Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
  • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
  • A61K47/551—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds one of the codrug's components being a vitamin, e.g. niacinamide, vitamin B3, cobalamin, vitamin B12, folate, vitamin A or retinoic acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
  • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
  • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
  • A61K47/58—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
  • C08F2/00—Processes of polymerisation
  • C08F2/38—Polymerisation using regulators, e.g. chain terminating agents, e.g. telomerisation
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
  • C08F293/00—Macromolecular compounds obtained by polymerisation on to a macromolecule having groups capable of inducing the formation of new polymer chains bound exclusively at one or both ends of the starting macromolecule
  • C08F293/005—Macromolecular compounds obtained by polymerisation on to a macromolecule having groups capable of inducing the formation of new polymer chains bound exclusively at one or both ends of the starting macromolecule using free radical "living" or "controlled" polymerisation, e.g. using a complexing agent
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
  • C08F2438/00—Living radical polymerisation
  • C08F2438/03—Use of a di- or tri-thiocarbonylthio compound, e.g. di- or tri-thioester, di- or tri-thiocarbamate, or a xanthate as chain transfer agent, e.g . Reversible Addition Fragmentation chain Transfer [RAFT] or Macromolecular Design via Interchange of Xanthates [MADIX]
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/35—Nature of the modification
  • C12N2310/351—Conjugate
  • C12N2310/3513—Protein; Peptide

Definitions

• each R 1 is independently a targeting ligand, which is selected from: a) a cyclic polypeptide depicted by SEQ ID NO: 1 :C*SRNLIDC* (SEQ ID NO: 1), wherein * denotes a disulfide bond linking the two C residues to thereby form the cyclic polypeptide, and b) 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 the polypeptide depicted by SEQ ID NO: 1, under the proviso that both C* residues are present, and wherein * denotes a disulfide bond linking the two C residues to thereby form the cyclic polypeptide;
• L is a linking group; y is 1, 2, 3, 4 or 5; and each R 2 is independently an oligonucleotide, a label (e.g., a label derivable from fluorescein isothiocyanate (FITC) or Cy5), a phenyl group that is substituted with a formyl (-CHO) group, or a group of formula:
• Block B comprises one or more residues of monomers Bl, B2, and B3 and has a molecular weight of from about 1 kDa to about 25 kDa.
• siRNA delivery to hepatocytes has been demonstrated with this class of therapeutics, successful application to other cell types has been more problematic. Suitable receptors must be identified, which are expressed relatively selectively on the target cell surface and in sufficient numbers. Then, appropriate ligands must be identified that bind with high specificity and affinity to the target receptor. A significant third hurdle is endosomal escape; when bound to a receptor, the ligand conjugate is taken up by the cell and entrapped in an endosome, from which it must escape to reach the cytoplasm. Despite lacking an active endosomal escape mechanism, GalNAc conjugates appear to mediate activity regardless, possibly because the receptor is so abundantly expressed and quickly recycled ( ⁇ 15 minutes) following uptake. The sheer number of conjugate molecules taken up by hepatocytes may obviate the need for an active endosomal escape. This has not been true for other ligand-based siRNA conjugate systems.
• Hepatic stellate cells play a key role in the progression of fibrosis: In response to chronic liver damage, they become activated, and are largely responsible for collagen deposition and scarring of the liver.
• RNAi strategies include RNAi strategies.
• siRNA conjugates have been synthesized to target HSC via platelet-derived growth factor receptor (PDGFR) using a cyclic octapeptide motif.
• PDGFR platelet-derived growth factor receptor
• trivalent presentation of a pPB ligand outperforms monovalent presentation for binding and uptake.
• Gene silencing experiments also demonstrated that trivalent ligand structures are particularly effective, e.g., when compared to mono and divalent conjugates.
• the invention also provides pPB conjugates of an endosome release polymer (ERP), the conjugate also being referred to as pPB-ERP.
• ERP endosome release polymer
• ERT endosome release polymer
• diblock polymer are used interchangeably herein.
• the specific ERT as provided by the present invention optionally characterized by presence of a functional group Z or alternatively by presence of a moiety T-L2-Y’-X’ as described herein, is obtainable by the process according to the present invention.
• the ERT according to the present invention is conjugated to a specific ligand T, such as e.g. specific targeting ligand, as described herein.
• the disease is a liver disease.
• the disease is a kidney disease.
• the disease is non-alcoholic steatohepatitis (NASH)
• the disease is kidney fibrosis.
• the disease is clear cell renal cell carcinoma.
• the disease is alcoholic steatohepatitis (ASH)
• HSC hepatic stellate cell
• HSCs hepatic stellate cells
• Targeted delivery of therapeutic agents to HSCs may be important for the successful treatment of liver fibrosis.
• a number of protein markers have been found to be overexpressed in activated HSCs, and their ligands have been used to specifically deliver various antifibrotic agents, (see, e.g., Chen et al., Journal of Pharmacology and Experimental Therapeutics, 2019, 370 (3) 695-702).
• delivery of therapeutics using other systems is needed as other means for delivering therapeutic agents to HSCs.
• Liver fibrosis is caused by the formation of an abnormally large amount of scar tissue in the liver. Liver fibrosis occurs when the liver attempts to repair and replace damaged cells. Various disorders and drugs can damage the liver and cause fibrosis.
• Nonalcoholic fatty liver disease is a condition in which triglycerides accumulate in the liver.
• Nonalcoholic steatohepatitis is a type of NAFLD.
• NASH is associated with inflammatory changes and liver cell damage. NASH is a leading cause of liver disease and often progresses to liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC).
• Non-alcoholic steatohepatitis (NASH) and alcoholic steatohepatitis (ASH) have a similar pathogenesis and histopathology but a different etiology and epidemiology.
• NASH and ASH are advanced stages of non-alcoholic fatty liver disease (NAFLD) and alcoholic fatty liver disease (AFLD).
• Alcoholic steatohepatitis is a chronic, progressive liver disease characterized by fibrosis of the liver as well as possible necrosis of the liver tissue, brought on by excessive, prolonged alcohol use. Women are more susceptible to the disease because alcohol metabolism is lower in women than in men.
• Liver fibrosis is an important underlying cause of liver dysfunction and predicts mortality. Progression to cirrhosis and HCC leads to ultimate liver failure and thus liver transplantation is required.
• the current US prevalence of NASH-related fibrosis (F2 and later) is about 3.8 million patients. Doctors typically recommend weight loss to treat NAFLD and NASH. While weight loss can reduce fat in the liver, inflammation, and fibrosis, no medicines have been approved to treat NAFLD and NASH. Specifically, no medicines have been approved to treat 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) Accordingly, new therapeutic treatment options, including delivery options, are needed for the treatment of liver fibrosis, e.g., in the context of NASH or ASH.
• conjugate includes compounds of formula (I) that comprise an oligonucleotide (e.g., an siRNA molecule) linked to a targeting ligand.
• oligonucleotide e.g., an siRNA molecule
• conjugate may be used herein interchangeably.
• small -interfering RNA or “siRNA” as used herein refers to double stranded
• RNA i.e., duplex RNA
• the siRNA may have substantial or complete identity to the target gene or sequence, or may comprise a region of mismatch (i.e., a mismatch motif).
• the siRNAs may be about 19-25 (duplex) nucleotides in length, and are preferably about 20-24, 21-22, or 21-23 (duplex) nucleotides in length.
• siRNAs are chemically synthesized.
• siRNA can also be generated by cleavage of longer dsRNA (e.g., dsRNA greater than about 25 nucleotides in length) with the E. coll RNase III or Dicer. These enzymes process the 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.
• Control samples may be assigned a value of 100%.
• silencing, inhibition, or reduction of expression of a target gene is achieved when the value of the test sample relative to the control sample (e.g., buffer only, an siRNA sequence that targets a different gene, a scrambled siRNA sequence, etc. ⁇ 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%.
• Suitable assays include, without limitation, examination of protein or mRNA levels using techniques known to those of skill in the art, such as, e.g., dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic assays known to those of skill in the art.
• nucleic acid refers to a polymer containing at least two nucleotides (/. ⁇ ., deoxyribonucleotides or ribonucleotides) in either single- or double-stranded form and includes DNA and RNA.
• Nucleotides contain a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate groups.
• Bases include purines and pyrimidines, which further include natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications which place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkylhalides.
• Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and which have similar binding properties as the reference nucleic acid.
• RNA may be in the form, for example, of small interfering RNA (siRNA), Dicer-substrate dsRNA, small hairpin RNA (shRNA), small activating RNA (saRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), tRNA, rRNA, tRNA, viral RNA (vRNA).
• siRNA small interfering RNA
• Dicer-substrate dsRNA small hairpin RNA
• saRNA small activating RNA
• aiRNA asymmetrical interfering RNA
• miRNA microRNA
• tRNA tRNA
• rRNA tRNA
• viral RNA viral RNA
• oligonucleotide refers to a polymer or oligomer of nucleotide or nucleoside monomers consisting of naturally-occurring bases, sugars and intersugar (backbone) linkages.
• oligonucleotide also includes polymers or
• label includes groups that enable the compound to be detected in vitro or in vivo.
• labeling agent is used interchangeably with the term label.
• a label may be selected from the group comprising a fluorophore, a chromophore, and a radionucleotide.
• the label can be detected by spectroscopy.
• the label can be detected by fluorescence spectroscopy.
• the label is a group that is derivable from fluorescein isothiocyanate (FITC) or Cy5.
• FITC fluorescein isothiocyanate
• a label can be detected by an apparatus suitable for detecting any one or more of alpha, beta particles, gamma rays, and x rays.
• alkynyl refers to an unsaturated alkyl radical having one or more triple bonds.
• unsaturated alkyl groups include vinyl, 2- propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(l,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers.
• alkylene by itself or as part of another substituent means a divalent radical derived from an alkane (including straight and branched alkanes), as exemplified by -CH2CH2CH2CH2- and -CH(CH 3 )CH 2 CH2-.
• cycloalkyl refers to hydrocarbon ringsystem having 3 to 20 overall number of ring atoms (e.g., 3-20 membered cycloalkyl is a cycloalkyl with 3 to 20 ring atoms, or C3-20 cycloalkyl is a cycloalkyl with 3-20 carbon ring atoms) and for a 3-5 membered cycloalkyl being fully saturated or having no more than one double bond between ring vertices and for a 6 membered cycloalkyl or larger being fully saturated or having no more than two double bonds between ring vertices.
• aryl means a carbocyclic aromatic group having 6-14 carbon atoms, whether or not fused to one or more groups.
• aryl groups include phenyl, naphthyl, biphenyl and the like unless otherwise stated.
• salts includes any anionic and cationic complex, such as the complex formed between a cationic lipid and one or more anions.
• anions include inorganic and organic anions, e.g., hydride, fluoride, chloride, bromide, iodide, oxalate (e.g., hemioxalate), phosphate, phosphonate, hydrogen phosphate, dihydrogen phosphate, oxide, carbonate, bicarbonate, nitrate, nitrite, nitride, bisulfite, sulfide, sulfite, bisulfate, sulfate, thiosulfate, hydrogen sulfate, borate, formate, acetate, benzoate, citrate, tartrate, lactate, acrylate, polyacrylate, fumarate, maleate, itaconate, glycolate, gluconate, malate, mandelate, tiglate, ascorbate,
• a peptide bond in case of backbone to backbone cyclization and e.g. disulfide bonds resulting from two residues containing thiol groups, such as cysteines which can form intramolecular disulfide bridges giving cyclic peptides.
• RNA, synthesizing RNA, hybridizing nucleic acids, making and screening cDNA libraries, and performing PCR are well known in the art (see, e.g., Gubler and Hoffman, Gene, 25:263-269 (1983); Sambrook et al., supra, Ausubel et al., supra), as are PCR methods (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 of skill in the art.
• siRNAs are chemically synthesized.
• the oligonucleotides that comprise the siRNA molecules of the invention can be synthesized using any of a variety of techniques known in the art, such as those described in 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).
• oligonucleotides makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5 ’-end and phosphoramidites at the 3 ’-end.
• small scale syntheses can be conducted on an Applied Biosystems synthesizer using a 0.2 pmol scale protocol.
• syntheses at the 0.2 pmol scale can be performed on a 96-well plate synthesizer from Protogene (Palo Alto, CA).
• Protogene Protogene
• siRNA molecules can be assembled from two distinct oligonucleotides, wherein one oligonucleotide comprises the sense strand and the other comprises the antisense strand of the siRNA.
• each strand can be synthesized separately and joined together by hybridization or ligation following synthesis and/or deprotection.
• the linking group can be variable provided the targeting conjugate functions as described herein.
• the linking group can vary in length and atom composition and for example can be branched or non-branched or cyclic or a combination thereof.
• the linking group may also modulate the properties of the targeted conjugate such as but not limited to solubility, stability and aggregation.
• the linker comprises about 3-5000 atoms. In one embodiment the linker comprises about 3-4000 atoms. In one embodiment the linker comprises about 3-2000 atoms. In one embodiment the linker comprises about 3-1000 atoms. In one embodiment the linker comprises about 3-750 atoms. In one embodiment the linker comprises about 3-500 atoms. In one embodiment the linker comprises about 3-250 atoms. In one embodiment the linker comprises about 3-100 atoms. In one embodiment the linker comprises about 3-50 atoms. In one embodiment the linker comprises about 3-25 atoms.
• the linker comprises about 10-5000 atoms. In one embodiment the linker comprises about 10-4000 atoms. In one embodiment the linker comprises about 10-2000 atoms. In one embodiment the linker comprises about 10-1000 atoms. In one embodiment the linker comprises about 10-750 atoms. In one embodiment the linker comprises about 10-500 atoms. In one embodiment the linker comprises about 10-250 atoms. In one embodiment the linker comprises about 10-100 atoms. In one embodiment the linker comprises about 10-50 atoms. In one embodiment the linker comprises about 10-25 atoms.
• the linker L comprises atoms selected from H, C, N, S and O.
• the linker L comprises atoms selected from H, C, N, S, P and O.
• the linker L comprises a branched or unbranched, saturated or unsaturated, hydrocarbon chain, having from about 1 to 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 is optionally replaced independently by -O-, -S, -N(R a )-, 3-7 membered heterocycle, 5-6-membered heteroaryl or carbocycle and wherein each chain, 3-7 membered heterocycle, 5-6-membered heteroaryl or carbocycle is optionally and independently substituted with one or more (e.g.
• the linker comprises a branched or unbranched, saturated or unsaturated, hydrocarbon chain, having from about 1 to 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 is optionally replaced independently by -O-, -S, -N(R a )-, wherein each R a is independently H or (Ci-Ce)alkyl.
• the linker L comprises a polyethylene glycol. In one embodiment the linker comprises a polyethylene glycol linked to the remainder of the targeted 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 repeat (e.g., -CH2CH2O-) 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).
• -CH2CH2O- repeat
• Rounded brackets denote the monomer moieties. Squared brackets denote Blocks A and B, respectively, of the diblock polymer. Stoichiometry (m and n; q, r and s) and molecular weights (v and w) are as defined in E25. E27. The process of E25 or E26 wherein Z is a functional group that is protected with a protecting group, wherein the process further comprises removing the protecting group from Z.
• BMA is butyl methacrylate residue
• PAA is propyl acrylic acid residue
• Rounded brackets denote the monomer moieties.
• Squared brackets denote Blocks A and B, respectively, of the diblock polymer.
• E32 The process of any one of E25-E31, wherein Z and Y are selected so that Y’Z’ comprises an oxime functionality.
• E33 The process of any one of E25-E31, wherein Z and Y are selected so that Y’Z’ comprises a triazole ring.
• E34 The process of any one of E25-E27 and E29-E32, wherein Z comprises an aminooxy group that is protected, and the protecting group is on the nitrogen of the aminooxy group.
• E35 The process of any one of E29-E32, wherein Z comprises an aminooxy group, Y comprises a ketone group or an aldehyde group, and Y’Z’ comprises an oxime functional group.
• E36 The process of any one of E29-E32, wherein Z comprises a ketone group or an aldehyde group, Y comprises an aminooxy group, and Y’Z’ comprises an oxime functional group.
• E37 The process of E29 or E33, wherein Z comprises an alkyne group, Y comprises an azide group, and Y’Z’ comprises a triazole ring.
• E38 The process of E29 or E33, wherein Z comprises an azide group, Y comprises an alkyne group, and Y’Z’ comprises a triazole ring.
• E39 The process of any one of any one of E29-E38, wherein T is a targeting ligand or a labeling agent.
• E40 The process of any one of E29-E38, wherein the targeting ligand is selected from the group of an oligonucleotide, a peptide, a saccharide, and a small molecule.
• L is a linking group; and y is 1, 2, 3, 4 or 5; and each R 2 is independently an endosomal release polymer.
• El 12. The compound or salt of any one of El 01 -El 07, wherein L is a branched or unbranched, saturated or unsaturated, hydrocarbon chain, having from about 1 to about 50 carbon atoms, wherein one or more of the carbon atoms is optionally replaced independently by -O-, -S, -N(R a )-, or R b , wherein each chain and R b is optionally and independently substituted with one or more (e.g.
• El 18 The compound or salt of any one of El 01 -El 17, wherein each R 2 is linked to L through a carbon-carbon bond.
• E121 The compound or salt of E101, wherein y is 1 and R 2 is an endosomal release polymer of formula XX:
• PEGMA is polyethyleneglycol methacrylate residue with 2-20 ethylene glycol units
• M 2 is a methacrylate residue selected from the group consisting of a (C4-Cis)alkyl-methacrylate residue; a (C4-Cis)branched alkyl- methacrylate residue; a cholesteryl methacrylate residue; a (C4-Cis)alkyl-methacrylate residue substituted with one or more fluorine atoms; and a (C4-Cis)branched alkyl-methacrylate residue substituted with one or more fluorine atoms;
• BMA is butyl methacrylate residue
• PAA is propyl acrylic acid residue
• E122 The compound or salt of E121, which is a compound of the following formula: or a salt thereof. Stoichiometry (m and n; q, r and s) and molecular weights (v and w) are as defined in E121.
• the invention provides a process for preparing a diblock polymer of formula (X):
• Prieve et al describe the synthesis of di -block polymers using RAFT polymerisation techniques, wherein the alpha end of the polymer features either a mannose or GalNac monosaccharide ligand. Details were limited, input and output amounts were not recorded, and no yields were given.
• the synthesis can require significant amounts of input starting material chain transfer agent (CTA) relative to the amount of output polymer. For example, in a synthesis using GalNAc CTA, 180g of input CTA was utilised to produce approximately 50 g of final polymer. This ratio of starting material to product seems to be consistent irrespective of the scale of the synthesis. In cases where ligands are relatively cheap and accessible, this material requirement is not necessarily an obstacle. However, in cases where the ligand may be significantly more expensive, this high input material requirement becomes prohibitive.
• a gradient polymer the content of one or more monomeric units increases or decreases in a gradient manner from the a-end of the polymer to the w-end.
• the brackets enclosing the constitutional units are not meant and are not to be construed to mean that the constitutional units themselves form blocks.
• the constitutional units within the square brackets may combine in any manner with the other constitutional units within the block, i.e., purely random, alternating random, regular alternating, regular block or random block configurations.
• the block copolymers described herein are, optionally, alternate, gradient or random block copolymers. Block copolymers and methods for their preparation are described in International Patent Application Publication Number W02015/017519, which is hereby incorporated herein by reference in its entirety.
• ligands which are not susceptible to RAFT polymerization such as in a non-limiting example oligonucleotides and peptides, such as the pPB peptide.
• one ligand (T), or one moiety bearing a natural number of targeting ligands (T) is being incorporated into every one polymer, i.e. according to the present invention a typical stoichiometry is 1, or a multiplicity thereof.
• the process of the present invention is thus differentiated over W02015/017519 at least in higher specificity of ligand incorporation and in allowing incorporating certain ligands that the process of W02015/017519 does not allow for.
• a further advantage of the present invention results from the fact that the ligand T is always placed on the hydrophilic end of the polymer, as opposed to incorporation at a random position as taught in W02015/017519.
• Block B comprises one or more residues of monomers B 1 , B2 and B3 and has a molecular weight of from about 2 kDa to about 15 kDa. In another embodiment, Block B comprises one or more residues of monomers Bl, B2 and B3 and has a molecular weight of from about 2 kDa to about 10 kDa. In another embodiment, Block B comprises one or more residues of monomers Bl, B2 and B3 and has a molecular weight of from about 2 kDa to about 5 kDa. In another embodiment, Block B comprises one or more residues of monomers B 1, B2 and B3 and has a molecular weight of from about 5 kDa to about 20 kDa.
• Block B comprises one or more residues of monomers Bl, B2 and B3 and has a molecular weight of from about 5 kDa to about 15 kDa. In another embodiment, Block B comprises one or more residues of monomers B 1, B2 and B3 and has a molecular weight of from about 5 kDa to about 10 kDa. In another embodiment, Block B comprises one or more residues of monomers Bl, B2 and B3 and has a molecular weight of from about 10 kDa to about 25 kDa. In another embodiment, Block B comprises one or more residues of monomers Bl, B2 and B3 and has a molecular weight of from about 10 kDa to about 20 kDa.
• Block B comprises one or more residues of monomers Bl, B2 and B3 and has a molecular weight of from about 10 kDa to about 15 kDa.
• Z and Y are selected so that Z in the diblock polymer of formula (X and XI) will react with Y in a compound of formula (XII):
• the conjugate is a conjugate of formula (XIII):
• the aminoxy of Z can react with an aldehyde (R”’is H) or with a ketone (R”’is C) of Y to provide the corresponding Z’Y’ oxime.
• the aminoxy of Y can react with an aldehyde (R”is H) or with a ketone (R”is C) of Z to provide the corresponding Z’Y’ oxime.
• Nomenclature pPB (N- to C- terminus) H2N-CSNLIDC-C00H (disulphide between two Cys).
• Biotinylated versions of ligands were prepared for in vitro binding experiments, to gauge relative binding efficiencies (see Example 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 techniques and the final ligands were purified with reverse phase HPLC. Product confirmation was achieved with mass spectrometry and product purity determined by RPLC techniques.
• SPPS solid phase peptide synthesis
• pPB monovalent Biotin Compound 1). MW: Calc 1260.56. Found 1260.2. Purity (HPLC) >90%
• Example 2 Synthesis of peptide conjugated PEG 12 benzaldehydes
• the following pPB benzaldehyde ligands (18a and 18b) were synthesized to allow for conjugation to the endosome release polymer (27) once RAFT polymerization was completed.
• 18a+b pPB benzaldehyde ligands
• the endosome release polymer was synthesized using RAFT polymerization and terminated with a protected aminooxy functional group to allow for subsequent oxime conjugation to benzaldehyde peptide ligands (18a and 18b). Part 1) Synthesis of chain transfer agent tert-butyl ((6-cyano-6-methyl-9,15-dioxo-4-thioxo-
• Step 1 Removal of BOC from endosomal release polymer 25 1 ,044g of polymer was taken up in DCM (4 ml) and TF A (3 ,3ml) was added. The reaction was stirred at RT for 2 hr. The reaction was concentrated in-vacuo for 60 mins. The residual TFA salt was used in the subsequent conjugation step without additional processing.
• Step 2. Oxime conjugation. The residual material from the previous step was taken up in DCM (5 mL), 0.55 stoichiometric equivalents of the appropriate peptide benzaldehyde (18 a or b) added and the reaction stirred at RT for 72 hrs.
• Step 5 Synthesis of tert-butyl 3- ⁇ 2-[2-(2- ⁇ 3-[2-(2- ⁇ 2-[3-(tert-butoxy)-3-oxopropoxy]- ethoxy ⁇ ethoxy)ethoxy]-5-[(l,3-dioxoisoindol-2-yl)methyl]phenoxy ⁇ ethoxy)ethoxy]ethoxy ⁇ - propanoate
• 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 .
• Trimeric pPB (compound 58) can be prepared according to the procedure described in the scheme above.
• Trimeric C6 scrambled pPB (compound 64) Trimeric C6 scrambled pPB (compound 64) can be prepared using an analogous procedure to that described for compound 63 pPB conjugation and siRNA synthesis were conducted as described elsewhere to give trimeric pPB (compound 58), trimeric scrambled pPB (compound 59), trimeric C6 pPB (compound 63) and trimeric C6 scrambled pPB (compound 64)
• pPB-ERP Improves in vivo Gene Silencing by TripPB siRNA Conjugate in Mouse Liver Fibrosis Model.
• RNAlater fixed liver tissue was homogenized in Epicentre lysis buffer containing 1% proteinase K using the FastPrep®-24 homogenization instrument (4.0 m/s, 3 x 15 second bursts, MP Biomedicals). Samples were kept on ice both before and after the homogenization to prevent degradation. The lysates were cleared by centrifugation at 16,000 x at 16°C for 5 min and diluted in lysis working buffer to ensure all values would be in the assay linear range of detection before being subjected to 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. Results
• RNAlater fixed liver tissue was homogenized in Epicentre lysis buffer containing 1% proteinase K using the FastPrep®-24 homogenization instrument (4.0 m/s, 3 x 15 second bursts, MP Biomedicals). Samples were kept on ice both before and after the homogenization to prevent degradation. The lysates were cleared by centrifugation at 16,000 xg at 16°C for 5 min and diluted in lysis working buffer to ensure all values would be in the assay linear range of detection before being subjected to 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.
• Mannose-ERP 40 and GalNAc-ERP 41 are presented below.

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