 atoms. In one embodiment, the linker comprises about 3 to 500 atoms. In one embodiment, the linker comprises about 3 to 250 atoms. In one embodiment, the linker comprises about 3 to 100 atoms. In one embodiment, the linker comprises about 3 to 50 atoms. In one embodiment, the linker comprises about 3 to 25 atoms.

[0067] In one embodiment, the linker comprises about 10 to 5,000 atoms. In one embodiment, the linker comprises about 10 to 4,000 atoms. In one embodiment, the linker comprises about 10 to 2,000 atoms. In one embodiment, the linker comprises about 10 to 1,000 atoms. In one embodiment, the linker comprises about 10 to 750 atoms. In one embodiment, the linker comprises about 10 to 500 atoms. In one embodiment, the linker comprises about 10 to 250 atoms. In one embodiment, the linker comprises about 10 to 100 atoms. In one embodiment, the linker comprises about 10 to 50 atoms. In one embodiment, the linker comprises about 10 to 25 atoms.

[0068] In one embodiment, the linker L comprises atoms selected from H, C, N, S, and O.

[0069] In one embodiment, the linker L comprises atoms selected from H, C, N, S, P, and O.

[0070] In one embodiment, the linker L comprises a branched or unbranched, saturated or unsaturated hydrocarbon chain having about 1 to 1000 (or 1 to 750, 1 to 500, 1 to 250, 1 to 100, 1 to 50, 1 to 25, 1 to 10, 1 to 5, 5 to 1000, 5 to 750, 5 to 500, 5 to 250, 5 to 100, 5 to 50, 5 to 25, 5 to 10, or 2 to 5 carbon atoms), wherein one or more of the carbon atoms are optionally independently selected from -O-, -S, -N(R a )-, a 3- to 7-membered heterocycle, a 5- to 6-membered heteroaryl, or a carbocycle, each of which is optionally and independently selected from (C-C)alkyl, (C-C)alkoxy, (C-C)cycloalkyl, (C-C)alkanoyl, (C-C)alkanoyloxy, (C-C)alkoxycarbonyl, (C-C)alkylthio, azido, cyano, nitro, halo, -N(R a )2, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy, and each R a are independently H or (C1-C6) alkyl. In one embodiment, the linker comprises a branched or unbranched, saturated or unsaturated hydrocarbon chain having about 1-1000 (or 1-750, 1-500, 1-250, 1-100, 1-50, 1-25, 1-10, 1-5, 5-1000, 5-750, 5-500, 5-250, 5-100, 5-50, 5-25, 5-10, or 2-5 carbon atoms), wherein one or more of the carbon atoms are optionally independently selected from -O-, -S, -N(R a )- and each R a are independently H or (C1-C6) alkyl.

[0071] In one embodiment, the linker L comprises polyethylene glycol. In one embodiment, the linker comprises polyethylene glycol linked to the remainder of the targeting conjugate by a carbonyl group. In one embodiment, the polyethylene glycol comprises about 1 to about 500, or about 5 to about 500, or about 3 to about 100 repeating (e.g., —CHCHO—) units (Greenwald, R.B., et al., Poly(ethylene glycol) Prodrugs: Altered Pharmacokinetics and Pharmacodynamics, Chapter, 2.3.1., 283-338; Filpula, D., et al., Releasable PEGylation of proteins with customized linkers, Advanced Drug Delivery, 60, 2008, 29-49; Zhao, H., et al., Drug Conjugates with Poly(Ethylene Glycol), Drug Delivery in Oncology, 2012, 627-656).

[0072] In one embodiment, the linker L is -NH(CH2CH2O)4CH2CH2C(=O)-. In one embodiment, the linker L is -NH(CH2CH2O) n CH2CH2C(=O)-, where n is 1 to 500, 5 to 500, 3 to 100, 5 to 50, 1 to 50, 1 to 20, 1 to 10, 1 to 5, 2 to 50, 2 to 20, 2 to 10, 2 to 5, 3 to 50, 3 to 20, 3 to 10, 3 to 5, 4 to 50, 4 to 20, 4 to 10, and 4 to 5.

[0073] In one aspect, the linker L is —(CH 2 CH 2 O) 4 CH 2 CH 2 C(═O)—.

[0074] In one aspect, the linker L is a branched or unbranched, saturated or unsaturated hydrocarbon chain having from about 1 to about 500 carbon atoms, wherein one or more of the carbon atoms are optionally independently selected from -O-, -S, -N(R a)-, a 3-20 membered heterocycle, a 3-20 membered heteroaryl, or a 3-20 membered carbocycle, each of which is optionally and independently selected from (C-C) alkyl, (C-C) alkoxy, (C-C) cycloalkyl, (C-C) alkanoyl, (C-C) alkanoyloxy, (C-C) alkoxycarbonyl, azido, cyano, nitro, halo, -N(R a )2, hydroxy, oxo (=O), and carboxy; and each R a are independently H or (C1-C6) alkyl.

[0075] In one aspect, the linker L is a branched or unbranched, saturated or unsaturated hydrocarbon chain having from about 1 to about 100 carbon atoms, one or more of which may optionally be independently selected from -O-, -S, -N(R a )-, a 3-20 membered heterocycle, a 3-20 membered heteroaryl, or a 3-20 membered carbocycle, each of which is optionally and independently selected from (C-C) alkyl, (C-C) alkoxy, (C-C) cycloalkyl, (C-C) alkanoyl, (C-C) alkanoyloxy, (C-C) alkoxycarbonyl, azido, cyano, nitro, halo, -N(R a )2, hydroxy, oxo (=O), and carboxy; and each R a are independently H or (C1-C6) alkyl.

[0076] In one aspect, the linker L is a branched or unbranched, saturated or unsaturated hydrocarbon chain having from about 1 to about 100 carbon atoms, one or more of which may optionally be independently selected from -O-, -S, -N(R a)-, a 3-20 membered heterocycle, a 3-20 membered heteroaryl, or a 3-20 membered carbocycle, each of which is optionally and independently substituted with one or more (e.g., 1, 2, 3, 4, 5, or more) substituents independently selected from the group consisting of azido, halo, and oxo (=O), and carboxy; and each R a are independently H or (C1-C6) alkyl.

[0077] In one aspect, the linker L is a branched or unbranched, saturated or unsaturated hydrocarbon chain having from about 1 to about 50 carbon atoms, one or more of which may optionally be independently selected from -O-, -S, -N(R a )-, a 3-20 membered heterocycle, a 3-20 membered heteroaryl, or a 3-20 membered carbocycle, each of which is optionally and independently substituted with one or more (e.g., 1, 2, 3, 4, 5, or more) substituents independently selected from the group consisting of azido, halo, and oxo (=O), and carboxy; and each R a are independently H or (C1-C6) alkyl.

[0078] In one aspect, the linker L is a branched or unbranched, saturated or unsaturated hydrocarbon chain having from about 1 to about 50 carbon atoms, one or more of which may optionally be independently selected from -O-, -S, -N(R a )-, or R b and each chain and R b is optionally and independently substituted with one or more (e.g., 1, 2, 3, 4, 5, or more) substituents independently selected from the group consisting of azido, halo, and oxo (=O), and carboxy; and each R b are independently H or (C-C) alkyl, and each R b is the following: TIFF2025531872000011.tif46128.

[0079] In one aspect, the linker L is -C(=O)(CH2) a It contains N(H)C(=O)-, where a is 3, 4, 5, 6, 7, or 8.

[0080] In one aspect, the linker L is -(CH2CH2O) b CH2CH2C(=O)- or -(CH2CH2O) b It includes CH2CH2N(H)C(=O)-, and b is 2 to 50.

[0081] In one embodiment, the linker L comprises phen-1,3-diyl or phen-1-1,3,5-triyl.

[0082] In one aspect, the linker L is: Includes TIFF2025531872000012.tif45128.

[0083] In one aspect, each R 1 is linked to L via the carbonyl group of L.

[0084] In one aspect, each R 2 is linked to L via a carbon-carbon bond.

[0085] In one aspect, the linker L is: TIFF2025531872000013.tif212144TIFF2025531872000014.tif185139.

[0086] In one aspect, the linker is comprised of a moiety of the formula: L2-Y'Z'-L1, where L2, Y'Z', and L1 are each as defined herein. Optionally, Y and Z are selected such that Y'Z' together comprise an oxime functional group. Alternatively, Y and Z are selected such that Y'Z' together comprise a triazole ring.

[0087] Aspects of the present invention One aspect of the present invention is a compound of Formula I, or a salt thereof, as described in the Summary of the Invention.

[0088] In a first aspect (Embodiment 1; abbreviated as "E1"), the present invention provides a compound of formula (I): TIFF2025531872000015.tif13128 or a salt thereof, wherein x is 2, 3, 4, or 5; Each R 1 is independently a targeting ligand, and the targeting ligand is a) SEQ ID NO: 1: C * SRNLIDC * (SEQ ID NO: 1) A cyclic polypeptide represented by the formula: * represents a disulfide bond linking two C residues thereby forming a cyclic polypeptide, and b) C * Any cyclic polypeptide having at least 80% sequence identity (e.g., at least 85%; e.g., at least 87.5%; e.g., at least 90%; e.g., at least 95%; e.g., at least 99% sequence identity) with a polypeptide set forth in SEQ ID NO:1, provided that both residues are present, wherein: * exhibits a disulfide bond linking two C residues thereby forming a cyclic polypeptide. Selected from L is a linking group, y is 1, 2, 3, 4, or 5; Each R 2 are independently an oligonucleotide, a label (e.g., a label derived from fluorescein isothiocyanate (FITC) or Cy5), a phenyl group substituted with a formyl (-CHO) group, or a group of the formula: Based on TIFF2025531872000016.tif17128, The present invention provides a compound or a salt thereof.

[0089] In one aspect, R 1 is a polypeptide, preferably a cyclic polypeptide having 6 to 10 amino acid residues, for example, 7 to 9 amino acid residues, preferably about 8 amino acid residues.

[0090] In one embodiment, the polypeptide defined in b) is identical to the polypeptide defined in a), except that exactly one of the eight amino acid residues has been replaced with another amino acid and is therefore not identical. In that case, the sequence identity of the polypeptide defined in b) is 7 (= identical amino acid) / 8 (all positions). * 100=87.5%. Thus, in a preferred embodiment, the polypeptide of b) described herein has 87.5% sequence identity with the polypeptide defined in a). In one embodiment, the polypeptide has 7 to 10 amino acid residues, preferably about 8 or 9 amino acid residues, and most preferably 8 amino acid residues.

[0091] Alternatively, in one embodiment, R1 can be described as a polypeptide having 7 to 10 amino acid residues, preferably about 8 or 9 amino acid residues, and most preferably 8 amino acid residues, which polypeptide has the amino acid sequence set forth in SEQ ID NO:1, except for one or more amino acid residues being inserted, deleted, or replaced. Preferably, one to three amino acid residues are inserted, deleted, or replaced, more preferably two or fewer amino acid residues are inserted, deleted, or replaced, and most preferably one amino acid residue is inserted, deleted, or replaced. The polypeptide has at least two functional groups capable of forming bonds with each other, e.g., thiol groups capable of forming disulfide bonds to form a cyclic polypeptide, e.g., the thiol group of a cysteine ​​residue. The functional groups capable of forming bonds with each other are preferably located at or near the N-terminus and at or near the C-terminus, respectively.

[0092] In one aspect, R1 is a polypeptide having a core as defined in a) or b) above, plus an extension of the polypeptide sequence at the N-terminus and / or C-terminus of the polypeptide defined above, such that the entire R1 is a longer polypeptide including, but not limited to, R1.

[0093] In the cyclic polypeptides described above, cyclization typically occurs by the formation of a disulfide bridge between cysteine ​​residues of the peptide, for example, between the amino acid at position 1 and the amino acid at position 8 of the amino acid sequences described herein, particularly in a) and b) above. Alternatively, an intramolecular bond may be formed in the peptides described herein by a cyclization other than Cys-Cys cyclization. Thus, in another aspect, the present invention also contemplates peptides in which one or both of the cysteine ​​moieties in the peptides described herein are replaced with a moiety other than Cys. In that aspect, one or both of the Cys residues at the first and last positions (e.g., positions 1 and 8) of the amino acid sequences described herein may be replaced with an amino acid capable of forming an intramolecular bond in the peptide. Preferably, the bond is formed between the amino acids at the first and last positions (e.g., positions 1 and 8) of the amino acid sequences described herein.

[0094] Further aspects (E2 to E68) are described below. E2. The compound or salt according to E1, wherein x is 2 or 3. Such compounds are particularly advantageous, for example, when R2 is an oligonucleotide, e.g., an siRNA (see, e.g., Example 12 for non-limiting exemplary embodiments).

[0095] E3. The compound or salt according to E1, wherein x is 3.

[0096] E4. Each R 1 is covalently bonded to L (i) via the N-terminus of the polypeptide, or (ii) via the C-terminus of the polypeptide, or (iii) via a side chain of an amino acid in the polypeptide.

[0097] E5. Each R 1 is covalently bonded to L via the N-terminus of the polypeptide.

[0098] E6. Each R 1 but independently, the formula: The compound or salt according to any one of E1 to E3, which is a targeting ligand of TIFF2025531872000017.tif69128.

[0099] E7. Each R 2 are independently an oligonucleotide.

[0100] E8. Each R 2 is independently an siRNA. Such compounds are particularly advantageous (see, e.g., Example 12 for non-limiting exemplary embodiments).

[0101] E9. The compound or salt of any one of E7-E8, wherein each oligonucleotide or each siRNA is attached to L via the phosphate P(=O)(OH)2-O- of each oligonucleotide or each siRNA.

[0102] E10. Each R 2 but independently, the formula: The compound or salt according to any one of E1 to E6, which is a group represented by TIFF2025531872000018.tif17128.

[0103] E11. Each R 2 is independently 3-formylphenyl or 4-formylphenyl.

[0104] E12. L is a branched or unbranched, saturated or unsaturated hydrocarbon chain having about 1 to about 500 carbon atoms, wherein one or more of the carbon atoms are optionally independently selected from -O-, -S, -N(R a)-, a 3-20 membered heterocycle, a 3-20 membered heteroaryl, a 6-14 membered aryl, or a 3-20 membered carbocycle, wherein each chain, the 3-20 membered heterocycle, the 3-20 membered heteroaryl, the 6-14 membered aryl, and the 3-20 membered carbocycle are optionally and independently selected from (C-C)alkyl, (C-C)alkoxy, (C-C)cycloalkyl, (C-C)alkanoyl, (C-C)alkanoyloxy, (C-C)alkoxycarbonyl, azido, cyano, nitro, halo, -N(R a )2, hydroxy, oxo (=O), and carboxy; and each R a is independently H or (C1-C6) alkyl.

[0105] E13. L is a branched or unbranched, saturated or unsaturated hydrocarbon chain having about 1 to about 100 carbon atoms, wherein one or more of the carbon atoms are optionally independently selected from -O-, -S, -N(R a )-, a 3-20 membered heterocycle, a 3-20 membered heteroaryl, a 6-14 membered aryl, or a 3-20 membered carbocycle, wherein each chain, the 3-20 membered heterocycle, the 3-20 membered heteroaryl, the 6-14 membered aryl, and the 3-20 membered carbocycle are optionally and independently selected from (C-C)alkyl, (C-C)alkoxy, (C-C)cycloalkyl, (C-C)alkanoyl, (C-C)alkanoyloxy, (C-C)alkoxycarbonyl, azido, cyano, nitro, halo, -N(R a )2, hydroxy, oxo (=O), and carboxy; and each R a is independently H or (C1-C6) alkyl.

[0106] E14. L is a branched or unbranched, saturated or unsaturated hydrocarbon chain having about 1 to about 100 carbon atoms, wherein one or more of the carbon atoms are optionally independently selected from -O-, -S, -N(R a )-, 3-20 membered heterocycle, 3-20 membered heteroaryl, 6-14 membered aryl, or 3-20 membered carbocycle, each of which is optionally and independently substituted with one or more (e.g., 1, 2, 3, 4, 5, or more) substituents independently selected from the group consisting of azido, halo, and oxo (=O), and carboxy; a is independently H or (C1-C6) alkyl.

[0107] E15. L is a branched or unbranched, saturated or unsaturated hydrocarbon chain having about 1 to about 50 carbon atoms, one or more of the carbon atoms being optionally independently -O-, -S, -N(R a )-, 3-20 membered heterocycle, 3-20 membered heteroaryl, 6-14 membered aryl, or 3-20 membered carbocycle, each of which is optionally and independently substituted with one or more (e.g., 1, 2, 3, 4, 5, or more) substituents independently selected from the group consisting of azido, halo, and oxo (=O), and carboxy; a is independently H or (C1-C6) alkyl.

[0108] E16. L is a branched or unbranched, saturated or unsaturated hydrocarbon chain having about 1 to about 50 carbon atoms, wherein one or more of the carbon atoms are optionally independently selected from -O-, -S, -N(R a )-, or R b and each chain and R bis optionally and independently substituted with one or more (e.g., 1, 2, 3, 4, 5, or more) substituents independently selected from the group consisting of azido, halo, ═N(OH), —O(NH) and oxo (═O), and carboxy; and each R b is independently H or (C-C) alkyl, and each R b But the following: The compound or salt according to any one of E1 to E11, independently selected from the group consisting of TIFF2025531872000019.tif60128.

[0109] E17. L is -C(=O)(CH2) a The compound or salt of any one of E1 to E11, which contains N(H)C(=O)-, and a is 3, 4, 5, 6, 7, or 8.

[0110] E18. L is -(CH2CH2O) b CH2CH2C(=O)- or -(CH2CH2O) b The compound or salt according to any one of E1 to E11, comprising CH2CH2N(H)C(=O)-, wherein b is 2 to 50.

[0111] E19. The compound or salt of any one of E1 to E11, wherein L comprises phen-1,3-diyl or phen-1-1,3,5-triyl.

[0112] E20. L is as follows: The compound or salt according to any one of E1 to E11, including TIFF2025531872000020.tif60128.

[0113] E21. Each R 1 is linked to L via the carbonyl group of L.

[0114] E22. Each R 2is linked to L via a carbon-carbon bond.

[0115] E23. L is as follows: The compound or salt according to any one of E1 to E11, selected from the group consisting of TIFF2025531872000021.tif203166TIFF2025531872000022.tif234128.

[0116] E24. x is 1, 2 or 3 and the compound is: TIFF2025531872000023.tif169128TIFF2025531872000024.tif197103TIFF2025531872000025.tif226150TIFF2025531872000026.tif141156.

[0117] For clarity, in embodiment E24, compounds where x=1 are also contemplated. All definitions and moieties are as defined in E1, except for x, which in embodiment E24 may be selected to be 1. These embodiments, i.e., embodiments where x is optionally 1, are also contemplated in R 2 are independently a label (e.g., a label derived from fluorescein isothiocyanate (FITC) or Cy5), a phenyl group substituted with a formyl (-CHO) group, or a group of the formula: In other words, the embodiment, i.e., the embodiment where x is optionally 1, is particularly applicable when R 2 is not an oligonucleotide. 2 is not an oligonucleotide. In a preferred embodiment of E24, is selected to be 1.

[0118] In a twenty-fifth embodiment (Embodiment 25; abbreviated as "E25"), the present invention provides a compound of formula (X): 1. A process for preparing a diblock polymer of TIFF2025531872000028.tif8128, comprising: a) reacting a compound of structure Va, Vb, Vc, or Vd to obtain a first product: R 27 However, (C1-C 12 ) alkyl TIFF2025531872000029.tif16128; R 28 However, (C1-C 12 ) alkyl TIFF2025531872000030.tif16128; R 25 and R 26 However, independently, H, (C1-C 12 ) alkyl, aryl, or heteroaryl; TIFF2025531872000031.tif25128; TIFF2025531872000032.tif27128, as follows: i) polyethylene glycol methacrylate (PEGMA) having 2 to 20 ethylene glycol units; and ii) M, a methacrylate 2 wherein the methacrylate is optionally (C4-C 18 ) alkyl methacrylate, (C4-C 18 ) branched alkyl methacrylates, cholesteryl methacrylate, (C4-C 18 ) alkyl methacrylates, and (C4-C 18 ) Branched alkyl methacrylate Selected from M 2 and one or more A monomers selected from the group consisting of: contacting in the presence of free radicals; b) reacting the first product with Equations B1, B2, and B3: one or more B monomers of TIFF2025531872000033.tif75128; contacting in the presence of free radicals; and c) optionally contacting the second product with a free radical source (e.g., AIBN) to remove the chain transfer agent and obtain the diblock polymer of formula (X). Including, During the ceremony, block A comprises one or more residues of an A monomer and has a molecular weight of about 1 kDa to about 25 kDa; block B comprises one or more residues of monomers B1, B2, and B3 and has a molecular weight of about 1 kDa to about 25 kDa; L1 is a linking moiety, Z is optionally protected by a protecting group and has the formula (XII): T-L2-Y (XII) is a functional group capable of reacting with Y of a compound of formula T is a ligand (optionally a targeting ligand); Y is a functional group capable of reacting with Z to form a conjugate; L2 is absent or a linking moiety; Provide a process.

[0119] The term "free radical source" as used herein is not particularly limited. Suitable free radical sources include, but are not limited to, azobisisobutyronitrile (AIBN), 1,1-azobis(cyanocyclohexane) (ACHN / VAZO-88), 4,4'-azobis(4-cyanovaleric acid) (ACVA), and potassium persulfate (KSO).

[0120] As used herein, the term "chain transfer agent" (CTA) refers to a molecular entity suitable for controlling the molecular weight and polydispersity produced during free radical polymerization. Representative CTAs suitable in the present invention include, but are not limited to, thiocarbonylthio compounds or analogs. CTAs are also sometimes referred to in the art as "RAFT agents." As introduced above, compounds Va, Vb, Vc, and Vd are non-limiting examples of CTAs suitable in the process according to the present invention.

[0121] Although step c) is optional, it is preferred that the process of the present invention actually also includes step c). The result of a process that includes step c) is a compound of formula (I) that is free of CTA moieties. In another embodiment in which step c) is absent, the resulting polymer still contains moieties derived from CTA, but is otherwise identical to the compound of formula (I).

[0122] The product of this process, with or without step c), is useful for coupling to a T moiety, as described herein below. Typically, a T moiety is conjugated to the polymer obtained by the above process in a post-polymerization conjugation step.

[0123] This process and all its aspects are particularly useful for preparing polymers having a T moiety that is resistant to RAFT polymerization, as described, for example, in WO 2015 / 017519 A1. This is the case, for example, when T is selected to be an oligonucleotide or a peptide, such as a cyclic peptide described herein. In particular, the process according to the present invention is characterized in that T is conjugated to the polymer after the polymerization reaction (also referred to as RAFT polymerization). Therefore, the process according to the present invention may also be referred to as post-polymerization conjugation. The inventors have made available, through the present invention, an endosomal releasing polymer (ERP) featuring such a T moiety. Accordingly, the present invention provides a process for producing an endosomal releasing polymer (ERP), characterized in that the ERP obtainable by such a process optionally contains a ligand that is an oligonucleotide or a peptide. While oligonucleotides and peptides, among other ligands, are resistant to RAFT polymerization, the present invention makes available an ERP having such a ligand. The process of the present invention is also an economical process for producing an ERP containing a relatively expensive or valuable ligand, optionally selected from oligonucleotides and peptides. Conjugating such expensive or valuable ligands at the end of the synthesis, as opposed to at the beginning of the RAFT polymerization process, significantly reduces overall production costs, since the yield of the RAFT polymerization does not affect the amount of ligand required to make the final product.

[0124] The second product, i.e., the product resulting from step b), has attached thereto a radical starter moiety resulting from the compound of structure Va, Vb, Vc, or Vd, and when the second product is contacted with one or more free radicals, a diblock polymer of formula (X) is produced. Thus, the addition of the free radicals removes this group, converting the polymer end groups to protons. Preferably, the contact of the second product with one or more free radicals is carried out under protonic conditions. This is advantageous for converting the polymer end groups to substitute the RAFT agent with protons.

[0125] The process for making the diblock polymer of formula (X) described in E25 and any of its embodiments described herein may be referred to as "reversible addition-fragmentation chain transfer" (or RAFT) polymerization. According to the present invention, RAFT is used in the synthesis of the ethylene backbone polymers of the present invention. RAFT is a living polymerization process. RAFT involves a free-radical degenerative chain transfer process. In some embodiments, the RAFT procedure for preparing the polymers described herein uses thiocarbonylthio compounds, including but not limited to dithioesters, dithiocarbamates, trithiocarbonates, and xanthates, to mediate polymerization via a reversible chain transfer mechanism. In certain cases, stabilized radical intermediates are formed upon reaction of a polymer radical with the C=S group of any of the aforementioned compounds. Typically, these stabilized radical intermediates do not undergo the termination reaction typical of standard radical polymerization; instead, they reintroduce radicals capable of reinitiation or propagation with monomer, reorganizing the C=S bond in the process. In most cases, this cycle of addition to the C=S bond, followed by subsequent radical fragmentation, continues until all monomers are consumed or the reaction is quenched. Generally, the low concentration of active radicals at any particular time limits successful termination reactions.

[0126] In some embodiments, the polydispersity index (PDI or DI) or chain length difference of the polymers of the present invention is small. The polydispersity index can be determined in any suitable manner, for example, by dividing the weight average molecular weight of a polymer chain by its number average molecular weight. The number average molecular weight is the sum of the molecular weights of the individual chains divided by the number of chains. The weight average molecular weight is proportional to the square of the molecular weight divided by the number of molecules of that molecular weight. Since the weight average molecular weight is always greater than the number average molecular weight, the polydispersity is always greater than or equal to 1. As the numbers approach the same, i.e., as the polydispersity approaches a value of 1, the polymer approaches monodispersity, with all chains having exactly the same number of units. Polydispersity values ​​approaching 1 are achievable using radical living polymerization. Methods for determining polydispersity are well known in the art, including, but not limited to, size exclusion chromatography, dynamic light scattering, matrix-assisted laser desorption / ionization chromatography, and electrospray mass chromatography. In some embodiments, the polymers provided herein (e.g., membrane destabilizing polymers) have a polydispersity index (PDI) of less than 2.0, or less than 1.8, or less than 1.6, or less than 1.5, or less than 1.4, or less than 1.3, or less than 1.2. In some embodiments, the polymer is a block copolymer (e.g., an endosomal releasing copolymer) comprising a hydrophilic block and a hydrophobic block and having a polydispersity index (PDI) of less than 2.0, or less than 1.8, or less than 1.6, or less than 1.5, or less than 1.4, or less than 1.3, or less than 1.2.

[0127] The polymerization process described herein optionally occurs in any suitable solvent or mixture thereof. Suitable solvents include water, alcohols (e.g., methanol, ethanol, n-propanol, isopropanol, butanol), tetrahydrofuran (THF), dimethylsulfoxide (DMSO), dimethylformamide (DMF), acetone, acetonitrile, hexamethylphosphoramide, acetic acid, formic acid, hexane, cyclohexane, benzene, toluene, dioxane, methylene chloride, ethers (e.g., diethyl ether), chloroform, and ethyl acetate. In one aspect, the solvent includes water and mixtures of water and water-miscible organic solvents, such as DMF.

[0128] E26. PEGMA, TIFF2025531872000034.tif16128, and / or M 2 but, TIFF2025531872000035.tif16128, process as described in E25.

[0129] In other words, in this embodiment, the A monomer is selected from the group consisting of monomers of formulae A1 and A2.

[0130] In one aspect, the process of E26 comprises reacting a compound of formula (XI): This gives the compound TIFF2025531872000036.tif91158. The parentheses indicate the monomer moieties. The square brackets indicate block A and block B of the diblock polymer, respectively. The stoichiometry (m and n; q, r, and s) and molecular weight (v and w) are as defined in E25.

[0131] E27. The process according to E25 or E26, wherein Z is a functional group protected with a protecting group, and the process further comprises removing the protecting group from Z.

[0132] E28. The process according to E25 or E26, wherein Z is a functional group that is not protected by a protecting group.

[0133] E29. A diblock polymer of formula (XI) Formula (XII): T-L2-Y (XII) and a compound of Z reacts with Y to form a group Y'Z' of the formula: reacting under conditions that result in a conjugate of TIFF2025531872000037.tif4128 The process of E27 or E28, further comprising:

[0134] In one embodiment, this process results in an endosomally releasing polymer according to E49 and any of its embodiments.

[0135] In one embodiment, the conjugate has formula (XIII): This is a combination of TIFF2025531872000038.tif91160. The parentheses indicate the monomer moieties. The square brackets indicate block A and block B of the diblock polymer, respectively.

[0136] E30. The process according to E29, wherein block A has a molecular weight of about 1 kDa to about 25 kDa, and block B has a molecular weight of about 1 kDa to about 25 kDa.

[0137] E31. Block A has the formula: -[PEGMA m -M 2 n ] v - wherein PEGMA is a polyethylene glycol methacrylate residue having 2 to 20 ethylene glycol units; M 2 but, (C4-C 18 ) alkyl methacrylate residue, (C4-C 18 ) branched alkyl methacrylate residues, cholesteryl methacrylate residue, (C4-C 18) alkyl-methacrylate residues, and (C4-C 18 ) Branched alkyl methacrylate residue is a methacrylate residue selected from the group consisting of: v is 1 to 25 kDa; Block B has the formula: -[DMAEMA q -PAA r -BMA s ] w wherein BMA is the residue of butyl methacrylate; PAA is a propylacrylic acid residue, DMAEMA is the residue of dimethylaminoethyl methacrylate; m and n are each a mole fraction greater than 0, m is greater than n, and m+n=1; q is a mole fraction between 0.2 and 0.75; r is a mole fraction between 0.05 and 0.6; s is a mole fraction of 0.2 to 0.75; q+r+s=1, w is 1 to 25 kDa; Process described in E29.

[0138] E31 has the following formula: It can also be represented as TIFF2025531872000039.tif15140. The round brackets indicate the monomer moieties, and the square brackets indicate block A and block B of the diblock polymer, respectively.

[0139] E32. The process of any one of E25-E31, wherein Z and Y are selected such that Y'Z' comprises an oxime functional group.

[0140] E33. The process of any one of E25-E31, wherein Z and Y are selected such that Y'Z' comprises a triazole ring.

[0141] E34. The process according to any one of E25 to E27 and E29 to E32, wherein Z comprises a protected aminooxy group, the protecting group being on the nitrogen of the aminooxy group.

[0142] E35. The process of any one of E29-E32, wherein Z comprises an aminooxy group, Y comprises a ketone or aldehyde group, and Y'Z' comprises an oxime functional group.

[0143] E36. The process of any one of E29-E32, wherein Z comprises a ketone or aldehyde group, Y comprises an aminooxy group, and Y'Z' comprises an oxime functional group.

[0144] E37. The process according to E29 or E33, wherein Z comprises an alkyne group, Y comprises an azide group, and Y'Z' comprises a triazole ring.

[0145] E38. The process according to E29 or E33, wherein Z comprises an azide group, Y comprises an alkyne group, and Y'Z' comprises a triazole ring.

[0146] E39. The process of any one of E29-E38, wherein T is a targeting ligand or labeling agent.

[0147] E40. The process of any one of E29-E38, wherein the targeting ligand is selected from the group of an oligonucleotide, a peptide, a sugar, and a small molecule.

[0148] E41. The process of any one of E29-E38, wherein the targeting ligand comprises a cyclic peptide.

[0149] E42. The process of any one of E29-E38, wherein the targeting ligand comprises a cyclic peptide described in E1.

[0150] E43. The process of any one of E29-E38, wherein the targeting ligand comprises a cyclic peptide described in E6.

[0151] E44. The process of any one of E29-E38, wherein the labeling agent is selected from the group consisting of a fluorophore, a chromophore, and a radionucleotide.

[0152] E45. The process according to any one of E29 to E38, wherein the labeling substance is derived from fluorescein isothiocyanate (FITC) or Cy5.

[0153] E46. A product prepared by the process according to any one of E25 to E45.

[0154] E47. A conjugate prepared by the process according to any one of E29 to E45.

[0155] E48. A composition comprising a plurality of conjugates as defined in any one of E29 to E45.

[0156] E49. The composition of E48, wherein at least one of the conjugates comprises a labeling substance.

[0157] In a fiftieth embodiment (Embodiment 50; abbreviated as "E50"), the present invention provides a compound of formula (I): TIFF2025531872000040.tif13128 or a salt thereof, wherein x is 1, 2, 3, 4, or 5; Each R 1 is independently a targeting ligand, and the targeting ligand is a) SEQ ID NO: 1: C * SRNLIDC * (SEQ ID NO: 1) A cyclic polypeptide represented by the formula: *represents a disulfide bond linking two C residues thereby forming a cyclic polypeptide, and b) C * Any cyclic polypeptide having at least 80% sequence identity (e.g., at least 85%; e.g., at least 87.5%; e.g., at least 90%; e.g., at least 95%; e.g., at least 99% sequence identity) with a polypeptide set forth in SEQ ID NO:1, provided that both residues are present, wherein: * exhibits a disulfide bond linking two C residues thereby forming a cyclic polypeptide. Selected from L is a linking group, y is 1, 2, 3, 4, or 5; Each R 2 are independently an endosomal release polymer, or a salt thereof.

[0158] Preferably, the compound or salt of E50 is obtainable by the process of E29 and any of its embodiments.

[0159] E51. An endosomal release polymer having the following formula: [Block A]-[Block B] and a diblock polymer comprising a block A and a block B having the formula: block A comprises one or more residues of monomers A1 and A2 as described in E25 and has a molecular weight of about 1 kDa to about 25 kDa; Block B comprises one or more residues of monomers B1, B2, and B3 as described in E25 and has a molecular weight of about 1 kDa to about 25 kDa; Compounds or salts of E50.

[0160] In one aspect, compounds of formula (I) are obtainable by the processes described herein.

[0161] E52. The endosomal release polymer has the formula XX: -[PEGMA m -M 2 n ] v -[DMAEMA q -PAA r -BMA s ] w (XX) wherein PEGMA is a polyethylene glycol methacrylate residue having 2 to 20 ethylene glycol units; M 2 but, (C4-C 18 ) alkyl methacrylate residue, (C4-C 18 ) branched alkyl methacrylate residues, cholesteryl methacrylate residue, (C4-C 18 ) alkyl-methacrylate residues, and (C4-C 18 ) Branched alkyl methacrylate residue is a methacrylate residue selected from the group consisting of: BMA is the residue of butyl methacrylate; PAA is a propylacrylic acid residue, DMAEMA is the residue of dimethylaminoethyl methacrylate; m and n are each a mole fraction greater than 0, m is greater than n, and m+n=1; q is a mole fraction between 0.2 and 0.75; r is a mole fraction between 0.05 and 0.6; s is a mole fraction of 0.2 to 0.75; q+r+s=1, v is 1 to 25 kDa; w is 1 to 25 kDa; Compounds or salts of E50.

[0162] E53. A compound or salt according to E51, wherein block A comprises one or more residues of monomers of formulae A1 and A2 as described in E26.

[0163] E54. A pharmaceutical composition comprising a compound according to any one of E1 to E24 and E50 to E53 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0164] E55. Each R 2 are independently an oligonucleotide; and The second compound according to any one of E50 to E53 or a pharmaceutically acceptable salt thereof A pharmaceutical composition comprising:

[0165] E56. A method for delivering an oligonucleotide to an animal, comprising administering to the animal a compound according to any one of E1 to E24, or a pharmaceutically acceptable salt thereof.

[0166] E57. A method for delivering an oligonucleotide to a cell expressing a platelet-derived growth factor receptor (PDGFR) in an animal, comprising administering to the animal a compound described in any one of E1 to E24 or a pharmaceutically acceptable salt thereof.

[0167] E58. The method according to E57, wherein the platelet-derived growth factor receptor (PDGFR) is platelet-derived growth factor receptor alpha (PDGFRα).

[0168] E59. The method according to E57, wherein the platelet-derived growth factor receptor (PDGFR) is platelet-derived growth factor receptor β (PDGFRβ).

[0169] E60. The method of E57, wherein the PDGFR-expressing cell is a hepatic stellate cell (HSC), an endothelial cell, a fibroblast, or a tumor cell.

[0170] E61. A method for treating a disease involving PDGFR-expressing cells (e.g., liver fibrosis, non-alcoholic steatohepatitis (NASH), clear cell renal cell carcinoma, renal fibrosis, or alcoholic steatohepatitis (ASH)), comprising administering to an animal a compound described in any one of E1 to E24 or a pharmaceutically acceptable salt thereof.

[0171] E62. The compound according to any one of E1 to E24 or a pharmaceutically acceptable salt thereof for delivering an oligonucleotide to an animal.

[0172] E63. The compound according to any one of E1 to E24 or a pharmaceutically acceptable salt thereof for delivering an oligonucleotide to a cell expressing a platelet-derived growth factor receptor (PDGFR).

[0173] E64. The compound according to any one of E1 to E24 or a pharmaceutically acceptable salt thereof for the preventive or therapeutic treatment of a disease involving PDGFR-expressing cells (e.g., liver fibrosis, non-alcoholic steatohepatitis (NASH), clear cell renal cell carcinoma, renal fibrosis, or alcoholic steatohepatitis (ASH)).

[0174] E65. Use of a compound according to any one of E1 to E24, or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for delivering an oligonucleotide to an animal.

[0175] E66. Use of a compound according to any one of E1 to E24 or a pharmaceutically acceptable salt thereof for the preparation of a pharmaceutical agent for delivering an oligonucleotide to cells expressing platelet-derived growth factor receptor (PDGFR).

[0176] E67. Use of a compound according to any one of E1 to E24 or a pharmaceutically acceptable salt thereof for the preparation of a medicament for treating a disease involving PDGFR-expressing cells (e.g., liver fibrosis, non-alcoholic steatohepatitis (NASH), clear cell renal cell carcinoma, renal fibrosis, or alcoholic steatohepatitis (ASH)).

[0177] E68. Each R 2 are independently an oligonucleotide; and a second compound according to any one of E50 to E53; including the combined use of A method of use, a compound, or a use thereof according to any one of E56 to E67.

[0178] In another embodiment (embodiment 101; abbreviated as "E101"), the present invention provides a compound of formula (I): TIFF2025531872000041.tif13128 or a salt thereof, wherein x is 1, 2, 3, 4, or 5; Each R 1 but independently, the formula: TIFF2025531872000042.tif73128 targeting ligand, L is a linking group, y is 1, 2, 3, 4, or 5; Each R 2 are independently an oligonucleotide, a label (e.g., a label derived from fluorescein isothiocyanate (FITC) or Cy5), a phenyl group substituted with a formyl (-CHO) group, or a group of the formula: TIFF2025531872000043.tif17128 or y is 1, 2, 3, 4, or 5; R 2 is an endosomal release agent comprising a polymer and a peptide; The present invention provides a compound or a salt thereof.

[0179] Further aspects (E102 to E122) are described below.

[0180] E102. Each R 1 but independently, the formula: The compound or salt according to E101, which is a targeting ligand of TIFF2025531872000044.tif69128.

[0181] E103. Each R 2 is independently an oligonucleotide.

[0182] E104. Each R 2 is independently an siRNA.

[0183] E105. The compound according to E103 or E104, wherein each oligonucleotide or each siRNA is attached to L via a phosphate P(=O)(OH)2-O- of each oligonucleotide or each siRNA.

[0184] E106. Each R 2 but independently, the formula: The compound or salt according to E101 or E102, which is a group represented by TIFF2025531872000045.tif17128.

[0185] E107. Each R 2 A compound or salt according to E101 or E102, wherein is independently 3-formylphenyl or 4-formylphenyl.

[0186] E108. L is a branched or unbranched, saturated or unsaturated hydrocarbon chain having from about 1 to about 500 carbon atoms, one or more of which are optionally independently -O-, -S, -N(R a)-, a 3-20 membered heterocycle, a 3-20 membered heteroaryl, a 6-14 membered aryl, or a 3-20 membered carbocycle, wherein each chain, the 3-20 membered heterocycle, the 3-20 membered heteroaryl, the 6-14 membered aryl, and the 3-20 membered carbocycle are optionally and independently selected from (C-C)alkyl, (C-C)alkoxy, (C-C)cycloalkyl, (C-C)alkanoyl, (C-C)alkanoyloxy, (C-C)alkoxycarbonyl, azido, cyano, nitro, halo, -N(R a )2, hydroxy, oxo (=O), and carboxy; and each R a is independently H or (C1-C6) alkyl.

[0187] E109. L is a branched or unbranched, saturated or unsaturated hydrocarbon chain having from about 1 to about 100 carbon atoms, one or more of which are optionally independently -O-, -S, -N(R a )-, a 3-20 membered heterocycle, a 3-20 membered heteroaryl, a 6-14 membered aryl, or a 3-20 membered carbocycle, wherein each chain, the 3-20 membered heterocycle, the 3-20 membered heteroaryl, the 6-14 membered aryl, and the 3-20 membered carbocycle are optionally and independently selected from (C-C)alkyl, (C-C)alkoxy, (C-C)cycloalkyl, (C-C)alkanoyl, (C-C)alkanoyloxy, (C-C)alkoxycarbonyl, azido, cyano, nitro, halo, -N(R a )2, hydroxy, oxo (=O), and carboxy; and each R a is independently H or (C1-C6) alkyl.

[0188] E110. L is a branched or unbranched, saturated or unsaturated hydrocarbon chain having about 1 to about 100 carbon atoms, one or more of the carbon atoms being optionally independently -O-, -S, -N(R a )-, 3-20 membered heterocycle, 3-20 membered heteroaryl, 6-14 membered aryl, or 3-20 membered carbocycle, each of which is optionally and independently substituted with one or more (e.g., 1, 2, 3, 4, 5, or more) substituents independently selected from the group consisting of azido, halo, and oxo (=O), and carboxy; a is independently H or (C1-C6) alkyl.

[0189] E111. L is a branched or unbranched, saturated or unsaturated hydrocarbon chain having about 1 to about 50 carbon atoms, one or more of which are optionally independently -O-, -S, -N(R a )-, 3-20 membered heterocycle, 3-20 membered heteroaryl, 6-14 membered aryl, or 3-20 membered carbocycle, each of which is optionally and independently substituted with one or more (e.g., 1, 2, 3, 4, 5, or more) substituents independently selected from the group consisting of azido, halo, and oxo (=O), and carboxy; a is independently H or (C1-C6) alkyl.

[0190] E112. L is a branched or unbranched, saturated or unsaturated hydrocarbon chain having about 1 to about 50 carbon atoms, one or more of which are optionally independently -O-, -S, -N(R a )-, or R b and each chain and R bis optionally and independently substituted with one or more (e.g., 1, 2, 3, 4, 5, or more) substituents independently selected from the group consisting of azido, halo, ═N(OH), —O(NH) and oxo (═O), and carboxy; and each R b is independently H or (C-C) alkyl, and each R b But the following: The compound or salt according to any one of E101 to E107, independently selected from the group consisting of TIFF2025531872000046.tif60128.

[0191] E113. L is -C(=O)(CH2) a The compound or salt according to any one of E101 to E112, which contains N(H)C(=O)-, and a is 3, 4, 5, 6, 7, or 8.

[0192] E114. L is -(CH2CH2O) b CH2CH2C(=O)- or -(CH2CH2O) b The compound or salt according to any one of E101 to E113, comprising CH2CH2N(H)C(=O)-, wherein b is 2 to 50.

[0193] E115. The compound or salt according to any one of E101 to E114, wherein L comprises phen-1,3-diyl or phen-1-1,3,5-triyl.

[0194] E116. L is: A compound or salt according to any one of E101 to E115, including TIFF2025531872000047.tif60128.

[0195] E117. Each R 1 is linked to L via the carbonyl group of L.

[0196] E118. Each R 2is linked to L via a carbon-carbon bond.

[0197] E119. L is as follows: The compound or salt according to any one of E101 to E118, selected from the group consisting of TIFF2025531872000048.tif60166TIFF2025531872000049.tif199164TIFF2025531872000050.tif197133.

[0198] E120. Below: The compound according to E101 or E102, selected from the group consisting of TIFF2025531872000051.tif251103TIFF2025531872000052.tif241142TIFF2025531872000053.tif237156.

[0199] E121. y is 1 and R 2 But, formula XX: -[PEGMA m -M 2 n ] v -[DMAEMA q -PAA r -BMA s ] w (XX) an endosomally released polymer of the formula: PEGMA is a polyethylene glycol methacrylate residue having 2 to 20 ethylene glycol units; M 2 but, (C4-C 18 ) alkyl methacrylate residue, (C4-C 18 ) branched alkyl methacrylate residues, cholesteryl methacrylate residue, (C4-C 18 ) alkyl-methacrylate residues, and (C4-C 18 ) Branched alkyl methacrylate residue is a methacrylate residue selected from the group consisting of: BMA is the residue of butyl methacrylate; PAA is a propylacrylic acid residue, DMAEMA is the residue of dimethylaminoethyl methacrylate; m and n are each a mole fraction greater than 0, m is greater than n, and m+n=1; q is a mole fraction between 0.2 and 0.75; r is a mole fraction between 0.05 and 0.6; s is a mole fraction of 0.2 to 0.75; q+r+s=1, v is 1 to 25 kDa; w is 1 to 25 kDa; A compound or salt according to E101.

[0200] E122. The following formula: The compound or salt according to E121, which is a compound or salt of TIFF2025531872000054.tif71162. The stoichiometry (m and n; q, r and s) and molecular weight (v and w) are as defined in E121.

[0201] The process of the present invention In one aspect, the present invention provides a compound of formula (X): A process for preparing a diblock polymer of TIFF2025531872000055.tif8128 is provided.

[0202] Prieve et al. (Mol Ther 2018) described the synthesis of diblock polymers using RAFT polymerization, characterized by a mannose or GalNAc monosaccharide ligand at the α-terminus of the polymer. Details were limited; input and output quantities were not recorded, and yields were not given. When preparing similar GalNAc polymers, the synthesis can require significant amounts of input starting material chain transfer agent (CTA) relative to the amount of output polymer. For example, a synthesis using GalNAc CTA utilized 180 g of input CTA to produce approximately 50 g of final polymer. This starting material-to-product ratio appears consistent regardless of the scale of synthesis. If the ligand is relatively inexpensive and readily available, the need for this material is not necessarily a barrier. However, if the ligand can be quite expensive, the need for this large amount of input material can become very costly.

[0203] Furthermore, while RAFT polymerization methodology has been reported to tolerate a variety of functional groups, it does not necessarily extend to all possible functional groups, including those that may be present in peptides, where each amino acid residue may have different groups of potentially different reactivity on its side chain. The incorporation of protecting group strategies to address these complications would likely add significant technical complexity to the synthesis and further increase the material requirements and costs associated with attempting to perform RAFT polymerization on ligands more complex than simple sugars.

[0204] One potential solution to address these shortcomings would be to couple the ligand after polymerization has taken place. This would simultaneously significantly reduce the amount of ligand required (since the MW of the ligand is typically about 10% of the polymer MW) and reduce or eliminate the need for protecting group strategies for conjugating the ligand. Choosing a conjugation strategy is not simple. The chemical reaction must be mild, easy to perform, and essentially quantitative, and the resulting functional groups must be relatively biologically inert (not metabolized in vivo and not reactive with proteins and genetic material). It is also necessary to avoid distinct sizes and structures that could adversely affect the hydrophilic / hydrophobic balance of the polymer and the resulting micelle formation. These criteria severely limit the available options.

[0205] Conjugation via oxime formation is one possible option. In fact, oxime formation proceeds to a significant extent, especially in the presence of an acid catalyst (e.g., trifluoroacetic acid (TFA)). Therefore, oxime chemistry is particularly suitable for ligands (functional groups and protecting groups) that are not acid-sensitive.

[0206] For example, GalNAc can be conjugated to polymers via processes known in the art, but is not suitable for conjugation in the new process according to the present invention due to its potential for acid-catalyzed hydrolysis at the glycometric center, resulting in ligand elimination. There are numerous ligands, such as those described herein, including the peptides described herein, that are particularly suitable for conjugation by the process according to the present invention, but which cannot be conjugated to a satisfactory degree by processes previously known in the art.

[0207] Additionally, polymer synthesis is complicated by the lack of useful non-destructive characterization methods for reaction monitoring and determination of reaction completion. The process of the present invention allows for accurate determination of reaction endpoints, confirmation of ligand attachment, and provides a reaction purification approach that removes unwanted reagents from the reaction without the need for additional precipitation or dialysis, which can significantly affect the recovery of the final polymer.

[0208] As used herein, the term "block copolymer" refers to two or more homopolymer or copolymer subunits linked by covalent bonds. Block copolymers using two or three different blocks are referred to as diblock and triblock copolymers, respectively. A schematic generalization of a diblock copolymer is represented by the formula [F;G,Hh...]g-[JjKxLi...]: where each letter represents a constitutional unit derived from the polymerization of the corresponding monomer, each subscript on a constitutional unit represents the mole fraction of that unit in a particular block, three dots indicate that there may be more (or less) constitutional units in each block, and q and r indicate the molecular weight of each block in the diblock copolymer. As suggested by the schematic, in some cases, the number and content of each constitutional unit are controlled separately for each block. The schematic is not intended to, and should not be construed to, show any relationship between the number of constitutional units in each block and the number of different types of constitutional units. The schematic diagrams are not intended to represent any particular number or arrangement of constitutional units within a particular block. Within each block, the constitutional units may be arranged in a purely random, alternating random, regular alternating, regular block, or random block arrangement, unless expressly specified otherwise. A purely random arrangement may have, for example, the non-limiting form: ffghfgghghhh.... An exemplary alternating random arrangement may have the non-limiting form: fgfhgfghgfh...., and an exemplary regular alternating arrangement may have the non-limiting form: fgh-fg-hfgh.... An exemplary regular block arrangement may have the following non-limiting arrangement: ...fffggghhhff..., whereas an exemplary random block arrangement may have the non-limiting arrangement: fffhhffggghhhffhhh....In a gradient polymer, the content of one or more monomeric units increases or decreases in a gradient from the a-end to the w-end of the polymer. None of the foregoing general examples imply any particular juxtaposition of individual building blocks or blocks, nor the number of building blocks within a block, nor should they be interpreted in any way to relate to or limit the actual structure of the block copolymers described herein. As used herein, brackets surrounding building blocks are not intended to mean that the building blocks themselves form blocks, and should not be interpreted as meaning that the building blocks themselves form blocks. That is, building blocks within brackets can be combined with other building blocks in any manner, i.e., in pure random, alternating random, regular alternating, regular block, or random block configurations. The block copolymers described herein are optionally alternating, gradient, or random block copolymers. Block copolymers and methods for preparing them are described in International Patent Application Publication No. WO 2015 / 017519, the entire contents of which are incorporated herein by reference.

[0209] However, according to the present invention, the ligand (e.g., targeting ligand) is not incorporated during polymerization, e.g., by incorporating monomer A4 (containing targeting moiety T2) into block A of the polymer, e.g., during the polymerization contemplated in WO 2015 / 017519. Conversely, according to the present invention, the ligand T (e.g., targeting ligand) is incorporated after polymerization. This, among other aspects, allows for control of the stoichiometry of the ligand incorporated per copolymer, and also allows for the incorporation of ligands that are not susceptible to RAFT polymerization, such as, in non-limiting examples, oligonucleotides and peptides, e.g., pPB peptides. In representative embodiments, one ligand (T) or one moiety having a whole number of targeting ligands (T) is incorporated into every polymer. That is, according to the present invention, the typical stoichiometry is one or multiples thereof. Thus, the process of the present invention is distinguished from WO2015 / 017519 at least in terms of the high specificity of the ligand incorporation and in allowing the incorporation of certain specific ligands, which is not possible with the process of WO2015 / 017519. A further advantage of the present invention arises from the fact that the ligand T is always located at the hydrophilic end of the polymer, as opposed to being incorporated at random positions as disclosed in WO2015 / 017519. In other words, a compound of formula (I): TIFF2025531872000056.tif7128, where 1 and R 2 and L are defined as specified herein, x is selected from the integers 1, 2, 3, 4, or 5, and y is selected from the integers 1, 2, 3, 4, or 5. The liganded endosomal releasing polymers (ERTs) of the present invention comprise R 1 and R 2 The stoichiometry is defined as

[0210] Examples of block copolymers according to the present invention include block copolymers in which block A is a first block that is a random copolymer formed from monomers of formulae A1, A2, and A3, as described herein. Further examples of block copolymers include block copolymers in which block A is a first block that is a random copolymer formed from monomers comprising formulae A1 and A2, as described above. Further examples of block copolymers include block copolymers in which A is a first block that is a polymer formed from monomer A2, as described above. Further examples of block copolymers include block copolymers in which block A is a first block that is a random copolymer comprising residues of a monomer of formula A1. Further examples of block copolymers include block copolymers in which block A is a first block that is a random copolymer comprising residues of a monomer of formula A2. Further examples of block copolymers include block copolymers in which block A is a first block that is a random copolymer comprising residues of a monomer of formula A3.

[0211] Examples of block copolymers include block copolymers in which block B is a second block that is a random copolymer formed from monomers of formulas B1, B2, and B3 as described herein. Further examples of block copolymers include block copolymers in which block B is a second block that is a random copolymer comprising residues of a monomer of formula B1. Further examples of block copolymers include block copolymers in which block B is a second block that is a random copolymer comprising residues of a monomer of formula B2. Further examples of block copolymers include block copolymers in which block B is a second block that is a random copolymer comprising residues of a monomer of formula B3.

[0212] In the diblock polymers of formula (X and XI), block A comprises residues derived from the polymerization of monomers of formula A1 and A2. As used herein, the term "residue" refers to the portion of a monomer remaining in a polymer after polymerization. In one embodiment, block A comprises one or more residues of monomers A1 and A2 and has a molecular weight of about 1 kDa to about 25 kDa. In another embodiment, block A comprises one or more residues of monomers A1 and A2 and has a molecular weight of about 2 kDa to about 20 kDa. In another embodiment, block A comprises one or more residues of monomers A1 and A2 and has a molecular weight of about 2 kDa to about 15 kDa. In another embodiment, block A comprises one or more residues of monomers A1 and A2 and has a molecular weight of about 2 kDa to about 10 kDa. In another embodiment, block A comprises one or more residues of monomers A1 and A2 and has a molecular weight of about 2 kDa to about 5 kDa. In another embodiment, block A comprises one or more residues of monomers A1 and A2 and has a molecular weight of about 5 kDa to about 20 kDa. In another embodiment, block A comprises one or more residues of monomers A1 and A2 and has a molecular weight of about 5 kDa to about 15 kDa. In another embodiment, block A comprises one or more residues of monomers A1 and A2 and has a molecular weight of about 5 kDa to about 10 kDa. In another embodiment, block A comprises one or more residues of monomers A1 and A2 and has a molecular weight of about 10 kDa to about 25 kDa. In another embodiment, block A comprises one or more residues of monomers A1 and A2 and has a molecular weight of about 10 kDa to about 20 kDa. In another embodiment, block A comprises one or more residues of monomers A1 and A2 and has a molecular weight of about 10 kDa to about 15 kDa.

[0213] In the diblock polymers of formula (X and XI), block B comprises residues derived from the polymerization of monomers of formulae B1, B2, and B3. As used herein, the term "residue" refers to the portion of a monomer remaining in a polymer after polymerization. In one embodiment, block B comprises one or more residues of monomers B1, B2, and B3 and has a molecular weight of about 1 kDa to about 25 kDa. In another embodiment, block B comprises one or more residues of monomers B1, B2, and B3 and has a molecular weight of about 2 kDa to about 20 kDa. In another embodiment, block B comprises one or more residues of monomers B1, B2, and B3 and has a molecular weight of about 2 kDa to about 15 kDa. In another embodiment, block B comprises one or more residues of monomers B1, B2, and B3 and has a molecular weight of about 2 kDa to about 10 kDa. In another embodiment, block B comprises one or more residues of monomers B1, B2, and B3 and has a molecular weight of about 2 kDa to about 5 kDa. In another embodiment, block B comprises one or more residues of monomers B1, B2, and B3 and has a molecular weight of about 5 kDa to about 20 kDa. In another embodiment, block B comprises one or more residues of monomers B1, B2, and B3 and has a molecular weight of about 5 kDa to about 15 kDa. In another embodiment, block B comprises one or more residues of monomers B1, B2, and B3 and has a molecular weight of about 5 kDa to about 10 kDa. In another embodiment, block B comprises one or more residues of monomers B1, B2, and B3 and has a molecular weight of about 10 kDa to about 25 kDa. In another embodiment, block B comprises one or more residues of monomers B1, B2, and B3 and has a molecular weight of about 10 kDa to about 20 kDa. In another embodiment, block B comprises one or more residues of monomers B1, B2, and B3 and has a molecular weight of about 10 kDa to about 15 kDa.

[0214] According to the process of the present invention, a compound of formula (XII): T-L2-Y (XII) In the compound of formula (X and XI), Z in the diblock polymer of formula (X and XI) reacts with Y to form a group Y'Z', Z and Y are chosen to yield the conjugate TIFF2025531872000057.tif4128.

[0215] In one embodiment, the conjugate has formula (XIII): It is a combination of TIFF2025531872000058.tif79136.

[0216] Suitable non-limiting examples of Z and Y, and the corresponding groups Y'Z' remaining in the conjugate of formula (XIII) after reaction of Y with Z, are set out in Table A below.

[0217] Table A: Embodiments of X and Y and the resulting conjugation products X'-Y' according to certain exemplified embodiments of the present invention TIFF2025531872000059.tif164153

[0218] An aminooxy Z in the first row of the table can react with an aldehyde (R''' is H) or ketone (R''' is C) Y to give the corresponding Z'Y' oxime. An aminooxy Y in the second row of the table can react with an aldehyde (R''' is H) or ketone (R''' is C) Z to give the corresponding Z'Y' oxime.

[0219] It may be desirable to prepare a compound of formula (X) in which Z is protected with a protecting group. In such cases, the protecting group may be removed from Z before reacting with a compound of formula (XII). It may also be desirable to prepare a compound of formula (XII) in which Y is protected with a protecting group. In such cases, the protecting group may be removed from Y before reacting with a compound of formula (X). A compound of formula (X) in which Z is protected with a protecting group is a useful intermediate for preparing a conjugate of the invention (e.g., a conjugate of formula (XIII)). Such protected compounds of formula (X) are an embodiment of the invention. A compound of formula (XII) in which Y is protected with a protecting group is a useful intermediate for preparing a conjugate of the invention (e.g., a conjugate of formula (XIII)). Such protected compounds of formula (XII) are an embodiment of the invention. [Example]

[0220] The present invention will now be described in further detail by way of specific examples. The following examples are given for illustrative purposes and are not intended to limit the present invention in any way. Those skilled in the art will readily recognize a variety of non-critical parameters that can be changed or modified to yield essentially the same results.

[0221] name pPB = (N- to C-terminus) H2N-CSNLIDC-COOH (disulfide between two Cys). Scrambled pPB = (N- to C-terminus) H2N-CIDNLSRC-COOH (disulfide between two Cys). Used as a control for pPB activity ligand.

[0222] Example 1: Biotinylated Ligand Biotinylated versions of the ligands were prepared for in vitro binding experiments to measure relative binding efficiencies (see Examples 6, 7, and 8). The following biotinylated ligands were prepared using standard solid-phase peptide synthesis (SPPS). Biotin was coupled using standard organic chemistry amide coupling methods, and the final ligands were purified by reverse-phase HPLC. The products were confirmed by mass spectrometry, and the purity of the products was determined by RPLC. TIFF2025531872000060.tif58128pPB Monovalent Biotin (Compound 1) MW: Calculated 1260.56. Found 1260.2. Purity (HPLC) >90% TIFF2025531872000061.tif76128 Scrambled pPB monovalent biotin (compound 2) MW: Calculated 1260.56. Found 1260.2. Purity (HPLC) >90% TIFF2025531872000062.tif93128pPB Trivalent Biotin (Compound 3) MW: Calculated 3437.97, Found 3438.89, Purity (HPLC) >90% TIFF2025531872000063.tif108128 Scrambled pPB trivalent biotin (compound 4) MW: Calculated 3438.97, Found 3438.39, Purity (HPLC) >90% TIFF2025531872000064.tif74128pPB Bivalent Biotin "1A" (Compound 5) MW: Calculated 2462.87, Found 2463.37, Purity (HPLC >90%) TIFF2025531872000065.tif70128pPB Bivalent Biotin "1B" (Compound 6) MW: Calculated 2689.18, Found 2690.32, Purity (HPLC >90%) TIFF2025531872000066.tif90128pPB divalent "2A" (compound 7) MW: calculated 2490.92. Found 2491.2. Purity (HPLC>90%) TIFF2025531872000067.tif85129pPB divalent "2B" (compound 8) MW: calculated 2717.24, found 2718.15, purity (HPLC>90%) TIFF2025531872000068.tif90128pPB divalent "2C" (compound 9) MW: calculated 2405.82. Found 2406.22. Purity (HPLC>90%) TIFF2025531872000069.tif72132pPB divalent "3B" (compound 10) MW: calculated 2632.13. Found 2632.54. Purity (HPLC > 90%)

[0223] Example 2: Synthesis of peptide-linked PEG12 benzaldehyde The following pPB benzaldehyde ligands (18a and 18b) were synthesized for conjugation with the endosomally releasing polymer (27) upon completion of RAFT polymerization. TIFF2025531872000070.tif54161

[0224] a. Step 1 Synthesis of perfluorophenyl 3-(1,3-dioxolan-2-yl)benzoate (13) 3-(1,3-Dioxolan-2-yl)benzoic acid 11 (7.9 g, 41 mmol) and (TEA 11.4 mL, 81 mmol) were dissolved in DCM and stirred at 0 °C. 2,3,4,5,6-Pentafluorophenyl 2,2,2-trifluoroacetate 12 (7.0 mL, 41 mmol) was added dropwise and the reaction was allowed to warm to RT over 2 h. The reaction was concentrated under reduced pressure, dissolved in EtOAc (200 mL), washed with NaHCO (2 × 100 mL) and brine (1 × 100 mL), dried (MgSO), filtered, and concentrated under reduced pressure to give perfluorophenyl 3-(1,3-dioxolan-2-yl)benzoate 13 (13.6 g, 92.8%), which was used without purification.

[0225] b. Step 2 Synthesis of 1-{[3-(1,3-dioxolan-2-yl)phenyl]formamide}-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-oic acid (15) 1-Amino-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-oic acid (14) (7 g, 11.3 mmol), 2,3,4,5,6-pentafluorophenyl 3-(1,3-dioxolan-2-yl)benzoate (13) (4.5 g, 12.5 mmol), and DMAP (69 mg, 0.6 mmol) were dissolved in DCM (200 ml) and stirred at RT for 16 h. The reaction was concentrated under reduced pressure and purified by automated flash chromatography (0–10% MeOH / DCM) to give 1-{[3-(1,3-dioxolan-2-yl)phenyl]formamide}-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-oic acid (15) (5.1 g, 56.7%).

[0226] c. Step 3 Synthesis of 4-nitrophenyl 1-{[3-(1,3-dioxolan-2-yl)phenyl]formamide}-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatetriacont-39-noate (17) 1-{[3-(1,3-dioxolan-2-yl)phenyl]formamide}-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatetriacontan-39-oic acid (15) (5.1 g, 6.4 mmol), TEA (1.4 mL, 9.6 mmol), and bis(4-nitrophenyl)carbonate (16) (2.15 g, 7.1 mmol) were dissolved in DCM (100 mL) and stirred at RT for 16 h. The reaction was diluted with DCM (100 mL), washed with saturated NaHCO3 (5 x 75 mL), concentrated under reduced pressure, and the residue was purified by automated flash chromatography (0-10% MeOH / DCM) to give 4-nitrophenyl 1-{[3-(1,3-dioxolan-2-yl)phenyl]formamide}-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacon-39-noate (17) (5.1 g, 86.8%).

[0227] d. Step 4 Synthesis of pPB PEG 12 benzaldehyde (18a+b) pPB and scrambled pPB peptides were prepared using standard solid-phase peptide synthesis. After global deprotection of the peptides, amide conjugation was performed with activated carboxylic acids (n). Dioxalane deprotection, purification by reversed-phase RPLC, and subsequent lyophilization afforded the peptide-linked PEG-benzaldehydes (18a+b). TIFF2025531872000071.tif42157 Monovalent pPB PEG12 benzaldehyde (18a) MW: Calculated 1652.92, Found 1653.92, Purity (RP-HPLC) >95% TIFF2025531872000072.tif43157 Scrambled monovalent pPB PEG12 benzaldehyde (18b) MW: Calculated 1652.92, Found 1653.85, Purity (RP-HPLC) >95%

[0228] Example 3: Synthesis of Endosomally Released Polymers Endosomally releasing polymers were synthesized using RAFT polymerization and terminated with protected aminooxy functionalities for later oxime conjugation with benzaldehyde peptide ligands (18a and 18b).

[0229] Part 1) Synthesis of the chain transfer agent tert-butyl ((6-cyano-6-methyl-9,15-dioxo-4-thioxo-3,5-dithia-10,14-diazahexadecan-16-yl)oxy)carbamate (compound 24) TIFF2025531872000073.tif94161

[0230] a. Step 1 Synthesis of perfluorophenyl 4-cyano-4-(((ethylthio)carbonothioyl)thio)-pentanoate (20) 4-Cyano-4-{[(ethylsulfanyl)methanethioyl]sulfanyl}-4-methylbutanoic acid (19) (26 g, 99 mmol) and triethylamine (12 g, 118 mmol) were dissolved in anhydrous DCM (500 mL) and stirred at 0 °C. 2,3,4,5,6-Pentafluorophenyl 2,2,2-trifluoroacetate (12) (33 g, 118.5 mmol) was added slowly over 15 min. The solution was stirred at 0 °C until complete. The reaction was washed with saturated NaHCO (3 × 250 mL), dried (MgSO), filtered, and concentrated under reduced pressure to give perfluorophenyl 4-cyano-4-(((ethylthio)carbonothioyl)thio)pentanoate (20) (Quant), which was used without further purification.

[0231] b. Step 2 Synthesis of tert-butyl(2-((3-(((benzyloxy)carbonyl)amino)propyl)amino)-2-oxoethoxy)carbamate (22) {[(tert-Butoxycarbonyl)amino]oxy}acetic acid (21) (20 g, 105 mmol), DIPEA (55 mL, 314 mmol), HATU (40 g, 105 mmol), and benzyl N-3-[amino]propylcarbamimate HCl salt (26 g, 105 mmol) were dissolved in DCM (350 mL) and stirred at RT for 16 h. DCM (150 mL) was added, and the reaction was washed with saturated NaHCO and brine, dried (MgSO), concentrated under reduced pressure, and purified by automated flash chromatography (0–5% MeOH in DCM) to give tert-butyl N-{[(3-{[(benzyloxy)carbonyl]amino}propyl)carbamoyl]methoxy}carbamate (22) (31 g, 78.2%).

[0232] c. Step 3 Synthesis of tert-butyl (2-((3-aminopropyl)amino)-2-oxoethoxy)-carbamate (23) tert-Butyl N-{[(3-{[(benzyloxy)carbonyl]amino}propyl)carbamoyl]methoxy}-carbamate (22) (31 g, 81 mmol) was subjected to catalytic hydrogenation with 10% palladium on carbon (cat.) in MeOH (200 mL) at RT for 16 h. Upon completion, the solution was filtered through Celite and concentrated to dryness to give tert-butyl (2-((3-aminopropyl)amino)-2-oxoethoxy)carbamate (23) (19.4 g, 91.5%), which was used without further purification.

[0233] d. Step 4 Synthesis of tert-butyl ((6-cyano-6-methyl-9,15-dioxo-4-thioxo-3,5-dithia-10,14-diazahexadecan-16-yl)oxy)carbamate (24) tert-Butyl N-{[(3-aminopropyl)carbamoyl]methoxy}carbamate (23) (18 g, 73 mmol), 2,3,4,5,6-pentafluorophenyl 4-cyano-4-{[(ethylsulfanyl)methanethioyl]sulfanyl}-4-methylbutanoate (20) (31 g, 73 mmol), and DMAP (178 mg, 1.5 mmol) were dissolved in DCM (100 mL) and stirred at RT until completion. The reaction was concentrated under reduced pressure, dissolved in EtOAc (250 mL), washed with saturated NaHCO (2 × 100 mL), dried (MgSO), filtered, and concentrated under reduced pressure. The residue was purified by automated flash chromatography (0-5% MeOH / DCM) to give tert-butyl ((6-cyano-6-methyl-9,15-dioxo-4-thioxo-3,5-dithia-10,14-diazahexadecan-16-yl)oxy)carbamate (24) (28 g, 78.1%). TIFF2025531872000074.tif19156

[0234] Part 2) Synthesis of "BOC-gly" Endosomal Release Polymer (Compound 25) TIFF2025531872000075.tif101162

[0235] Prieve, M.G., Harvie, P., Monahan, S.D., Roy, D., Li, A.G., Blevins, T.L., Paschal, A.E., Waldheim, M., Bell, E.C., Galperin, A., Ella-Menye, J.R., et al. (2018). Targeted mRNA Therapy for Ornithine Transcarbamylase Deficiency. Mol Ther 26, 801-813. Similar to a similar synthesis described in 10.1016 / j.ymthe.2017.12.024, the "BOC-Gly" endosomally released polymer (25) was prepared from chain transfer agent (24) using RAFT polymerization. The final released material had the following observed monomer composition and average molecular weight: TIFF2025531872000076.tif951622'-O-methyl nucleotides are indicated by an "m" capital letter. 2'-fluoro nucleotides are indicated by an "f" capital letter. Phosphorothioate linkers are indicated by an "s."

[0236] Part 3) Synthesis of oxime-linked ligand-functionalized endosomally released polymers, exemplified by the following general reaction protocol TIFF2025531872000077.tif236163

[0237] a. Step 1. Removal of BOC from Endosomally Released Polymer 25 1.044 g of polymer was dissolved in DCM (4 ml) and TFA (3.3 ml) was added. The reaction was stirred at RT for 2 h. The reaction was concentrated under reduced pressure for 60 min. The remaining TFA salt was used in the subsequent conjugation step without further treatment.

[0238] b. Step 2. Oxime conjugation The material remaining from the previous step was dissolved in DCM (5 mL), 0.55 stoichiometric equivalents of the appropriate peptide benzaldehyde (18a or b) was added, and the reaction was stirred at RT for 72 h. An additional 0.55 stoichiometric equivalents of the appropriate peptide benzaldehyde was added, and stirring was continued for another 72 h. Resin-bound sequestering agent PS-TsNHNH2 (PS-tosylhydrazide) resin (3 equivalents) was added, and the reaction was left for 24 h. The resin was removed by filtration washing with additional DCM. The solvent was removed by vacuum drying until a constant weight was reached. The remaining material was dissolved in acetone and dialyzed against methanol for 2 days, followed by dialysis against water for 2 days. Lyophilization yielded the peptide oxime-conjugated endosomally released polymer 28a or b.

[0239] Example 4: Ligands and Chemistry Used to Form siRNA-Ligand Conjugates The following ligands and corresponding siRNA conjugates were synthesized to evaluate their in vitro (Example 10) and in vivo (Example 11) activities in combination with targeted endosomal release polymers (Example 3). First, the peptide ligands were synthesized using standard solid-phase peptide synthesis protocols and then functionalized with an appropriate conjugation motif (e.g., maleimide or DBCO) to facilitate coupling to the 3'-end of the sense strand of the siRNA molecule. Correspondingly, the phosphate at the 3'-end of the siRNA was also modified (e.g., 3'C6 amino or 3'C6 sulfhydryl) for coupling to the ligand. TIFF2025531872000078.tif52152 Monovalent pPB (compound 29) TIFF2025531872000079.tif46148 Scrambled monovalent pPB (compound 30) TIFF2025531872000080.tif213136TIFF2025531872000081.tif155150Divalent pPBmaleimide (Compound 42)

[0240] a. Step 1: Synthesis of tert-butyl 3-(2-(2-(2-((methylsulfonyl)oxy)ethoxy)ethoxy)-ethoxy)propanoate tert-Butyl-3-{2-[2-(2-hydroxyethoxy)ethoxy]ethoxy}propanoate (25 g, 90 mmol) and TEA (16.0 mL, 113 mmol) were dissolved in DCM and stirred at 0 °C. Methanesulfonyl chloride (11.3 g, 99 mmol) dissolved in DCM was added dropwise, and the reaction was stirred at RT for 1.5 h. The organic compound was washed with saturated NaHCO, water, and brine, dried (MgSO), and concentrated under reduced pressure to give tert-butyl 3-(2-{2-[2-(methanesulfonyloxy)ethoxy]ethoxy}ethoxy)propanoate (32.1 g, Quant), which was used without purification.

[0241] b. Step 2: Synthesis of tert-butyl 3-(2-(2-(2-bromoethoxy)ethoxy)ethoxy)propanoate tert-Butyl 3-(2-{2-[2-(methanesulfonyloxy)ethoxy]ethoxy}ethoxy)propanoate (33 g, 92 mmol) and TBAB (39 g, 120 mmol) were heated at reflux for 2 hours. The reaction was cooled, washed with water, and back-extracted from water with hexanes. The combined organic compounds were washed with brine, dried (NaSO), concentrated under reduced pressure, and the residue was purified by automated flash chromatography (35% EtOAc / hex) to give tert-butyl 3-{2-[2-(2-bromoethoxy)ethoxy]ethoxy}propanoate (27.4 g, 87.1%).

[0242] c. Step 3: Synthesis of 2-(3,5-dimethoxybenzyl)isoindoline-1,3-dione (3,5-Dimethoxyphenyl)methanamine and isobenzofuran-1,3-dione were dissolved in AcOH and heated at reflux for 16 hours. The reaction was concentrated under reduced pressure, and the residue was dissolved in DCM and washed with 1 M HCl, NaHCO, water, and brine, dried (NaSO), and concentrated under reduced pressure. The residue was purified by automated flash chromatography (35% EtOAc / Hex) to give 2-[(3,5-dimethoxyphenyl)methyl]isoindole-1,3-dione (9.03 g, 45.6%).

[0243] d. Step 4: Synthesis of 2-(3,5-dihydroxybenzyl)isoindoline-1,3-dione 2-[(3,5-Dimethoxyphenyl)methyl]isoindole-1,3-dione (9 g, 30 mmol) was dissolved in DCM and stirred at 0 °C. BBr (11.7 mL, 121 mmol) was added dropwise, and the reaction was stirred for 16 h, warming to RT. The reaction was cooled to 0 °C, quenched with MeOH, and concentrated under reduced pressure. The residue was dissolved in EtOAc, washed successively with 1 M HCl, saturated NaHCO, water, and brine, dried (MgSO), and concentrated under reduced pressure to give 2-[(3,5-dihydroxyphenyl)methyl]isoindole-1,3-dione (7.8 g 95.7%), which was used without further purification.

[0244] e. Step 5: Synthesis of tert-butyl 3-{2-[2-(2-{3-[2-(2-{2-[3-(tert-butoxy)-3-oxopropoxy]-ethoxy}ethoxy)ethoxy]-5-[(1,3-dioxoisoindol-2-yl)methyl]phenoxy}ethoxy)ethoxy]ethoxy}-propanoate 2-[(3,5-Dihydroxyphenyl)methyl]isoindole-1,3-dione (10 g, 37 mmol), tert-butyl 3-{2-[2-(2-bromoethoxy)ethoxy]ethoxy}propanoate (30.4 g, 89 mmol), and KCO were dissolved in MeCN and heated at reflux for 16 h. The reaction was cooled to RT, filtered, washed with additional MeCN, and concentrated under reduced pressure. The residue was purified by automated flash chromatography (60% EtOAc / hex) to give tert-butyl 3-{2-[2-(2-{3-[2-(2-{2-[3-(tert-butoxy)-3-oxopropoxy]ethoxy}ethoxy)ethoxy]-5-[(1,3-dioxoisoindol-2-yl)methyl]phenoxy}ethoxy)ethoxy]ethoxy}propanoate (14 g, 47.7%).

[0245] f. Step 6: Synthesis of tert-butyl 3-[2-(2-{2-[3-(aminomethyl)-5-[2-(2-{2-[3-(tert-butoxy)-3-oxopropoxy]ethoxy}ethoxy)ethoxy]phenoxy]ethoxy}ethoxy)ethoxy]propanoate tert-Butyl 3-{2-[2-(2-{3-[2-(2-{2-[3-(tert-butoxy)-3-oxopropoxy]ethoxy}ethoxy)-ethoxy]-5-[(1,3-dioxo-3a,7a-dihydroisoindol-2-yl)methyl]phenoxy}ethoxy)ethoxy]-ethoxy}propanoate (14 g, 17.7 mmol) and hydrazine hydrate (4.3 ml, 88 mmol) were dissolved in MeOH and stirred at 50° C. for 16 hours. The resulting precipitate was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by automated flash chromatography (10% MeOH / DCM) to give tert-butyl 3-[2-(2-{2-[3-(aminomethyl)-5-[2-(2-{2-[3-(tert-butoxy)-3-oxopropoxy]ethoxy}ethoxy)ethoxy]phenoxy]ethoxy}ethoxy)ethoxy]propanoate (7.71 g, 66.1%).

[0246] g. Step 7: Synthesis of tert-butyl 3-{2-[2-(2-{3-[2-(2-{2-[3-(tert-butoxy)-3-oxopropoxy]ethoxy}ethoxy)ethoxy]-5-{[3-(2,5-dioxopyrrol-1-yl)propanamido]methyl}phenoxy}ethoxy)ethoxy]ethoxy}propanoate tert-Butyl-3-[2-(2-{2-[3-(aminomethyl)-5-[2-(2-{2-[3-(tert-butoxy)-3-oxopropoxy]ethoxy}ethoxy)ethoxy]phenoxy]ethoxy}ethoxy)ethoxy]propanoate (7.71 g, 11.7 mmol), 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxopyrrol-1-yl)propanoate (3.1 g, 11.7 mmol), and NMM (1.41 ml, 12.9 mmol) were dissolved in DCM and stirred at RT for 36 h. The reaction was concentrated under reduced pressure and the residue was purified by automated flash chromatography (2.5% MeOH / DCM) to give tert-butyl 3-{2-[2-(2-{3-[2-(2-{2-[3-(tert-butoxy)-3-oxopropoxy]ethoxy}ethoxy)ethoxy]-5-{[3-(2,5-dioxopyrrol-1-yl)propanamido]methyl}phenoxy}ethoxy)ethoxy]ethoxy}propanoate (9.0 g, 95.1%).

[0247] h. Step 8: Synthesis of 3-[2-(2-{2-[3-(2-{2-[2-(2-carboxyethoxy)ethoxy]ethoxy}ethoxy)-5-{[3-(2,5-dioxopyrrolidin-1-yl)propanamido]methyl}phenoxy]ethoxy}ethoxy)ethoxy]propanoic acid tert-Butyl 3-{2-[2-(2-{3-[2-(2-{2-[3-(tert-butoxy)-3-oxopropoxy]ethoxy}ethoxy)ethoxy]-5-{[3-(2,5-dioxopyrrolidin-1-yl)propanamido]methyl}phenoxy}ethoxy)ethoxy]-ethoxy}propanoate (9.0 g, 11.1 mmol) was dissolved in formic acid and stirred at RT for 16 h. The reaction was concentrated under reduced pressure, azeotroped twice with toluene, and dried under high vacuum to give quantitative 3-[2-(2-{2-[3-(2-{2-[2-(2-carboxyethoxy)ethoxy]ethoxy}ethoxy)-5-{[3-(2,5-dioxopyrrolidin-1-yl)propanamido]methyl}phenoxy]ethoxy}ethoxy)ethoxy]propanoic acid, which was used for purification.

[0248] i. Step 9: Synthesis of 2,5-dioxopyrrolidin-1-yl 3-(2-{2-[2-(3-{[3-(2,5-dioxopyrrol-1-yl)propanamido]methyl}-5-{2-[2-(2-{3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3-oxopropoxy}ethoxy)ethoxy]ethoxy}phenoxy)ethoxy]ethoxy}ethoxy)propanoate 3-[2-(2-{2-[3-(2-{2-[2-(2-carboxyethoxy)ethoxy]ethoxy}ethoxy)-5-{[3-(2,5-dioxopyrrol-1-yl)propanamido]methyl}phenoxy]ethoxy}ethoxy)ethoxy]propanoic acid (8 g, 11.5 mmol), N-hydroxysuccinimide (3.6 g, 31.4 mmol), and DMAP were dissolved in DCM and stirred at RT for 5 h. The reaction was concentrated under reduced pressure and purified by automated flash chromatography (10% MeOH / DCM) to give 2,5-dioxopyrrolidin-1-yl 3-(2-{2-[2-(3-{[3-(2,5-dioxopyrrol-1-yl)propanamido]methyl}-5-{2-[2-(2-{3-[(2,5-dioxopyrrolidin-1-yl)oxy]-3-oxopropoxy}ethoxy)ethoxy]ethoxy}phenoxy)ethoxy]-ethoxy}ethoxy)propanoate (42) (3.7 g, 36.2%).

[0249] pPB conjugation and siRNA synthesis were performed as described elsewhere to yield aromatic dimeric pPB (compound 43) and aromatic dimeric scrambled pPB (compound 44).

[0250] TIFF2025531872000082.tif77161TIFF2025531872000083.tif126166 Trimeric pPB (Compound 58) Following the procedures described in the above scheme, trimeric pPB (compound 58) can be prepared.

[0251] TIFF2025531872000084.tif78128 Trimeric scrambled pPB (compound 59) Using a procedure similar to that described for compound 58, trimeric scrambled pPB (compound 59) can be prepared.

[0252] TIFF2025531872000085.tif218169 Trimeric C6 pPB (Compound 63) Following the procedures described in the above scheme, trimeric C6 pPB (compound 63) can be prepared.

[0253] TIFF2025531872000086.tif106128 Trimeric C6 Scrambled pPB (Compound 64) Using a procedure similar to that described for compound 63, trimeric C6 scrambled pPB (compound 64) can be prepared.

[0254] pPB conjugation and siRNA synthesis were performed as described elsewhere to obtain trimeric pPB (compound 58), trimeric scrambled pPB (compound 59), trimeric C6 pPB (compound 63), and trimeric C6 scrambled pPB (compound 64).

[0255] Example 5: Ligand siRNA conjugates As described in Example 3, the following siRNA-ligand conjugates were prepared using automated oligonucleotide synthesis using standard procedures. The 3'-modified sense strand and the corresponding complementary antisense strand were obtained by nucleotide deprotection and cleavage from the solid support. After purification by dual HPLC, the ligand-conjugated siRNA duplex was formed by annealing. Table 1 lists the siRNA sequences, IDs, and mass spectrometry.

[0256] (Table 1) siRNA conjugate TIFF2025531872000087.tif227166TIFF2025531872000088.tif133166A = adenine; G = guanine; U = uracil; C = cytosine; mN = 2'OMe-ribose modification; fN = 2'fluoro-ribose modification; s = phosphorothioate (PS) modification

[0257] Example 6. Uptake of monovalent and trivalent pPB ligands by HSC-T6 and NIH3T3 cells in vitro HSC-T6 is an immortalized rat hepatic stellate cell line, and NIH3T3 is a mouse embryonic fibroblast cell line. Both cell lines express PDGFRB, the target receptor of pPB, with NIH3T3 cells showing higher expression (Figure 1).

[0258] Preparation of biotin-pPB / AF488-streptavidin complex Biotinylated monovalent or trivalent pPB ligands were reconstituted in DMSO and functionalized by overnight incubation at 4°C with Alexa Fluor 488-labeled streptavidin in Tyrode's buffer (containing 10 mM HEPES, 5.6 mM glucose, 10 mM KCl, 35 mM NaCl, 0.4 mM MgCl, 1.0 mM CaCl, and 0.1% BSA, pH 7.3) at a molar ratio of biotinylated ligand:biotin-binding site of 4.5:1.

[0259] cell culture HSC-T6 cells were cultured in rat HSC medium (DMEM-high glucose without sodium pyruvate containing 10% FBS and 2.5 mM L-glutamine).NIH3T3 cells were cultured in MEM containing 10% FBS and 2.5 mM L-glutamine.

[0260] Western blot analysis of PDGFRB expression Cultured HSC-T6 and NIH3T3 cells were lysed in RIPA buffer. Cell lysates were clarified by centrifugation at 13,000 × g for 15 min, separated by SDS-PAGE, transferred to PVDF membranes, and probed with anti-PDGFRB (Cell Signaling) and anti-β-actin antibodies (Cell Signaling) in 5% BSA Tris-buffered saline and Tween-20 buffer.

[0261] Uptake of biotin-pPB / AF488-streptavidin complex HSC-T6 or NIH3T3 cells were seeded in sterile 96-well plates (20,000 cells / well) and cultured at 37°C for 48 hours. The previously prepared biotin-pPB / AF488-streptavidin complex was diluted in Tyrode's buffer to 2.22 μM, 0.556 μM, 0.136 μM, 0.035 μM, and 0.009 μM (based on streptavidin molar concentration) and placed on ice. Prior to incubation with the complex, the cells were washed three times with ice-cold Tyrode's buffer, and functionalized streptavidin was added to each well and incubated at 4°C for 1.5 hours. After incubation, the cells were washed three times with ice-cold Tyrode's buffer to remove unbound ligand complexes. Then, pre-warmed complete medium was added to the wells, and the cells were immediately incubated at 37°C for 1 hour to allow endocytosis of the receptor-bound ligand complexes. After incubation, cells were washed once with DPBS, and 50 μL of 0.25% trypsin-EDTA was added to each well. After 3-4 minutes of incubation at 37°C, 100 μL of medium was added to each well to inactivate the trypsin and mixed well to detach the cells. Cells were transferred to a V-bottom 96-well plate, spun at 1200 RPM for 5 minutes, washed twice with DPBS, and resuspended in DPBS containing Live / Dead Red stain. After 30 minutes at 4°C, cells were centrifuged, and the pellet was resuspended in staining buffer before flow cytometry analysis.

[0262] Flow cytometry analysis Analysis was performed on a FACS-Canto II using FACS Diva software. Cells were stained with Live / Dead Red as a viability marker. Forward and side scatter gates were set to include all live cells. Approximately 10,000–15,000 cells were counted for each sample, and binding / uptake was determined as the increase in green fluorescence intensity at 488 nm detected in the FL1 channel. Binding / uptake was determined by subtracting the MFI of cells incubated without functionalized streptavidin (free fluorophore only) from the mean fluorescence intensity (MFI) of cells incubated with functionalized streptavidin.

[0263] result We observed uptake of the monovalent and trivalent pPB ligand complexes in both HSC-T6 and NIH3T3 cells, as evidenced by a dose-dependent increase in AF488 fluorescence in treated cells (Figure 2). The trivalent pPB ligand exhibited approximately 60-fold higher binding affinity than the monovalent pPB ligand. Ligand complexes containing scrambled monovalent pPB peptides exhibited approximately 4-fold lower potency than the monovalent pPB ligand complexes. This difference in potency increased to approximately 60-fold with the trivalent ligand. The mean fluorescence intensity (MFI) from the ligands in NIH3T3 cells was higher than that in HSC-T6 cells. This correlated with the expression levels of the PDGFRB receptor in these two cell lines. These results demonstrated effective uptake of the pPB ligand in PDGFRB-expressing cells.

[0264] Example 7. Uptake of bivalent and trivalent pPB ligands by primary human hepatic stellate cells in vitro material and method Preparation of biotin-pPB / AF488-streptavidin complex Biotinylated bivalent, trivalent pPB, or scrambled trivalent pPB ligands were reconstituted in DMSO and functionalized by overnight incubation at 4°C with Alexa Fluor 488-labeled streptavidin in Tyrode's buffer (containing 10 mM HEPES, 5.6 mM glucose, 10 mM KCl, 35 mM NaCl, 0.4 mM MgCl, 1.0 mM CaCl, and 0.1% BSA, pH 7.3) at a molar ratio of biotinylated ligand:biotin-binding site of 4.5:1.

[0265] cell culture pHHSCs were cultured using ScienCell's Human Hepatic Stellate Cell Culture Kit.

[0266] Uptake of biotin-pPB / AF488-streptavidin complex pHHSCs were seeded into sterile 96-well plates (6,000 cells / well) and cultured at 37°C for 48 hours. The previously prepared biotin-pPB / AF488-streptavidin complex was diluted with Tyrode's buffer to 3 μM, 1 μM, 0.3 μM, and 0.1 μM (based on streptavidin molar concentration) and placed on ice. Prior to incubation with the complex, cells were washed three times with ice-cold Tyrode's buffer, and functionalized streptavidin was added to each well and incubated at 4°C for 1.5 hours. After incubation, cells were washed three times with ice-cold Tyrode's buffer to remove unbound ligand complexes. Complete medium prewarmed to 37°C was then added, and the cells were immediately incubated at 37°C for 1 hour to allow endocytosis of the receptor-bound ligand-complex. After incubation, cells were washed once with DPBS, and 50 μL of 0.025% trypsin-EDTA was added to each well. After 3-4 minutes of incubation at 37°C, 100 μL of trypsin neutralizing solution (TNS) was added to each well to inactivate the trypsin and mixed thoroughly to detach the cells. The cells were transferred to a V-bottom 96-well plate, spun at 1200 RPM for 5 minutes, washed twice with staining buffer, and then resuspended in staining buffer containing DAPI (4',6-diamidino-2-phenylindole) before flow cytometry analysis.

[0267] Flow cytometry analysis Analysis was performed using FACS Diva software on a FACS-Canto II. Cells were stained with DAPI as a viability marker. Forward and side scatter gates were set to include all live cells. Approximately 10,000–15,000 cells were counted for each sample, and binding / uptake was determined as the increase in green fluorescence intensity at 488 nm detected in the FL1 channel. Binding / uptake was calculated by subtracting the mean fluorescence intensity (MFI) of cells incubated with functionalized streptavidin from the MFI of cells incubated without functionalized streptavidin (free fluorophore only).

[0268] result We observed uptake of bivalent and trivalent pPB ligand complexes in pHHSCs, as evidenced by a dose-dependent increase in AF488 fluorescence in treated cells (Figure 3). The bivalent pPB ligand exhibited slightly lower binding activity than the trivalent pPB ligand, but was significantly more potent than the scrambled trivalent pPB ligand. These results demonstrate that the pPB ligand titer affects binding affinity.

[0269] Example 8. Uptake of trivalent pPB ligand by NIH3T3, LX-2, and primary human HSCs in vitro LX-2 is an immortalized human hepatic stellate cell line, and NIH3T3 is a mouse embryonic fibroblast cell line. All three cell lines expressed PDGFRB, the target receptor for pPB, with NIH3T3 cells showing the highest expression (Figure 4).

[0270] material and method Preparation of biotin-pPB / AF488-streptavidin complex The biotinylated trivalent pPB ligand was reconstituted in DMSO and functionalized by overnight incubation at 4°C with Alex Fluor 488-labeled streptavidin dissolved in Tyrode's buffer (containing 10 mM HEPES, 5.6 mM glucose, 10 mM KCl, 35 mM NaCl, 0.4 mM MgCl, 1.0 mM CaCl, and 0.1% BSA, pH 7.3) at a molar ratio of biotinylated ligand:biotin-binding site of 4.5:1.

[0271] cell culture LX-2 cells were cultured in DMEM-high glucose containing 10% FBS and 2.5 mM L-glutamine. NIH3T3 cells were cultured in MEM containing 10% FBS and 2.5 mM L-glutamine. pHHSCs were cultured using ScienCell's human hepatic stellate cell culture kit.

[0272] Western blot analysis of PDGFRB expression Cultured LX-2, NIH3T3, and pHHSC cells were lysed in RIPA buffer. Cell lysates were clarified by centrifugation at 13,000 × g for 15 min, separated by SDS-PAGE, transferred to PVDF membranes, and probed with anti-PDGFRB (Cell Signaling) and anti-β-actin antibodies (Cell Signaling) in 5% BSA Tris-buffered saline and Tween-20 buffer.

[0273] Uptake of biotin-pPB / AF488-streptavidin complex LX-2, NIH3T3, or pHHSC cells were seeded in sterile 96-well plates (20,000 cells / well) and cultured at 37°C for 48 hours. The previously prepared biotin-pPB / AF488-streptavidin complex was diluted in Tyrode's buffer to 3 μM, 1 μM, 0.3 μM, and 0.1 μM (based on streptavidin molar concentration) and placed on ice. Prior to incubation with the complex, the cells were washed three times with ice-cold Tyrode's buffer, and functionalized streptavidin was added to each well and incubated at 4°C for 1.5 hours. After incubation, the cells were washed three times with ice-cold Tyrode's buffer to remove unbound ligand complexes. Then, pre-warmed complete medium was added to the wells and the cells were immediately incubated at 37°C for 1 hour to allow endocytosis of the receptor-bound ligand complexes. After incubation, cells were washed once with DPBS, and 50 μL of 0.25% trypsin-EDTA was added to each well. After 3-4 minutes of incubation at 37°C, 100 μL of medium was added to each well to inactivate the trypsin and mix well to detach the cells. Cells were transferred to a V-bottom 96-well plate, spun at 1200 RPM for 5 minutes, washed twice with staining buffer, and resuspended in staining buffer containing DAPI before flow cytometry analysis.

[0274] Flow cytometry analysis Analysis was performed using FACS Diva software on a FACS-Canto II. Cells were stained with DAPI as a viability marker. Forward and side scatter gates were set to include all live cells. Approximately 10,000–15,000 cells were counted for each sample, and binding / uptake was determined as the increase in green fluorescence intensity at 488 nm detected in the FL1 channel. Binding / uptake was calculated using the mean fluorescence intensity (MFI) of cells incubated with functionalized streptavidin minus the MFI of cells incubated without functionalized streptavidin (free fluorophore only).

[0275] result We observed uptake of the trivalent pPB-ligand complex in HSC-T6, LX-2, and pHHSC, as evidenced by a dose-dependent increase in fluorescence intensity in the treated cells (Figure 5). The mean fluorescence intensity (MFI) in the three cell types correlated with the expression level of the target receptor (PDGFRB). These results demonstrate the effective uptake of pPB-ligand in PDGFRB-expressing cells, including human hepatic stellate cells.

[0276] Example 9. Trivalent pPB-conjugated siRNA enabled gene silencing in NIH3T3 cells in vitro when treated with chloroquine PPIB is a widely expressed protein-coding gene and was used as a surrogate siRNA target in this study. NIH3T3 cells express high levels of PDGFRB and showed the best pPB ligand uptake efficiency in previous experiments. To address the limited endosomal release during siRNA conjugate delivery, we stimulated cells with chloroquine after siRNA conjugate treatment.

[0277] material and method cell culture NIH3T3 cells were cultured in MEM containing 10% FBS and 2.5 mM L-glutamine.

[0278] siRNA treatment in NIH3T3 cells NIH3T3 cells were seeded (10,000 / well) into sterile 96-well plates and cultured at 37°C for 24 hours. The cells were then washed with 100 μL of OptiMEM and then incubated with 0.4 μM, 0.1 μM, or 0.025 μM of TripPB-siPPIB, Tri-Scr-pPB-siPPIB, unconjugated siPPIB, or siLuc2 dissolved in OptiMEM for 4 hours. The siRNA or conjugate was then removed, and the cells were treated with 50 μM chloroquine dissolved in complete culture medium for an additional 20 hours. After treatment was complete, the culture medium was removed, and the cells were lysed with QuantiGene Lysis Mixture for subsequent gene expression quantification.

[0279] QuantiGene branched DNA assay To evaluate gene silencing activity, cell lysates collected after siRNA treatment were subjected to the QuantiGene branched DNA assay according to the manufacturer's protocol. Briefly, cell lysates were incubated with capture probes targeting mPpib (target gene) and mGapdh (endogenous control) at 55°C for 18–20 hours. After washing, the plate was incubated with pre-amplification and amplification probes to amplify the signal. Excess probe was washed away, assay substrate was added, and luminescence was quantified using a plate reader. Ppib signals were normalized to the signal from Gapdh.

[0280] result Dose-dependent target gene (PPIB) silencing was observed in cells treated with TripPB-siPPIB, Tri-Scr-pPB-siPPIB, and unconjugated PPIB, but not in siLuc2-treated cells. This demonstrates that targeted gene silencing is mediated by siPPIB (Figure 6). Importantly, TripPB-conjugated siRNA conjugated to siPPIB conjugates exhibited the highest activity compared with Tri-Scr-pPB-siPPIB and unconjugated PPIB. This suggests that the TripPB ligand enables more efficient siRNA delivery. Consistent with the ligand conjugation results, Tri-Scr-pPB ligand conjugation moderately improved RNAi activity compared with unconjugated siRNA. Unconjugated siRNA was only active at the highest tested dose (0.40 μM). This may be due to a passive uptake delivery mechanism. These results demonstrate that trivalent pPB ligand conjugation can enhance siRNA delivery to NIH3T3 cells.

[0281] Example 10. Trivalent pPB-conjugated siRNA and pPB-functionalized endosomal-releasing polymers enabled gene silencing in pHHSC cells in vitro material and method The polymer was dissolved to 40 mg / mL in 10 mM phosphate / 200 mM sucrose PBS (pH 7) followed by serial filtration (3-4 times) through 0.2 micron sterile filters.

[0282] cell culture pHHSCs were cultured using ScienCell's Human Hepatic Stellate Cell Culture Kit.

[0283] Treatment of pHHSC cells with siRNA pHHSC cells were seeded (5000 / well) into sterile 96-well plates and cultured at 37°C for 24 hours. The cells were then washed with 100 μL of OptiMEM and then treated with 0.3 μM or 0.1 μM TripPB-siPPIB or unconjugated siPPIB dissolved in OptiMEM for 4 hours. GalNAc- or pPB-functionalized ERP was then added to the culture at a concentration of 30 μg / mL. 24 hours after siRNA treatment, the cells were supplemented with 10% FBS. 72 hours after siRNA treatment, the culture medium was removed, and the cells were lysed with QuantiGene Lysis Mixture for subsequent gene expression quantification.

[0284] QuantiGene branched DNA assay To evaluate gene silencing activity, cell lysates collected after siRNA treatment were subjected to the QuantiGene branched DNA assay according to the manufacturer's protocol. Briefly, cell lysates were incubated with capture probes targeting human PPIB (target gene) and GAPDH (endogenous control) at 55°C for 18–20 hours. After washing, the plate was incubated with pre-amplification and amplification probes to amplify the signal. Excess probe was then washed away, assay substrate was added, and luminescence was quantified using a plate reader. The signal from PPIB was normalized to the signal from GAPDH.

[0285] result When combined with 30 μg / mL pPB-ERP, TripPB-siPPIB enabled 79% and 65% target gene KD at 0.3 μM and 0.1 μM treatments, respectively (Figure 7). In contrast, when combined with 30 μg / mL pPB-ERP, unconjugated siPPIB exhibited low gene silencing activity (<40%), with no discernible dose-response effect. RNAi activity was also observed in the TripPB-siPPIB + GalNAc-ERP combo treatment group, but GalNAc-ERP was less effective than pPB-ERP. siRNA alone, whether unconjugated or conjugated with TripPB, had no effect on target gene silencing, nor did pPB-ERP alone. These results suggest that pPB ligands can improve the delivery of both siRNA and ERP to pHHSCs in vitro.

[0286] Example 11. pPB-ERP improves in vivo gene silencing by TripPB siRNA conjugates in a mouse liver fibrosis model To evaluate RNAi activity in targeted cell populations, we selected Hsp47, an HSC-specific gene, as the siRNA target.

[0287] material and method Evaluation of TripPB siRNA conjugates and pPB-ERP in a mouse liver fibrosis model To induce HSC activation, 6-8 week-old Balb / c female mice (n = 4-5 per group) were intraperitoneally injected with 2.5 mL / kg of 20% CCl4 dissolved in corn oil twice weekly for 2 weeks. One day after the final CCl4 dose, animals were subcutaneously (SQ) injected in the scapular region with a single dose of vehicle control (saline), pPB-ERP alone (40 mg / kg), TripPB-siHsp47 (10 mg / kg) and pPB-ERP (40 mg / kg), or unconjugated siHsp47 (10 mg / kg) and pPB-ERP (40 mg / kg) using a volume of 5 mL per kg of body weight. In one group of animals, a single intravenous (IV) dose of TripPB-siHsp47 (10 mg / kg) and pPB-ERP (40 mg / kg) was also tested. Two days after treatment with siRNA and ERP, livers from treated animals were harvested and fixed in RNAlater for a minimum of 16 hours at 4°C. The RNAi activity of the siRNA conjugates was assessed by quantifying target gene expression using the Quantigene assay.

[0288] QuantiGene branched DNA assay 20–25 mg of RNAlater-fixed liver tissue was homogenized in Epicentre lysis buffer containing 1% proteinase K using a FastPrep®-24 homogenization device (4.0 m / s, 3 × 15-second bursts, MP Biomedicals). Samples were placed on ice before and after homogenization to prevent degradation. Lysates were clarified by centrifugation at 16,000 × g for 5 minutes at 16°C and diluted with lysis working buffer to ensure all assay values ​​were within the linear detection range. Then, they were subjected to the bDNA assay using the Affymetrix QuantiGene 2.0 assay kit according to the manufacturer's protocol. The bDNA probes were specifically designed to target mouse Hsp47 and mouse Gapdh. The signal from Hsp47 was normalized to the signal from Gapdh.

[0289] result Combined treatment of TripPB-siHsp47 and pPB-ERP in the tested dosing regimens resulted in 47% target gene KD with subcutaneous administration (Figure 8) and 68% gene suppression with intravenous administration. The improved RNAi activity with IV administration is likely related to the high bioavailability of this dosing method. In contrast, minimal (25%) gene silencing was observed in animals treated with unconjugated siHsp47 and pPB-ERP. pPB-ERP treatment alone did not induce any reduction in target gene expression. Notably, Hsp47 is an HSC-specific gene. Together, these data demonstrate that combining trivalent pPB ligand-conjugated siRNA with pPB-ERP enables target gene silencing in HSCs in a liver fibrosis model.

[0290] Example 12. Dose-response of pPB-ERP in HSC-targeted RNAi in a mouse liver fibrosis model The HSC-specific gene Hsp47 was selected as an siRNA target to evaluate RNAi activity in targeted cell populations.

[0291] material and method Assessment of pPB-ERP dose response in a mouse liver fibrosis model To induce HSC activation, 6-8 week-old Balb / c female mice (n = 4-5 per group) were intraperitoneally injected with 2.5 mL / kg of 20% CCl4 dissolved in corn oil twice weekly for 2 weeks. One day after the final CCl4 dose, animals were subcutaneously injected in the scapular region with a single dose of 12 mg / kg, 20 mg / kg, or 40 mg / kg of vehicle control (saline), TripPB-siHsp47 (3 mg / kg), pPB-ERP alone (40 mg / kg), or TripPB-siHsp47 (3 mg / kg) and pPB-ERP, using a volume of 5 mL per kg of body weight. Two days after treatment with siRNA and ERP, livers from treated animals were harvested and fixed in RNAlater for a minimum of 16 hours at 4°C. RNAi activity of the siRNA conjugates was assessed by quantifying target gene expression using the Quantigene assay.

[0292] QuantiGene branched DNA assay 20–25 mg of RNAlater-fixed liver tissue was homogenized in Epicentre lysis buffer containing 1% proteinase K using a FastPrep®-24 homogenization device (4.0 m / s, 3 × 15-second bursts, MP Biomedicals). Samples were placed on ice before and after homogenization to prevent degradation. Lysates were clarified by centrifugation at 16,000 × g for 5 min at 16°C and diluted with lysis working buffer to ensure all assay values ​​were within the linear detection range. Then, they were subjected to the bDNA assay using the Affymetrix QuantiGene 2.0 assay kit according to the manufacturer's protocol. The bDNA probes were specifically designed to target mouse Hsp47 and mouse Gapdh. The signal from Hsp47 was normalized to the signal from Gapdh.

[0293] result No gene silencing was observed in either the TripPB-siHsp47 or pPB-ERP single-treatment groups (Figure 9). This is consistent with the results in other examples reported in this application. A dose-response effect of pPB-ERP treatment was evident in the TripPB-siHsp47 combination treatment group, with 12 mg / kg, 20 mg / kg, and 40 mg / kg of pPB-ERP reducing gene expression of the HSC target Hsp47 by 15%, 30%, and 53%, respectively. These data demonstrate the importance of including an endosomal release element.

[0294] Example 13. Bivalent pPB-siRNA conjugates exhibited comparable activity to TripPB siRNA conjugates in a mouse liver fibrosis model This example demonstrates in vivo gene silencing mediated by bivalent and trivalent pPB-conjugate siRNAs and pPB-ERP in a CCl4-induced mouse liver fibrosis model. The HSC-specific gene Hsp47 was selected as the siRNA target to evaluate RNAi activity in targeted cell populations.

[0295] material and method Evaluation of DipPB and TripPB siRNA conjugates and pPB-ERP in a mouse liver fibrosis model To induce HSC activation, 6-8 week-old Balb / c female mice (n = 4-5 per group) were intraperitoneally injected with 2.5 mL / kg of 20% CCl4 dissolved in corn oil twice weekly for 2 weeks. One day after the final CCl4 dose, animals were subcutaneously injected in the scapular region with a single dose of vehicle control (saline), DipPB-siHsp47 (3 mg / kg) and pPB-ERP (40 mg / kg), or TripPB-siHsp47 (3 mg / kg) and pPB-ERP (40 mg / kg) using a volume of 5 mL per kg of body weight. Two days after treatment with siRNA and ERP, livers from treated animals were harvested and fixed in RNAlater for a minimum of 16 hours at 4°C. RNAi activity of the siRNA conjugates was assessed by quantifying target gene expression using the Quantigene assay.

[0296] QuantiGene branched DNA assay 20–25 mg of RNAlater-fixed liver tissue was homogenized in Epicentre lysis buffer containing 1% proteinase K using a FastPrep®-24 homogenization device (4.0 m / s, 3 × 15-second bursts, MP Biomedicals). Samples were placed on ice before and after homogenization to prevent degradation. Lysates were clarified by centrifugation at 16,000 × g for 5 min at 16°C and diluted with lysis working buffer to ensure all assay values ​​were within the linear detection range. Then, they were subjected to the bDNA assay using the Affymetrix QuantiGene 2.0 assay kit according to the manufacturer's protocol. The bDNA probes were specifically designed to target mouse Hsp47 and mouse Gapdh. The signal from Hsp47 was normalized to the signal from Gapdh.

[0297] result Combined treatment of TripPB-siHsp47 and pPB-ERP in the tested dosing regimen demonstrated 46% target gene KD. Replacing TripPB-siHsp47 with DipPB-siHsp47 resulted in 58% gene silencing, which was not statistically different from the TripPB-siHsp47 group. Overall, the trivalent and bivalent pPB-siRNA conjugates in this study demonstrated comparable activity.

[0298] Synthesis of endosomally released polymers Endosomal release polymers (mannose-ERP40 and GalNAc-ERP41) were synthesized by Syngene International LTD using a synthetic method similar to that described in Prieve, MG, Harvie, P., Monahan, SD, Roy, D., Li, AG, Blevins, TL, Paschal, AE, Waldheim, M., Bell, EC, Galperin, A., Ella-Menye, JR, et al. (2018). Targeted mRNA Therapy for Ornithine Transcarbamylase Deficiency. Mol Ther 26, 801-813. 10.1016 / j.ymthe.2017.12.024. The structures of mannose-ERP40 and GalNAc-ERP41 are shown below. TIFF2025531872000089.tif103149·Block 1 MW 5.7 KDa ± 15% of target value (4.85-6.56 KDa) Block 2 MW 7.9 kDa ± 15% of target value (9.11 to 6.73 kDa) Incorporation of Block 1 monomer PEGMA 75% (71-79%) HMA 25% (21-29%) Incorporation of Block 2 monomers BMA 54% (47-57%) DMAEMA 35% (30-40%) PAA 12% (9-16%)

Claims

1. Formula (I): or a salt thereof, wherein x is 2, 3, 4, or 5; Each R 1 is independently a targeting ligand, and the targeting ligand is a) SEQ ID NO: 1: C * SRNLIDC * (SEQ ID NO:1) A cyclic polypeptide represented by the formula: * represents a disulfide bond linking two C residues thereby forming a cyclic polypeptide, and b) C * Any cyclic polypeptide having at least 80% sequence identity to a polypeptide set forth in SEQ ID NO:1, provided that both residues are present, wherein: * exhibits a disulfide bond linking two C residues thereby forming a cyclic polypeptide. Selected from L is a linking group, y is 1, 2, 3, 4, or 5; Each R 2 are independently an oligonucleotide, a label (e.g., a label derived from fluorescein isothiocyanate (FITC) or Cy5), a phenyl group substituted with a formyl (-CHO) group, or a group of the formula: The basis of A compound or a salt thereof.

2. 2. The compound or salt of claim 1, wherein x is 2 or 3.

3. 2. The compound or salt of claim 1, wherein x is 3.

4. Each R 1 is covalently attached to L (i) via the N-terminus of the polypeptide, or (ii) via the C-terminus of the polypeptide, or (iii) via a side chain of an amino acid of the polypeptide.

5. Each R 1 4. The compound or salt of claim 1, wherein is covalently attached to L via the N-terminus of the polypeptide.

6. Each R 1 but independently, the formula:

4. The compound or salt of claim 1, which is a targeting ligand of

7. Each R 2 The compound or salt of any one of claims 1 to 6, wherein is independently an oligonucleotide.

8. Each R 2 and (b) are independently siRNA.

9. Each oligonucleotide or each siRNA is phosphate P(=O)(OH) of each oligonucleotide or each siRNA. 2 9. The compound or salt of any one of claims 7 to 8, wherein L is attached via -O-.

10. Each R 2 but independently, the formula:

7. The compound or salt according to any one of claims 1 to 6, wherein the compound or salt is a group represented by the formula:

11. Each R 2 is independently 3-formylphenyl or 4-formylphenyl.

12. L is a branched or unbranched, saturated or unsaturated hydrocarbon chain having from about 1 to about 500 carbon atoms, one or more of which are optionally independently selected from -O-, -S, -N(R a )-, a 3-20 membered heterocycle, a 3-20 membered heteroaryl, a 6-14 membered aryl, or a 3-20 membered carbocycle, wherein each chain, 3-20 membered heterocycle, 3-20 membered heteroaryl, 6-14 membered aryl, and 3-20 membered carbocycle is optionally and independently replaced by (C 1 -C 6 ) alkyl, (C 1 -C 6 ) alkoxy, (C 3 -C 6 ) cycloalkyl, (C 1 -C 6 ) alkanoyl, (C 1 -C 6 ) alkanoyloxy, (C 1 -C 6 )alkoxycarbonyl, azido, cyano, nitro, halo, -N(R a ) 2 , hydroxy, oxo (=O), and carboxy; and each R a are independently H or (C 1 -C 6 12. The compound or salt of any one of claims 1 to 11, wherein:

13. L is a branched or unbranched, saturated or unsaturated hydrocarbon chain having from about 1 to about 100 carbon atoms, one or more of which are optionally independently selected from -O-, -S, -N(R a )-, a 3-20 membered heterocycle, a 3-20 membered heteroaryl, a 6-14 membered aryl, or a 3-20 membered carbocycle, wherein each chain, 3-20 membered heterocycle, 3-20 membered heteroaryl, 6-14 membered aryl, and 3-20 membered carbocycle is optionally and independently replaced by (C 1 -C 6 ) alkyl, (C 1 -C 6 ) alkoxy, (C 3 -C 6 ) cycloalkyl, (C 1 -C 6 ) alkanoyl, (C 1 -C 6 ) alkanoyloxy, (C 1 -C 6 )alkoxycarbonyl, azido, cyano, nitro, halo, -N(R a ) 2 , hydroxy, oxo (=O), and carboxy; and each R a are independently H or (C 1 -C 6 12. The compound or salt of any one of claims 1 to 11, wherein:

14. L is a branched or unbranched, saturated or unsaturated hydrocarbon chain having from about 1 to about 100 carbon atoms, one or more of which are optionally independently selected from -O-, -S, -N(R a )-, 3-20 membered heterocycle, 3-20 membered heteroaryl, 6-14 membered aryl, or 3-20 membered carbocycle, each of which is optionally and independently substituted with one or more (e.g., 1, 2, 3, 4, 5, or more) substituents independently selected from the group consisting of azido, halo, and oxo (=O), and carboxy; a are independently H or (C 1 -C 6 12. The compound or salt of any one of claims 1 to 11, wherein:

15. L is a branched or unbranched, saturated or unsaturated hydrocarbon chain having from about 1 to about 50 carbon atoms, one or more of which are optionally independently selected from -O-, -S, -N(R a )-, 3-20 membered heterocycle, 3-20 membered heteroaryl, 6-14 membered aryl, or 3-20 membered carbocycle, each of which is optionally and independently substituted with one or more (e.g., 1, 2, 3, 4, 5, or more) substituents independently selected from the group consisting of azido, halo, and oxo (=O), and carboxy; a are independently H or (C 1 -C 6 12. The compound or salt of any one of claims 1 to 11, wherein:

16. L is a branched or unbranched, saturated or unsaturated hydrocarbon chain having from about 1 to about 50 carbon atoms, one or more of which are optionally independently selected from -O-, -S, -N(R a )-, or R b and each chain and R b is optionally and independently substituted with one or more (e.g., 1, 2, 3, 4, 5, or more) substituents independently selected from the group consisting of azido, halo, ═N(OH), —O(NH) and oxo (═O), and carboxy; and each R b are independently H or (C 1 -C 6 ) alkyl, and each R b But independently, the following:

12. The compound or salt of any one of claims 1 to 11, independently selected from the group consisting of:

17. L is -C(=O)(CH 2 ) a 12. The compound or salt of any one of claims 1 to 11, comprising N(H)C(=O)-, wherein a is 3, 4, 5, 6, 7, or 8.

18. L is -(CH 2 CH 2 O) b CH 2 CH 2 C(=O)- or -(CH 2 CH 2 O) b CH 2 CH 2 12. The compound or salt of any one of claims 1 to 11, comprising N(H)C(=O)-, wherein b is 2 to 50.

19. 12. The compound or salt of any one of claims 1 to 11, wherein L comprises phen-1,3-diyl or phen1-1,3,5-triyl.

20. L is:

12. The compound or salt of any one of claims 1 to 11, comprising:

21. Each R 1 21. The compound or salt of any one of claims 1 to 20, wherein is linked to L through a carbonyl group of L.

22. Each R 2 21. The compound or salt of any one of claims 1 to 20, wherein is linked to L via a carbon-carbon bond.

23. L is:

12. The compound or salt of any one of claims 1 to 11, selected from the group consisting of:

24. x is 1, 2 or 3 and the compound is:

2. A compound essentially as defined in claim 1 selected from the group consisting of:

25. The following formula (X):

1. A process for preparing a diblock polymer of the formula: a) reacting a compound of structure Va, Vb, Vc, or Vd to obtain a first product: R 27 However, (C 1 -C 12 ) alkyl ; R 28 However, (C 1 -C 12 ) alkyl ; R 25 and R 26 However, independently, H,(C 1 -C 12 ) alkyl, aryl, or heteroaryl ; the following: iii) polyethylene glycol methacrylate (PEGMA) having 2 to 20 ethylene glycol units; and iv) M, a methacrylate 2 wherein the methacrylate is optionally (C 4 -C 18 ) alkyl methacrylate, (C 4 -C 18 ) branched alkyl methacrylates, cholesteryl methacrylate, substituted with one or more fluorine atoms (C 4 -C 18 ) alkyl-methacrylates, and substituted with one or more fluorine atoms (C 4 -C 18 ) Branched alkyl methacrylate Selected from M 2 and one or more A monomers selected from the group consisting of: contacting in the presence of free radicals; b) reacting the first product with Equations B1, B2, and B3: and one or more B monomers of contacting in the presence of free radicals; and c) optionally contacting the second product with a free radical source (e.g., AIBN) to remove the chain transfer agent and obtain the diblock polymer of formula (X). Including, During the ceremony, block A comprises one or more residues of an A monomer and has a molecular weight of about 1 kDa to about 25 kDa; block B comprises one or more residues of monomers B1, B2, and B3 and has a molecular weight of about 1 kDa to about 25 kDa; L 1 is the connecting part, Z is optionally protected by a protecting group and has the formula (XII): TL 2 -Y (XII) is a functional group capable of reacting with Y of a compound of formula T is a ligand (optionally a targeting ligand); Y is a functional group capable of reacting with Z to form a conjugate; L 2 is absent or is a linking moiety, process.

26. A monomer is selected from the group consisting of monomers of formula A1 and A2, PEGMA, and M 2 but, That is, 26. The process of claim 25.

27. 27. The process of claim 25 or 26, wherein Z is a functional group protected with a protecting group, and the process further comprises removing the protecting group from Z.

28. 27. The process of claim 25 or 26, wherein Z is a functional group that is not protected by a protecting group.

29. The diblock polymer of formula (X) Formula (XII): TL 2 -Y (XII) and a compound of Z reacts with Y to form a group Y'Z' of the formula: reacting under conditions to produce a conjugate of 29. The process of claim 27 or 28, further comprising:

30. 30. The process of claim 29, wherein block A has a molecular weight of about 1 kDa to about 25 kDa and block B has a molecular weight of about 1 kDa to about 25 kDa.

31. Block A has the formula: -[PEGMA m -M 2 n ] v - wherein PEGMA is a polyethylene glycol methacrylate residue having 2 to 20 ethylene glycol units; M 2 but, (C 4 -C 18 ) alkyl methacrylate residue, (C 4 -C 18 ) branched alkyl methacrylate residues, cholesteryl methacrylate residue, substituted with one or more fluorine atoms (C 4 -C 18 ) alkyl-methacrylate residues, and substituted with one or more fluorine atoms (C 4 -C 18 ) Branched alkyl methacrylate residue is a methacrylate residue selected from the group consisting of: v is 1 to 25 kDa; Block B has the formula: [DMAEMA q -PAA r -BMA s ] w wherein BMA is the residue of butyl methacrylate; PAA is a propylacrylic acid residue, DMAEMA is the residue of dimethylaminoethyl methacrylate; m and n are each a mole fraction greater than 0, m is greater than n, and m+n=1; q is a mole fraction between 0.2 and 0.75; r is a mole fraction between 0.05 and 0.6; s is a mole fraction between 0.2 and 0.75; q+r+s=1, w is 1 to 25 kDa; 30. The process of claim 29.

32. 32. The process of any one of claims 25 to 31, wherein Z and Y are selected such that Y'Z' comprises an oxime functional group.

33. 32. The process of any one of claims 25-31, wherein Z and Y are selected such that Y'Z' comprises a triazole ring.

34. 33. The process of any one of claims 25-27 and 29-32, wherein Z comprises a protected aminooxy group, the protecting group being on the nitrogen of the aminooxy group.

35. 33. The process of any one of claims 29-32, wherein Z comprises an aminooxy group, Y comprises a ketone or aldehyde group, and Y'Z' comprises an oxime functional group.

36. 33. The process of any one of claims 29-32, wherein Z comprises a ketone or aldehyde group, Y comprises an aminooxy group, and Y'Z' comprises an oxime functional group.

37. 34. The process of claim 29 or 33, wherein Z comprises an alkyne group, Y comprises an azide group, and Y'Z' comprises a triazole ring.

38. 34. The process of claim 29 or 33, wherein Z comprises an azide group, Y comprises an alkyne group, and Y'Z' comprises a triazole ring.

39. 39. The process of any one of claims 29 to 38, wherein T is a targeting ligand or a labeling agent.

40. 39. The process of any one of claims 29 to 38, wherein the targeting ligand is selected from the group of an oligonucleotide, a peptide, a sugar, and a small molecule.

41. 39. The process of any one of claims 29 to 38, wherein the targeting ligand comprises a cyclic peptide.

42. 39. The process of any one of claims 29 to 38, wherein the targeting ligand comprises a cyclic peptide according to claim 1.

43. 39. The process of any one of claims 29 to 38, wherein the targeting ligand comprises a cyclic peptide according to claim 6.

44. 39. The process of any one of claims 29 to 38, wherein the labeling agent is selected from the group consisting of a fluorophore, a chromophore, and a radionucleotide.

45. 39. The process of any one of claims 29 to 38, wherein the labeling substance is derived from fluorescein isothiocyanate (FITC) or Cy5.

46. 46. ​​A product prepared by the process of any one of claims 25 to 45.

47. 46. ​​A conjugate prepared by the process of any one of claims 29 to 45.

48. 46. ​​A composition comprising a plurality of conjugates as defined in any one of claims 29 to 45.

49. 49. The composition of claim 48, wherein at least one of the conjugates comprises a labeling substance.

50. Formula (I): or a salt thereof, x is 1, 2, 3, 4, or 5; Each R 1 is independently a targeting ligand, and the targeting ligand is a) SEQ ID NO: 1: C * SRNLIDC * (SEQ ID NO:1) A cyclic polypeptide represented by the formula: * exhibits a disulfide bond linking two C residues thereby forming a cyclic polypeptide, b) C * Any cyclic polypeptide having at least 80% sequence identity to a polypeptide set forth in SEQ ID NO:1, provided that both residues are present, wherein: * exhibits a disulfide bond linking two C residues thereby forming a cyclic polypeptide. Selected from L is a linking group, y is 1, 2, 3, 4, or 5; Each R 2 is independently an endosomally released polymer, A compound or a salt thereof.

51. The endosomal release polymer has the following formula: [Block A]-[Block B] and a diblock polymer comprising a block A and a block B having the formula: block A comprises one or more residues of the A monomer of claim 25 and has a molecular weight of about 1 kDa to about 25 kDa; Block B comprises one or more residues of monomers B1, B2, and B3 according to claim 25 and has a molecular weight of about 1 kDa to about 25 kDa.

51. The compound or salt of claim 50.

52. The endosomal release polymer has the formula (XX): -[PEGMA m -M 2 n ] v -[DMAEMA q -PAA r -BMA s ] w (XX) wherein PEGMA is a polyethylene glycol methacrylate residue having 2 to 20 ethylene glycol units; M 2 but, (C 4 -C 18 ) alkyl methacrylate residue, (C 4 -C 18 ) branched alkyl methacrylate residues, cholesteryl methacrylate residue, substituted with one or more fluorine atoms (C 4 -C 18 ) alkyl-methacrylate residues, and substituted with one or more fluorine atoms (C 4 -C 18 ) Branched alkyl methacrylate residue is a methacrylate residue selected from the group consisting of: BMA is the residue of butyl methacrylate; PAA is a propylacrylic acid residue, DMAEMA is the residue of dimethylaminoethyl methacrylate; m and n are each a mole fraction greater than 0, m is greater than n, and m+n=1; q is a mole fraction between 0.2 and 0.75; r is a mole fraction between 0.05 and 0.6; s is a mole fraction between 0.2 and 0.75; q+r+s=1, v is 1 to 25 kDa; w is 1 to 25 kDa; 51. The compound or salt of claim 50.

53. 52. The compound or salt of claim 51, wherein block A comprises one or more residues of monomers of formulae A1 and A2 of claim 26.

54. 54. A pharmaceutical composition comprising a compound according to any one of claims 1 to 24 and 50 to 53, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

55. Each R 2 is independently an oligonucleotide, or a pharmaceutically acceptable salt thereof; a second compound according to any one of claims 50 to 53 or a pharmaceutically acceptable salt thereof; 10. A pharmaceutical composition comprising:

56. 25. A method for delivering an oligonucleotide to an animal, comprising administering to said animal a compound of any one of claims 1 to 24, or a pharmaceutically acceptable salt thereof.

57. 25. A method for delivering an oligonucleotide to a cell expressing a platelet-derived growth factor receptor (PDGFR) in an animal, comprising administering to the animal a compound of any one of claims 1 to 24, or a pharmaceutically acceptable salt thereof.

58. 58. The method of claim 57, wherein the platelet-derived growth factor receptor (PDGFR) is platelet-derived growth factor receptor alpha (PDGFRalpha).

59. 58. The method of claim 57, wherein the platelet-derived growth factor receptor (PDGFR) is platelet-derived growth factor receptor beta (PDGFRβ).

60. 58. The method of claim 57, wherein the PDGFR-expressing cell is a hepatic stellate cell (HSC), an endothelial cell, a fibroblast, or a tumor cell.

61. 25. A method for treating a disease involving PDGFR-expressing cells (e.g., liver fibrosis, non-alcoholic steatohepatitis (NASH), clear cell renal cell carcinoma, renal fibrosis, or alcoholic steatohepatitis (ASH)), comprising administering to an animal a compound of any one of claims 1 to 24, or a pharmaceutically acceptable salt thereof.

62. 25. The compound of any one of claims 1 to 24, or a pharmaceutically acceptable salt thereof, for delivering an oligonucleotide to an animal.

63. 25. The compound of any one of claims 1 to 24, or a pharmaceutically acceptable salt thereof, for delivering an oligonucleotide to a cell expressing a platelet-derived growth factor receptor (PDGFR).

64. 25. The compound of any one of claims 1 to 24, or a pharmaceutically acceptable salt thereof, for the prophylactic or therapeutic treatment of a disease involving PDGFR-expressing cells (e.g., liver fibrosis, non-alcoholic steatohepatitis (NASH), clear cell renal cell carcinoma, renal fibrosis, or alcoholic steatohepatitis (ASH)).

65. 25. Use of a compound according to any one of claims 1 to 24, or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for delivering an oligonucleotide to an animal.

66. 25. Use of the compound of any one of claims 1 to 24, or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for delivering an oligonucleotide to cells expressing platelet-derived growth factor receptor (PDGFR).

67. 25. Use of the compound of any one of claims 1 to 24, or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for treating a disease involving PDGFR-expressing cells (e.g., liver fibrosis, non-alcoholic steatohepatitis (NASH), clear cell renal cell carcinoma, renal fibrosis, or alcoholic steatohepatitis (ASH)).

68. Each R 2 is independently an oligonucleotide; and a second compound according to any one of claims 50 to 53; including the combined use of 68. The method, compound, or use thereof of any one of claims 56 to 67.

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