WO2016149323A1 - Acides nucléiques sphériques immunomodulateurs - Google Patents

Acides nucléiques sphériques immunomodulateurs Download PDF

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Publication number
WO2016149323A1
WO2016149323A1 PCT/US2016/022579 US2016022579W WO2016149323A1 WO 2016149323 A1 WO2016149323 A1 WO 2016149323A1 US 2016022579 W US2016022579 W US 2016022579W WO 2016149323 A1 WO2016149323 A1 WO 2016149323A1
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fibrosis
nash
antagonist
nucleic acid
hcc
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PCT/US2016/022579
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English (en)
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Aleksandar Filip Radovic-Moreno
Sergei Gryaznov
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Exicure, Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • Modulating immunity in a targeted and specific manner is an attractive approach for treating disease. Promising early results have been observed in preclinical and clinical studies, particularly in infectious disease (1, 2), cancer (3-5), allergy (6-8), and autoimmune disease applications (9, 10). Several classes of agents are currently being evaluated for their ability to stimulate or repress immunity, but few are as versatile as nucleic acids. Nucleic acids can stimulate immunity by binding endosomal toll-like receptors (TLR 3, 7/8, 9), a finding that has been used to develop immunotherapies (11, 12).
  • TLR 3, 7/8, 9 endosomal toll-like receptors
  • Nucleic acids can also antagonize endosomal TLRs in order to regulate aberrant immunity and treat autoimmune disorders such as psoriasis (13) and systemic lupus erythematosus (14). Despite these advances, immunomodulatory nucleic acids have not yet reached their full potential in the clinic. A key challenge pertains to how therapeutic biological activity in humans can be safely increased.
  • Attempts to formulate immunomodulatory nucleic acid activity have primarily focused on constructs for immuno stimulation (IS) and include: complexing oligonucleotides with albumin (15), gold nanoparticles with two-component external coronas consisting of IS- oligonucleotides together with protein antigens (16), gold nanoparticle-IS -oligonucleotide conjugates (17), IS -oligonucleotides conjugated to polymeric nanoparticles (18), and IS- oligonucleotides in complex with lipids (19).
  • IS immuno stimulation
  • the invention relates to a method for treating or preventing nonalcoholic steatohepatitis (NASH), fibrosis or hepatocellular carcinoma (HCC) by
  • administering to a subject having NASH, fibrosis or HCC an effective amount to treat or prevent the NASH, fibrosis, or HCC a nanostructure comprising an antagonist of a nucleic acid-interacting complex, wherein the antagonist is selected from the group consisting of TLR 3, 7/8, and/or 9 antagonists.
  • the invention relates to a method for treating a disorder associated with immune modulation by administering to a subject having such a disorder an effective amount to treat the disorder, a nanostructure comprising an antagonist of a nucleic acid- interacting complex, wherein the antagonist is selected from the group consisting of TLR 3, 7/8, and/or 9 antagonists.
  • the antagonist of nucleic acid-interacting complex is a RNA or DNA. In other embodiments the antagonist of nucleic acid-interacting complex is a double stranded RNA or double stranded DNA. In yet other embodiments the antagonist of nucleic acid-interacting complex is a single stranded RNA.
  • the fibrosis in some embodiments is liver fibrosis.
  • the nanostructure has a core such as a lipid core or a metal core.
  • the antagonist of nucleic acid-interacting complex is an antagonist of a TLR.
  • the nanostructure includes at least two antagonists of different TLRs. In yet other embodiments the nanostructure includes at least three antagonists of different TLRs.
  • An additional therapeutic molecule such as a small molecule or antisense
  • oligonucleotide/siRNA may be included in the nanostructure.
  • the invention is a method for preventing or treating non-alcoholic steatohepatitis (NASH), fibrosis or hepatocellular carcinoma (HCC) comprising
  • administering to a subject having NASH, fibrosis or HCC an effective amount to prevent or treat the NASH, fibrosis, or HCC of a TNF antisense oligonucleotide.
  • IR-SNA Immunoregulatory SNAs
  • FIG. 3 Immunoregulatory effects of IR-SNAs confirmed by testing for production of TNF-a (points show mean + SD).
  • IR-SNAs show enhanced treatment of liver fibrosis in mice with NASH.
  • STAM mice were treated every other day from 6 to 9 wk of age.
  • NAS nonalcoholic fatty liver disease activity
  • FIG. 6 IR-SNA activity in vitro. 4084F-Ext or scrambled control (CTL) either formulated into a liposomal SNA or in PBS were tested for their ability to block RAW-Blue cell activation by TLR 9 agonist (CpG 1826-ps). The free oligonucleotide and SNA showed excellent activity in regulating activation by TLR 9 agonist.
  • CTL scrambled control
  • Figure 7 IR-SNA activity in vitro. 4084F-Ext or scrambled control (CTL) either formulated into a liposomal SNA or in PBS were tested for their ability to block RAW-Blue cell activation by TLR 7 agonist (imiquimod) in vitro. The free oligonucleotide and SNA showed excellent activity in regulating activation by TLR 7 agonist.
  • CTL scrambled control
  • FIG. 8 IR-SNA activity in prevention of liver fibrosis in STAM (NASH-like phenotype) mice in vivo.
  • mice show enhanced treatment of liver fibrosis in mice with NASH.
  • STAM mice were treated every other day from 6 to 9 wk of age.
  • Anti-TNF compound showed activity in both scores indicating excellent promise of these compounds in treating and/or preventing NASH and progression to fibrosis.
  • Spherical nucleic acids are a class of well-defined macromolecules, formed by organizing nucleic acids radially around a nanoparticle core (20). These structures exhibit the ability to enter cells without the need for auxiliary delivery vehicles or transfection reagents by engaging class A scavenger receptors (SR-A) and lipid rafts (21).
  • SR-A class A scavenger receptors
  • lipid rafts lipid rafts
  • SNAs Once inside the cell, the nucleic acid components of SNAs resist nuclease degradation, leading to longer intracellular lifetimes. Moreover, SNAs, due to their multi-functional chemical structures, have the ability to bind their targets in a multivalent fashion (24, 25). Therefore, in addition to demonstrating many of the characteristics that would be considered preferred for designing immunomodulatory therapies, SNAs also provide a chemical and structural scaffold for evaluating the structure-activity relationships for the two major components: the oligonucleotide shell and the nanoparticle core. Indeed, SNAs can be assembled in many different structural forms, with their cores consisting of inorganic compounds, such as gold, or organic materials such as ⁇ 50 nm liposomes (26, 27).
  • the nanostructures described herein may be useful in a wide variety of applications where there is a dysregulation of the immune response that is causing disease.
  • the enhanced activity of these structures positions them quite favorably among comparable agents, in the treatment of diseases such as fibrosis.
  • Other potential commercial applications include treatments for asthma, rheumatoid arthritis, non-alcoholic steatohepatitis, liver cirrhosis, diabetes, sepsis, atopic dermatitis, psoriasis, and a variety of other auto-immune disorders.
  • the nanostructures of the invention are typically composed of: (1) a core such as a lipid-containing core, which is formed by arranging non-toxic carrier lipids into a small hollow structure or a solid core such as a metal core, (2) a shell of oligonucleotides, which is formed by arranging oligonucleotides such that they point radially outwards from the core, and (3) optionally a hydrophobic (e.g. lipid) anchor group if the core is a lipid core, also referred to herein as a linker, which is attached to either the 5'- or 3 '-end of the
  • the anchor acts to drive insertion into the liposome and to anchor the oligonucleotides to the lipid-containing core.
  • the lipid-containing core can be constructed from a wide variety of lipids known to those in the art.
  • the exterior of the lipid-containing core has an oligonucleotide shell.
  • the oligonucleotide shell can be constructed from a wide variety of nucleic acids including, but not limited to: single- stranded deoxyribonucleotides, ribonucleotides, and other single- stranded oligonucleotides incorporating one or a multiplicity of modifications known to those in the art, double- stranded deoxyribonucleotides, ribonucleotides, and other double-stranded oligonucleotides incorporating one or a multiplicity of modifications known to those in the art, oligonucleotide triplexes incorporating deoxyribonucleotides, ribonucleotides, or oligonucleotides that incorporate one or a multiplicity of modifications known to those in the art.
  • the nucleic acids of the invention are antagonists of a nucleic acid-interacting complex, which refer to a molecule or complex of molecules that interact with a nucleic acid molecule and, for instance, are stimulated to produce an immune response in response to that interaction.
  • the molecule or complex of molecules may be a receptor.
  • a nucleic acid-interacting complex is a pattern recognition receptor (PRR) complex.
  • PRRs are a primitive part of the immune system composed of proteins expressed by cells of the innate immune system to identify pathogen-associated molecular patterns (PAMPs), which are associated with microbial pathogens or cellular stress, as well as damage-associated molecular patterns (DAMPs), which are associated with cell components released during cell damage.
  • PAMPs pathogen-associated molecular patterns
  • DAMPs damage-associated molecular patterns
  • PRRs include but are not limited to membrane-bound PRRs, such as receptor kinases, toll-like receptors (TLR), and C-type lectin Receptors (CLR) (mannose receptors and asialoglycoprotein receptors); Cytoplasmic PRRs such as RIG-I-like receptors (RLR), RNA Helicases, Plant PRRs, and NonRD kinases; and secreted PRRs.
  • membrane-bound PRRs such as receptor kinases, toll-like receptors (TLR), and C-type lectin Receptors (CLR) (mannose receptors and asialoglycoprotein receptors); Cytoplasmic PRRs such as RIG-I-like receptors (RLR), RNA Helicases, Plant PRRs, and NonRD kinases; and secreted PRRs.
  • Nucleic acid-interacting complexes include but are not limited to TLRs, RIG-I, transcription factors, cellular translation machinery, cellular transcription machinery, nucleic- acid acting enzymes, and nucleic acid associating autoantigens.
  • Nucleic acid molecules that are antagonists of a nucleic acid-interacting complex include but are not limited to TLR antagonists and antagonists of RIG-I, transcription factors, cellular translation machinery, cellular transcription machinery, nucleic-acid acting enzymes, and nucleic acid associating autoantigens.
  • an antagonist of a nucleic acid-interacting complex is a TLR antagonist.
  • a TLR antagonist as used herein is a nucleic acid molecule that interacts with and modulates, i.e. reduces, the activity of a TLR.
  • antagonists of TLR 7,8, or 9 include immunomodulatory nucleic acids, that include but are not limited to nucleic acids falling within the following formulas: 5'R n JGCN z 3', wherein each R is a nucleotide, n is an integer from about 0 to 10, J is U or T, each N is a nucleotide, and z is an integer from about 1 to about 100.
  • n is 0 and z is from about 1 to about 50.
  • N is 5'SiS 2 S 3 S 4 3', wherein S i, S 2 , S 3 , and S 4 are independently G, I, or 7-deaza- dG.
  • the nucleic acids are 4084F: 5 '-CCTGGATGGGAA-3 ' (fully phosphorothioate backbone) (SEQ ID NO: 2) or 4084F-Ext: 5'-
  • the invention also encompasses the use of TNF antisense oligonucleotides in the treatment and prevention of fibrosis.
  • TNFa antisense oligonucleotide refers to a nucleic acid based agent which interferes with TNFa activity.
  • the TNFa antisense inhibitors or TNFa antisense oligonucleotides of the invention reduce the expression of the TNFa gene.
  • Antisense nucleic acids typically include modified or unmodified RNA, DNA, or mixed polymer nucleic acids, and primarily function by specifically binding to matching sequences resulting in modulation of peptide synthesis. Antisense nucleic acids bind to target RNA by Watson Crick base-pairing and block gene expression by preventing ribosomal translation of the bound sequences either by steric blocking or by activating RNase H enzyme. Antisense molecules may also alter protein synthesis by interfering with RNA processing or transport from the nucleus into the cytoplasm.
  • antisense nucleic acid or "antisense oligonucleotide” describes a nucleic acid that hybridizes under physiological conditions to DNA comprising a particular gene or to an mRNA transcript of that gene in this case TNFa and, thereby, inhibits the transcription of that gene and/or the translation of that mRNA.
  • the antisense molecules are designed so as to interfere with transcription or translation of a target gene upon hybridization with the target gene or transcript.
  • “Inhibition of gene expression” refers to the absence or observable decrease in the level of protein and/or mRNA product from a target gene, such as the TNFa gene.
  • Specificity refers to the ability to inhibit the target gene without manifest effects on other genes of the cell. The consequences of inhibition can be confirmed by examination of the outward properties of the cell or organism or by biochemical techniques such as RNA solution hybridization, nuclease protection, Northern hybridization, reverse transcription, gene expression monitoring with a microarray, antibody binding, enzyme linked
  • ELISA immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescence activated cell analysis
  • the antisense oligonucleotides of the invention inhibit TNFa expression.
  • quantitation of the amount of gene expression allows one to determine a degree of inhibition which is greater than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% as compared to a cell not treated according to the present invention.
  • the efficiency of inhibition may be determined by assessing the amount of gene product in the cell.
  • antisense oligonucleotides typically have a length of 15-20 bases, which is generally long enough to have one complementary sequence in the mammalian genome. Additionally, antisense compounds having a length of at least 12, typically at least 15 nucleotides in length hybridize well with their target mRNA. Thus, the antisense
  • oligonucleotides of the invention are typically in a size range of 8-100 nucleotides, more preferably 12-50 nucleotides in length. In some embodiments of the invention the antisense oligonucleotides are of 18-19 nucleotides in length and comprise
  • the TNF antisense oligonucleotide is mUmGmGmGm AmGT *A*G*A*T*G* m AmGmGmUm AmC (SEQ ID NO: 1) where mN is the O-Methyl modification and * is the phosphorothioate linkage.
  • oligonucleotide and “nucleic acid” are used interchangeably to mean multiple nucleotides (i.e., molecules comprising a sugar (e.g., ribose or deoxyribose) linked to a phosphate group and to an exchangeable organic base, which is either a substituted pyrimidine (e.g., cytosine (C), thymidine (T) or uracil (U)) or a substituted purine (e.g., adenine (A) or guanine (G)).
  • a substituted pyrimidine e.g., cytosine (C), thymidine (T) or uracil (U)
  • a substituted purine e.g., adenine (A) or guanine (G)
  • Oligonucleosides i.e., a polynucleotide minus the phosphate
  • Oligonucleotides can be obtained from existing nucleic acid sources (e.g., genomic or cDNA), but are preferably synthetic (e.g., produced by nucleic acid synthesis).
  • Oligonucleotides associated with the invention can be modified such as at the sugar moiety, the phosphodiester linkage, and/or the base.
  • sugar moieties includes natural, unmodified sugars, including pentose, ribose and deoxyribose, modified sugars and sugar analogs. Modifications of sugar moieties can include replacement of a hydroxyl group with a halogen, a heteroatom, or an aliphatic group, and can include functionalization of the hydroxyl group as, for example, an ether, amine or thiol.
  • Modification of sugar moieties can include 2'-0-methyl nucleotides, which are referred to as "methylated.”
  • oligonucleotides associated with the invention may only contain modified or unmodified sugar moieties, while in other instances, oligonucleotides contain some sugar moieties that are modified and some that are not.
  • modified nucleomonomers include sugar- or backbone-modified ribonucleotides.
  • Modified ribonucleotides can contain a non-naturally occurring base such as uridines or cytidines modified at the 5'-position, e.g. , 5'-(2-amino)propyl uridine and 5'- bromo uridine; adenosines and guanosines modified at the 8-position, e.g. , 8-bromo guanosine; deaza nucleotides, e.g. , 7-deaza-adenosine; and N-alkylated nucleotides, e.g.
  • sugar-modified ribonucleotides can have the 2' -OH group replaced by an H, alkoxy (or OR), R or alkyl, halogen, SH, SR, amino (such as NH 2 , NHR, NR 2, ), or CN group, wherein R is lower alkyl, alkenyl, or alkynyl.
  • modified ribonucleotides can have the phosphodiester group connecting to adjacent ribonucleotides replaced by a modified group, such as a phosphorothioate group.
  • the sugar moiety can also be a hexose.
  • hydrophobic modifications refers to modification of bases such that overall hydrophobicity is increased and the base is still capable of forming close to regular Watson -Crick interactions.
  • base modifications include 5-position uridine and cytidine modifications like phenyl, 4-pyridyl, 2-pyridyl, indolyl, and isobutyl, phenyl (C 6 H 5 OH); tryptophanyl (C 8 H 6 N)CH 2 CH(NH 2 )CO), Isobutyl, butyl, aminobenzyl; phenyl; and naphthyl.
  • the nucleomonomers of an oligonucleotide of the invention are RNA nucleotides, including modified RNA nucleotides.
  • nucleoside includes bases which are covalently attached to a sugar moiety, preferably ribose or deoxyribose.
  • examples of preferred nucleosides include ribonucleosides and deoxyribonucleosides.
  • Nucleosides also include bases linked to amino acids or amino acid analogs which may comprise free carboxyl groups, free amino groups, or protecting groups. Suitable protecting groups are well known in the art (see P. G. M. Wuts and T. W. Greene, "Protective Groups in Organic Synthesis", 2 nd Ed., Wiley-Interscience, New York, 1999).
  • nucleotide includes nucleosides which further comprise a phosphate group or a phosphate analog.
  • linkage includes a naturally occurring, unmodified phosphodiester moiety (-0-(PO )-0-) that covalently couples adjacent nucleomonomers.
  • substitute linkage includes any analog or derivative of the native phosphodiester group that covalently couples adjacent nucleomonomers. Substitute linkages include phosphodiester analogs, e.g. , phosphorothioate, phosphorodithioate, and P- ethyoxyphosphodiester, P-ethoxyphosphodiester, P-alkyloxyphosphotriester,
  • methylphosphonate and nonphosphorus containing linkages, e.g. , acetals and amides.
  • linkages e.g. , acetals and amides.
  • Such substitute linkages are known in the art (e.g. , Bjergarde et al. 1991. Nucleic Acids Res.
  • non-hydrolizable linkages are preferred, such as phosphorothioate linkages.
  • oligonucleotides of the invention comprise 3 ' and 5' termini (except for circular oligonucleotides).
  • the 3 ' and 5' termini of an oligonucleotide can be substantially protected from nucleases, for example, by modifying the 3 Or 5' linkages (e.g. , U.S. Pat. No. 5,849,902 and WO 98/13526).
  • Oligonucleotides can be made resistant by the inclusion of a "blocking group.”
  • the term "blocking group” as used herein refers to substituents (e.g.
  • oligonucleotides or nucleomonomers that can be attached to oligonucleotides or nucleomonomers, either as protecting groups or coupling groups for synthesis (e.g. , FITC, propyl (CH 2 -CH 2 -CH 3 ), glycol (-O-CH 2 -CH 2 -O-) phosphate (PO 3 " ), hydrogen phosphonate, or phosphoramidite).
  • “Blocking groups” also include "end blocking groups” or “exonuclease blocking groups” which protect the 5' and 3 ' termini of the oligonucleotide, including modified nucleotides and non-nucleotide exonuclease resistant structures.
  • Exemplary end-blocking groups include cap structures (e.g. , a 7-methylguanosine cap), inverted nucleomonomers, e.g. , with 3 '-3 ' or 5 '-5' end inversions (see, e.g. , Ortiagao et al. 1992. Antisense Res. Dev. 2: 129), methylphosphonate, phosphoramidite, non-nucleotide groups (e.g. , non-nucleotide linkers, amino linkers, conjugates) and the like.
  • the 3 ' terminal nucleomonomer can comprise a modified sugar moiety.
  • the 3 ' terminal nucleomonomer comprises a 3 '-0 that can optionally be substituted by a blocking group that prevents 3 '- exonuclease degradation of the oligonucleotide.
  • the 3 '-hydroxyl can be esterified to a nucleotide through a 3 ' ⁇ 3' internucleotide linkage.
  • the alkyloxy radical can be methoxy, ethoxy, or isopropoxy, and preferably, ethoxy.
  • the 3 ' ⁇ 3 'linked nucleotide at the 3 ' terminus can be linked by a substitute linkage.
  • the 5' most 3 ' ⁇ 5' linkage can be a modified linkage, e.g. , a
  • the two 5' most 3 ' ⁇ 5' linkages are modified linkages.
  • the 5' terminal hydroxy moiety can be esterified with a phosphorus containing moiety, e.g. , phosphate, phosphorothioate, or P- ethoxypho sphate .
  • oligonucleotides can comprise both DNA and RNA.
  • At least a portion of the contiguous oligonucleotides are linked by a substitute linkage, e.g. , a phosphorothioate linkage.
  • a substitute linkage e.g. , a phosphorothioate linkage.
  • the oligonucleotides are preferably in the range of 6 to 100 bases in length. However, nucleic acids of any size greater than 6 nucleotides (even many kb long) are useful.
  • the nucleic acid is in the range of between 8 and 100 and in some embodiments between 8 and 50, 8 and 40, 8 and 30, 6 and 50, 6 and 40, or 6 and 30 nucleotides in size.
  • aspects of the invention relate to delivery of SNAs to a subject for therapeutic and/or diagnostic use.
  • the SNAs may be administered alone or in any appropriate pharmaceutical carrier, such as a liquid, for example saline, or a powder, for administration in vivo. They can also be co-delivered with larger carrier particles or within administration devices.
  • the SNAs may be formulated.
  • the formulations of the invention can be administered in
  • the SNAs associated with the invention are mixed with a substance such as a lotion (for example, aquaphor) and are administered to the skin of a subject, whereby the SNAs are delivered through the skin of the subject.
  • a lotion for example, aquaphor
  • an effective amount of the SNAs can be administered to a subject by any mode that delivers the SNAs to the desired cell.
  • Administering pharmaceutical compositions may be accomplished by any means known to the skilled artisan. Routes of administration include but are not limited to oral, parenteral, intramuscular, intravenous, subcutaneous, mucosal, intranasal, sublingual, intratracheal, inhalation, ocular, vaginal, dermal, rectal, and by direct injection.
  • IR-SNAs both Au-SNA and L-SNA
  • oligonucleotide 4084F (28).
  • TLR antagonist activity cells were first activated by incubation with PS CpG 1826 (at 0.5 ⁇ ) for 2 h. The cells were then treated with various antagonists: 4084F-derivatized Au-SNA, free 4084F all-PS oligonucleotide, or mismatched oligonucleotide controls. The results demonstrate that 4084F-derivatized Au-SNAs show a ⁇ 8-fold increase in potency as compared to the free 4084F-all-PS counterpart ( Figure 1). Similar trend in the activity was observed with L-SNA antagonist constructs designed with 4084F sequences ( Figure 2).
  • IR-SNAs Demonstrate Enhanced Antifibrotic Activity in a Mouse Model of NASH.
  • Nonalcoholic steatohepatitis is a fatty liver disease with prevalence as high as 3-5% in the United States. It is
  • livers of patients characterized by pathologic changes in livers of patients, including steatosis and
  • HCC hepatocellular carcinoma
  • the STAM mouse model has been developed as a small animal model that recapitulates important features of the human disease (29). Nearly 100% of STAM mice follow disease progression from steatosis to NASH to fibrosis to HCC, making this a model that is well suited to evaluate the ability of compounds to prevent disease progression. In this model, mice that are 6 wk old demonstrate evidence of NASH, and from 6 to 9 wk old progress to histological evidence of fibrosis.
  • IR-SNAs Neither IR-SNAs nor unformulated oligonucleotides showed any significant effect on nonalcoholic fatty liver disease score in this study. These results suggest that IR-SNAs can potentially be developed into compounds that modify rates of NASH progression to fibrosis, an advance that could reduce mortality from this disease.
  • immunomodulatory pharmacophores and they highlight the modular nature of the SNA therapeutic construct.
  • RAW-Blue cells and Ramos-Blue cells (InvivoGen), derivatives of RAW 264.7 macrophages and Ramos B cells, respectively, stably expressing a secreted alkaline phosphatase (SEAP) inducible by NF-kB, were cultured as recommended by the distributor.
  • SEAP secreted alkaline phosphatase
  • Human PBMCs collected from healthy donors were purchased from Sanguine (Sherman Oaks, CA) and kept frozen in liquid N 2 until use.
  • Spherical nucleic acids, reagents Oligonucleotides were synthesized using automated solid support phosphor amidite synthesis (sequences for activating and regulating immunity are included in the
  • oligonucleotide conjugated OVA was hybridized to the oligonucleotide shell of the SNAs. Diameters were measured by dynamic light scattering (DLS) using a Zetasizer Nano zs (Malvern Instruments, USA).
  • the IR-oligonucleotide 4084F was used as a model IR- oligonucleotide: 5'- CCTGGATGGGAA-3 ' (fully phosphorothioate backbone) (SEQ ID NO: 2).
  • SEQ ID NO: 3 To extend activity to TLR 7 blockade, the sequence 4084F-Ext was developed: 5'- TGCTTGACACCTGGATGGGAA-3 ' (fully phosphorothioate backbone) (SEQ ID NO: 3).
  • Control (CTL) sequences consisted of the corresponding sequence for each study but with the 5'-CpG-3' dinucleotide inverted (5'-GpC-3').
  • cells were plated in 96-well plates, stimulated with CpG 1826 PS for 2 h, then repressed by addition of the test reagent overnight.
  • Cell supernatants were collected and probed for SEAP using the QUANTI-Blue assay (InvivoGen) by following the manufacturer's recommended protocol.
  • Supernatants were probed for cytokine levels by ELISA (mouse TNF-alpha: Pierce #EMTNFA2, mouse total IL-12: Pierce #EMIL12TOT2, mouse IL-6: Pierce #EM2IL62) by following the appropriate manufacturer's recommended protocols.
  • IR-SNAs The activity of IR-SNAs was tested in the STAM mouse model (Stelic Institute & Co., Inc.) of non-alcoholic steatohepatitis (NASH).
  • STAM mouse model StepAM mouse model (Stelic Institute & Co., Inc.) of non-alcoholic steatohepatitis (NASH).
  • NASH non-alcoholic steatohepatitis
  • Compounds or control were administered (40 ⁇ oligo equivalent concentration) in 100 ⁇ ⁇ by intraperitoneal (i.p.) administration.
  • the impact of treatment administration on NASH and liver fibrosis was determined by collecting livers at 9 weeks of age and assessing NASH disease and fibrosis histologically.
  • H&E stained liver sections were scored for steatosis (0-3), lobular inflammation (0-3), and hepatocellular ballooning (0-2), after which a composite non-alcoholic fatty liver disease score (NAS) was assigned (scale of 0-8). Sections were also stained with Sirius Red for fibrosis and scored as % positive area according to established protocols.
  • NAS non-alcoholic fatty liver disease score
  • DNA-like class R inhibitory oligonucleotides preferentially block autoantigen-induced B-cell and dendritic cell activation in vitro and autoantibody production in lupus-prone MRL-Fas(lpr/lpr) mice in vivo.

Abstract

Acides nucléiques immunomodulateurs présentant un potentiel important pour traiter une maladie, bien que les progrès en clinique aient été limités par le manque d'outils pour augmenter l'activité chez des patients en toute sécurité. Acides nucléiques immunomodulateurs agissent par l'agonisation ou l'antagonisation de récepteurs de type toll endosomaux (TLR 3, 7/8, 9) une famille de protéines impliquées dans la signalisation immunitaire innée. Les nouveaux acides nucléiques sphériques immunomodulateurs (SNA) qui régulent (immunorégulateurs, IR-SNA) l'immunité par l'engagement d'oligonucléotides antisens TLR ou anti-TNT ont été conçus, synthétisés et caractérisés. Les SNA d'acide nucléique présentent, selon certains modes de réalisation, une multiplication par 8 ou plus de la puissance et une réduction de 30 % ou plus de la marque de fibrose chez un sujet ayant une stéatohépatite non alcoolique (NASH).
PCT/US2016/022579 2015-03-16 2016-03-16 Acides nucléiques sphériques immunomodulateurs WO2016149323A1 (fr)

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US11633503B2 (en) 2009-01-08 2023-04-25 Northwestern University Delivery of oligonucleotide-functionalized nanoparticles
US10837018B2 (en) 2013-07-25 2020-11-17 Exicure, Inc. Spherical nucleic acid-based constructs as immunostimulatory agents for prophylactic and therapeutic use
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WO2021183485A1 (fr) * 2020-03-09 2021-09-16 Emory University Acides nucléiques sphériques hétéromultivalents et leurs utilisations dans des applications thérapeutiques et diagnostiques

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