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WO2021118927A1 - Méthodes et compositions pour l'administration ciblée d'agents thérapeutiques par l'acide nucléique - Google Patents

Méthodes et compositions pour l'administration ciblée d'agents thérapeutiques par l'acide nucléique Download PDF

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WO2021118927A1
WO2021118927A1 PCT/US2020/063610 US2020063610W WO2021118927A1 WO 2021118927 A1 WO2021118927 A1 WO 2021118927A1 US 2020063610 W US2020063610 W US 2020063610W WO 2021118927 A1 WO2021118927 A1 WO 2021118927A1
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reagent
cell
nucleic acid
gene
targetmer
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Alan Gordon HERBERT
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Insideoutbio Inc
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Insideoutbio Inc
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    • 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
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • 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
    • A61K47/545—Heterocyclic 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
    • A61K47/54—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
    • A61K47/56—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
    • A61K47/59—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 otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/595—Polyamides, e.g. nylon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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

  • Another approach is to deliver a therapeutic directly to the cytoplasm using a membrane binding steroid linked to ethidium bromide that intercalates into any double-stranded region of a nucleic acid with only limited sequence specificity [13]. While this approach solves the delivery problem, the therapeutic is not targeted to any specific cell type.
  • Molecules that bind to the minor groove of a double-stranded nucleic acid structure offer a new way to attach targeting ligands to a nucleic acid therapeutic. They have many properties that are useful for the design of a delivery strategy for nucleic acid therapeutics.
  • Minor grove binders are specific for short sequence motifs of 4 or more bases allowing the number that bind the therapeutic to be determined from its sequence [14]. They also protect the therapeutic against attack by nuclease [15]. By making the nucleic acid stiffer and more rod-like, they are similar to other engineered DNA structures that show enhanced penetration into cells [16]. The minor groove binders do not induce aggregation of nucleic acids as occurs with other delivery reagents such as polyethylenimines where particle size determines distribution in the body and uptake by cells. Some minor groove binders also disrupt membrane structure [17], potentially facilitating escape of therapeutics from endosomes [18].
  • distamycin While distamycin is specific for 4 bases, a "distamycin trimer" connected head to tail by ⁇ -alanine linkers recognizes a 16 base sequence [24], [ 0009] While minor groove binders have been used to increase the strength of association of a triplex forming oligonucleotide (TFO’s) to the major groove of double- stranded DNA, their use in directing the import of nucleic acids into cells has not been reported [25, 26].
  • TFO triplex forming oligonucleotide
  • the present invention describes therapeutic compositions for the targeted delivery of nucleic acids to specific cells and tissues, and methods of their use to treat conditions and diseases as described herein. More specifically, the present invention encompasses methods and compositions for the targeted delivery of a therapeutic/drug/pharmaceutical agent, referred to herein as Reagent 1 , to a cell using a synthetic construct referred to herein as a “targetmer”.
  • Reagent 1 a therapeutic/drug/pharmaceutical agent
  • the invention enables methods known to those skilled in the art that activate, enhance, alter, or inhibit the expression of nucleic acid sequences in the host cell, regardless of whether the expressed sequences derive from Reagent lor from the host nucleic acids.
  • the nucleic acids used to construct Reagent 1 are suitable for use in these compositions are unencapsulated.
  • the present invention comprises a therapeutic composition comprising at least three components (also referred to herein as “Reagents”.
  • Reagent 1 (“reagent” is also referred to herein as component or agent) is a nucleic acid modified to reduce nuclease susceptibility and wherein the nucleic acid comprises a specific nucleotide sequence designed to elicit or produce a therapeutic effect within a targeted cell or tissue.
  • Reagent 1 has activity to, for example, activate, enhance, alter, or inhibit the expression of nucleic acid sequences in the host cell, thereby eliciting a therapeutic response in the cell or tissue.
  • Reagent 1 is bound/linked/ coupled to one or more copies of a “targetmer”, or to one or more copies of different “targetmers”, wherein a “targetmer” is described in FIG. 1.
  • Modifications to Reagent lto reduce nuclease sensitivity may include the use of modified bases [3]. It may also involve DNAs with covalently closed ends (FIG. 1) [28]. [ 0014 ] As described herein, the targetmer is composed of two parts (also referred to herein as components). The first part is a molecule (Reagent 2) that has sequence-specific binding to the minor-groove of a double-stranded region of a nucleic acid.
  • the second part of the molecule (Reagent 3) is comprised of a ligand that binds specifically to a targeted cell or tissue (e.g., a tumor cell), thereby delivering Reagent 1 to that cell or tissue, resulting in the expression of Reagent 1 in that cell or tissue, and whereby the expression of Reagent 1 results in the desired therapeutic activity of Reagent 1 in in that cell or tissue.
  • a targeted cell or tissue e.g., a tumor cell
  • Reagent 2 and Reagent 3 comprise the targetmer.
  • the combination of Reagent 1 and the targetmer(s) comprise the therapeutic construct/composition.
  • Reagent 1 and the targetmer(s) include design elements for the entry of reagent 1 into the targeted cell or tissue, its release from endosomal compartments, its delivery to the nucleus (if required for efficacy) and the synthesis of RNA it encodes (if required for efficacy).
  • reagent 2 is covalently coupled to reagent 3, either directly, or via a linker that connects one to the other (Fig. 2a).
  • a linker that connects one to the other (Fig. 2a).
  • reagent 2 is coupled to an agent that is has high affinity for another agent coupled to reagent 3, where the association is not covalent, but sufficient to form a targetmer from reagent 2 and reagent 3 (Fig. 2b).
  • the different possible methods for forming non-covalent associations between reagent 2 and reagent 3 are well known to those skilled in the art and are summarized in Table 1 of Schreiber and Smith [30].
  • the binding specificity of reagent 2 to reagent 1 is for a short sequence motif of about 4-16 nucleotides as described herein.
  • the binding kinetics of reagent 2 can be altered by adding nucleotides to reagent 2 so that bind the major groove through base-specific Watson-Crick and Hoogsteen hydrogen bonds.
  • nucleotides bound to minor groove binders are known to change the dissociation rate of a minor groove binder from the nucleic acid [25, 26],
  • reagent 2 is comprised of molecules known to specifically bind the minor groove, examples of which include distamycin and Hoehsct 33258 (see Fig. 3) [31, 32],
  • Another embodiment of the invention is when reagent 2 is synthesized using solid phase chemistry using pyrrole and imidazole polyamides and their derivatives [14, 21, 22],
  • reagent 3 is a small molecule that has specificity for its ligand and is chemically coupled to reagent 2 through a linker [35], one example being the conjugation of folate or its derivatives to target the folate receptor a. (Fig. 2c).
  • the targetmers are used to deliver Reagent 1 for the purposes of correcting genetic errors that lead to disease.
  • a listing of such errors is given in the online catalog of Mendelian Disease that is entitled “Online Mendelian Inheritance in Man” (known as OMIM, www.omim.org/).
  • reagent 3 specifically targets one of the following receptors: VEGFR1, VEGFR2, VEGFR3, HER2/neu, Muc-1, Nucleolin, Optoneurin, Integral, Prostate-Specific Membrane Antigen , AXL, Carcinoembryonic Antigen, folate receptor a , Protein Tyrosine Kinase 7, a checkpoint inhibitor like Cytotoxic T Cell Antigen-4 or Programmed Cell Death Molecule 1 (PD-1), (ASGPR), T Cell Receptors, OX40 and related Tumor Necrosis family members [36], whether with an aptamer [2], carbohydrate, peptide, lipid, steroid protein or small molecule (examples of conjugations to imidazoles are given in Midoux et al.
  • the present invention also encompasses methods for use of targetmers to engineer immune cells ex vivo, such as Chimeric Antigen Receptor (CAR) bearing cells (CAR- cells), then administered to patients to act in vivo.
  • CAR Chimeric Antigen Receptor
  • the present invention also encompasses methods to enhance the effectiveness of tumor-specific vaccines by using targetmers to direct expression of pro-inflammatory proteins IL-23, IL-36 ⁇ and OX40L encoded by reagent 1 in antigen presenting cells [38] so as to enhance an immune response against a tumor.
  • the present invention also encompasses methods to enhance the effectiveness of pathogen-specific vaccines by using targetmers to direct expression of pathogen- specific antigen and pro-inflammatory proteins IL-23, IL-36 ⁇ and OX40L encoded by reagent 1 in antigen presenting cells [38] so as to enhance an immune response against the pathogen.
  • the present invention encompasses methods to inhibit antigen-specific immune responses, such as those found in autoimmune and allergic diseases by using targetmers to direct expression of inhibitory molecule such as PD- 1 and other checkpoint inhibitors encoded by reagent 1 on cell surfaces bearing the immunostimulatory antigen.
  • Methods described herein include the construction of a therapeutic composition constructed from a nucleic acid encoding therapeutic molecules (reagent 1) that will bind a known number of targetmers.
  • Methods described herein allow the use of more than one targetmer for the delivery of reagent 1.
  • the targetmers used may differ in reagent 2, reagent 3 or in both, allowing the tuning of the therapeutic delivery for a particular use in a controlled fashion.
  • reagent 1 derives from a polynucleotide composed of either DNA or RNA that enables expression in the target cell of a desired molecule (Fig. 4).
  • the expression of the fusion protein may be limited to a particular cell type by use of appropriate promoters, enhancers or other regulatory sequences known to one skilled in the art.
  • the therapeutic comprising reagent 1 and the targetmer(s) may be delivered to the target cell by injection, electroporation or other mechanical or electrophysiological mechanisms either locally into a space like the spinal cord or the peritoneum where the therapeutic can contact the targeted tissue.
  • administration may be by systemic administration that involves transport by the blood or lymphatic systems, or after ex-vivo manufacture in the case of a cellular therapy.
  • the methods of the present invention include an expression vector (reagent 1) coupled to one, or more targetmer(s), wherein the vector comprises a nucleic acid construct that expresses a molecule(s) encoded by reagent 1.
  • reagent 1 is delivered to those cells that binds reagent 2 and results in the transport of reagent 1 to the nucleus.
  • the methods include delivery of a defined antigen along with immunosuppressive molecules like PD- 1 and TGF ⁇ decreases or suppresses immune responses associated with allergy and autoimmunity triggered by the specified antigen. Delivery of the defined antigen may be implemented in a number of ways as known to those experienced in the art and does not always depend upon reagent 1.
  • the subject of the methods of this invention is a mammal, and more particularly, the mammal is a human and can activate immunity using the approaches described.
  • a particular embodiment of the present invention encompasses methods of treating cancer in an individual, preventing metastasis of the cancer and protecting against reoccurrence of the cancer wherein administering to the individual an effective amount of the therapeutic agent that increases the expression of the encoded molecules targeted to the tumor cells or to cells in the tumor micro-environment.
  • the methods described herein using targeted delivery of reagent 1 by the targetmer(s) can be used to treat many different forms of cancers.
  • the cancer can be ovarian, breast, colon, renal, neural or lung cancer.
  • Another embodiment of the present invention is a vaccine composition, and method of using that vaccine composition, to vaccinate (i.e., elicit an immune response) a subject against tumors that express a defined antigen so as to provoke an immune response to protect an individual against that tumor type, including applications where the vaccine is delivered locally, to lymph nodes, to other tissues or systemically by injection and targeted to the tumor cells or to cells in the tumor micro-environment.
  • the vaccine composition can be used to vaccinate a subject against a pathogen that expresses a defined antigen by targeting the therapeutic to a set of immune cells and so provoke an immune response to protect an individual against any pathogen that bears the antigen.
  • the complementary cancer treatment can be selected from a therapy comprising checkpoint inhibitor; a proteasome inhibitor; immunotherapeutic agent; radiation therapy or chemotherapy.
  • Other suitable additional or complementary cancer therapies are known to those of skill in the art.
  • compositions comprising a therapeutically effective amount of the targeted therapeutic composed of reagents 1 , 2 and 3 as described herein.
  • the composition additionally can include a pharmaceutically acceptable medium, suitable as a carrier for the agent.
  • the compositions can also include other agents that improve delivery of the compositions to specific tumor sites.
  • One embodiment includes the administration of 4- methylumbelliferone to reduce the thickness of the glycocalyx present in tumors.
  • FIG. 1 Construction of Nucleic Acid Delivery Therapeutic from Three Reagents, where Reagent 1 is a nucleic acid with closed ends to reduce nuclease susceptibility, Reagent 2 is a molecule that binds to the minor groove of Reagent 1 in a sequence specific manner with micro-molar or sub-micro-molar affinity, Reagent 3 is a ligand for a receptor that is expressed on the targeted cell. Linkers are shown between Reagent 2 and 3 so that the length between each can be varied to improve interaction of Reagent 3 with its receptor.
  • FIG. 2a-c Examples where different variations of reagent 2 and reagent 3 are combined to form Nucleic Acid Delivery Therapeutic from reagent 1.
  • Reagent 2 is a molecule that binds to the minor groove of reagent 1 in a sequence specific manner (e.g. Distamycin, Hoechst 33428, netropsin or a polyamide synthesized from a heterocyclic or heteroaryl, aromatic amino acid) and may be covalently linked to reagent 3 or through a high affinity non-covalent interaction (e.g.
  • Reagent 3 is a ligand for the receptor on the targeted cell (e.g. aptamer, peptide, protein, drug, carbohydrate, vitamin etc.).
  • FIG. 3 Examples of compounds that bind the minor groove of nucleic acids
  • FIG. 4 Example of the various elements in Reagent 1; an enhancer, promoter,
  • DTS is a DNA targeting sequences that promotes nuclear localization of reagent 1 [39]. [ 0052 ] FIG.
  • the Fmoc solid phase synthetic scheme for polyamides 1 and 2 starting from commercially available Fmoc- ⁇ -alanine-Wang resin (i) 20% piperidine/NMP; (ii) Fmoc-Py acid, HBTU, DIEA; (iii) 20% piperidine /NMP; (iv) Fmoc-Py acid, HBTU, DIEA; (v) 20% piperidine/NMP ; (vi) Fmoc-Py acid, HBTU, DIEA; (vii) 20% piperidine/NMP; (viii) Fmoc-Py acid (for 1) or Fmoc-Im acid (for 2), HBTU, DIEA; (ix) 20% piperidine/NMP; (x) Fmoc- ⁇ -aminobutyric acid, HBTU, DIEA; (xi) 20% piperidine/NMP; (xii) Fmoc-Py acid, HBTU, DIEA; (x
  • FIG. 6 Example of using pyrrole and imidazole polyamides to synthesize a minor-groove binding agent with Carboxy- terminal reagent groups to allow coupling to reagent 3 or to a linker capable of a non-covalent attachment to reagent 3.
  • the solid-phase synthesis follows the method of Wurth et al. [16] as described in the legend to Fig. 5.
  • FIG. 7 A and B Chemistry for Fmoc protected Folate for solid phase peptide synthesis using click chemistry (from [40]).
  • FIG. 8 A targetmer for the Folate Receptor a consisting of folate linked to distamycin through a polyethylene glycol linker (molecular weight and is determined by application).
  • the term “and/or” includes any and all combinations of one or more of the associated listed items. Further, the singular forms and the articles “a”, “an” and “the” are intended to include the plural forms as well, unless expressly stated otherwise. It will be further understood that the terms: includes, comprises, including and/or comprising, when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. Further, it will be understood that when an element, including component or subsystem, is referred to and/or shown as being connected or coupled to another element, it can be directly connected or coupled to the other element or intervening elements may be present.
  • RNA polymerase mediated techniques e.g., NASBA
  • cell is used in reference to methods or systems that produce surfaces bearing targeted receptors with or without antigens and are used without respect to species.
  • vector means the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence.
  • Vectors typically comprise the DNA of a transmissible agent, into which foreign DNA encoding a protein is inserted by restriction enzyme technology.
  • a common type of vector is a "plasmid”, which generally is a self-contained molecule of double-stranded DNA that can readily accept additional (foreign) DNA and which can readily introduced into a suitable host cell.
  • plasmid and fungal vectors have been described for replication and/or expression in a variety of eukaryotic and prokaryotic hosts.
  • Non-limiting examples include pKK plasmids (Clonetech), pUC plasmids, pET plasmids (Novagen, Inc., Madison, Wis.), pRSET or pREP plasmids (Invitrogen, San Diego, Calif.), or pMAL plasmids (New England Biolabs, Beverly, Mass.), and many appropriate host cells, using methods disclosed or cited herein or otherwise known to those skilled in the relevant art.
  • Recombinant cloning vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g., antibiotic resistance, and one or more expression cassettes.
  • the nucleic acid of reagent 1 can be a replication competent vector capable of infecting only replicating tumor cells with particular mutations.
  • a replication competent vector comprises an internal ribosomal entry site (IRES) 5' to the heterologous polynucleotide encoding, e.g., a cytosine deaminase, miRNA, siRNA, cytokine, receptor, antibody or the like.
  • IRES internal ribosomal entry site
  • the heterologous polynucleotide encodes a non-translated RNA such as siRNA, miRNA or RNAi then no IRES is necessary, but may be included for another translated gene, and any kind of vector can be used.
  • the polynucleotide is 3' to a sequence that encodes an unrelated protein.
  • the vector is a capsid free AAV vector capable of nuclear retention as the targeted cell divides [28].
  • express and expression mean allowing or causing the information in a gene or DNA sequence to become manifest, for example producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence or RNA sequence.
  • a DNA sequence or RNA sequence is expressed in or by a cell to form an "expression product" such as a protein.
  • the expression product itself e.g. the resulting protein, may also be said to be “expressed” by the cell.
  • a polynucleotide or polypeptide is expressed recombinantly, for example, when it is expressed or produced in a foreign host cell under the control of a foreign or native promoter, or in a native host cell under the control of a foreign promoter.
  • RNA editing or “gene editing techniques” as described herein can include RNA-mediated interference (referred to herein as RNAi, or interfering RNA molecules), or Short Hairpin RNA (shRNA) or CRISPR-Cas9 and TALEN.
  • RNAi RNA-mediated interference
  • shRNA Short Hairpin RNA
  • CRISPR-Cas9 CRISPR-Cas9 and TALEN.
  • Gene therapy generally means a method of therapy wherein a desired gene/genetic sequence is inserted into a cell or tissue (along with other sequences necessary for the expression of the specific gene). See, for example, genetherapynet.com for description of gene therapy techniques.
  • subject can include a human subject for medical purposes, such as for the treatment of an existing disease, disorder, condition or the prophylactic treatment for preventing the onset of a disease, disorder, or condition or an animal subject for medical, veterinary purposes, or developmental purposes.
  • Suitable animal subjects include mammals including, but not limited to, primates, e.g., humans, monkeys, apes, gibbons, chimpanzees, orangutans, macaques and the like; bovines, e.g., cattle, oxen, and the like; o vines, e.g., sheep and the like; caprines, e.g., goats and the like; porcines, e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras, and the like; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, and the like; and rodents, including mice, rats, guinea pigs, and the like.
  • primates e.g., humans, monkeys, apes, gibbons, chimpanzees, orangutans, macaques and the like
  • an animal may be a transgenic animal.
  • the subject is a human including, but not limited to, fetal, neonatal, infant, juvenile, and adult subjects.
  • a "subject” can include a patient afflicted with or suspected of being afflicted with a disease, disorder, or condition.
  • Subjects also include animal disease models (e.g., rats or mice used in experiments, and the like).
  • cancer includes, but is not limited to, solid tumors and blood borne tumors. These terms include diseases of the skin, tissues, organs, bone, cartilage, blood and vessels. These terms further encompass primary and metastatic cancers.
  • minor groove refers to the narrower of the two grooves found in structures of double-stranded nucleic acids. Functionally, the minor groove presents a different binding surface for molecules than does the larger “major groove”. Some examples of molecules that specifically recognize the minor groove are given in Lauria et al. [31].
  • minor groove binder refers to a compound that binds to the minor groove of double-stranded nucleic acids (Fig. 3) [23, 31].
  • targetmer refers to the combination of a minor-groove binder specific for a double-stranded nucleic-acid (Reagent 2) and a receptor binding ligand (Reagent 3) regardless of how or where they associate.
  • the term “antigen” is defined as any molecule that a T-Cell or B-Cell receptor has specificity for, or any molecule targeted by Natural Killer Cells or other Innate Cells that specifically targets their effector function such as cytotoxic killing of cells, release of growth factors, lymphokines or cytokines. (Microbiology and Immunology On-line, Edited by Richard Hunt, PhD; www.microbiologvbook.org/maver/antigens2000 ).
  • CAR refers to any chimeric antigen receptor introduced into immune cells for therapeutic purposes [41].
  • the term “genetic disease” includes any of those listed in the online catalogue of Mendelian Disease that is entitled “Online Mendelian Inheritance in Man” (known as OMIM, www.omim.org/).
  • the methods and compositions of the present invention are suitable to treat a disease due to a nucleotide variant in the genome regardless of whether the variant is inherited or arises from a somatic DNA mutation. More specifgically, methods and compositions of the present invention enable treatment of many different types of genetic disease, either to cure the disease or to completely, or partially, ameliorate disease symptoms and its effects.
  • the methods and compositions of the present invention may be used to treat any type cancerous tumor or cancer cells.
  • Such tumors/cancers may be located anywhere in the body, including without limitation in a tissue selected from brain, colon, urogenital, lung, renal, prostate, pancreas, liver, esophagus, stomach, hematopoietic, breast, thymus, testis, ovarian, skin, bone marrow and/or uterine tissue.
  • Cancers that may treated by methods and compositions of the invention include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus.
  • the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acid
  • a “therapeutically effective” amount as used herein refers to an amount sufficient to have the desired biological effect (for example, an amount sufficient to express a molecule or molecules with the desired effect on the underlying disease state (for example, an amount sufficient to inhibit tumor growth in a subject, produce an immune response to an antigen or to inhibit autoimmune disease) in at least a subpopulation of cells in a subject at a reasonable benefit/risk ratio applicable to any medical treatment. Determination of therapeutically effective amounts of the agents used in this invention, can be readily made by one skilled in the art, by the use of known techniques and by observing results obtained under analogous circumstances.
  • the amounts/ dosages may be varied depending upon the requirements of the subject in the judgment of the treating clinician; the severity of the condition being treated and the particular composition being employed. In determining the therapeutically effective amount, a number of factors are considered by the treating clinician, including, but not limited to: the specific disease state; pharmacodynamic characteristics of the particular agent and its mode and route of administration; the desired time course of treatment; the species being treated; its size, age, and general health; the specific disease involved; the degree of or involvement or the severity of the disease; the response of the individual patient; the particular agent administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the kind of concurrent treatment (i.e., the interaction of the agent with other co-administered agents); and other relevant circumstances.
  • the treating clinician including, but not limited to: the specific disease state; pharmacodynamic characteristics of the particular agent and its mode and route of administration; the desired time course of treatment; the species being treated; its size, age, and general health; the specific disease involved; the degree of or
  • the sequence of reagent 1 may be changed to alter the number of binding sited for reagent 3.
  • the composition of reagent 2 may be changed to target different sequence motifs in reagent 1.
  • the composition of reagent 3 may be changed to target reagent 1 to different tissues or cells.
  • Different chemistries known to those skilled in the art may be used to link reagent 2 to either reagent 3, the preferred embodiment, or to reagent 1 (see for example, WO1996/026950A1, the teachings of which are incorporated herein by reference).
  • the agents described for use in this invention can be combined with other pharmacologically active compounds ("additional active agents") or peptide antigens (“antigens”) known in the art according to the methods and compositions provided herein.
  • Additional active agents can be large molecules (e.g., proteins, lipids, carbohydrates) or small molecules (e.g., synthetic inorganic, organometallic, or organic molecules).
  • additional active agents independently or synergistically help to treat cancer.
  • chemotherapeutic agent includes, without limitation, platinum-based agents, such as carboplatin and cisplatin; nitrogen mustard alkylating agents; nitrosourea alkylating agents, such as carmustine (BCNU) and other alkylating agents; antimetabolites, such as methotrexate; purine analog antimetabolites; pyrimidine analog antimetabolites, such as fluorouracil (5-FU) and gemcitabine; hormonal antineoplastics, such as goserelin, leuprolide, and tamoxifen; natural antineoplastics, such as taxanes (e.g., docetaxel and paclitaxel), aldesleukin, interleukin-2, etoposide (VP- 16), interferon alfa, and tretinoin (ATRA); antibiotic natural antineoplastics, such as bleomycin, dactinomycin,
  • antineoplastic agent may also be used in combination with an antineoplastic agent, even if not considered antineoplastic agents themselves: dactinomycin; daunorubicin HC1; docetaxel; doxorubicin HC1; epoetin alfa; etoposide (VP- 16); ganciclovir sodium; gentamicin sulfate; interferon alfa; leuprolide acetate; meperidine HC1; methadone HC1; ranitidine HC1; vinblastin sulfate; and zidovudine (AZT).
  • fluorouracil has recently been formulated in conjunction with epinephrine and bovine collagen to form a particularly effective combination.
  • reagent 1 may also be expressed from reagent 1 or be administered as protein therapeutics along with the therapeutic comprised of reagent 1, 2 and 3: checkpoint inhibitors that target for example, PD-1 and CTLA-4, interleukins 1 through 37, including mutants and analogues; interferons or cytokines, such as interferons .alpha., .beta., and .gamma.; hormones, such as luteinizing hormone releasing hormone (LHRH) and analogues and, gonadotropin releasing hormone (GnRH); growth factors, such as transforming growth factor-. beta.
  • checkpoint inhibitors that target for example, PD-1 and CTLA-4, interleukins 1 through 37, including mutants and analogues
  • interferons or cytokines such as interferons .alpha., .beta., and .gamma.
  • hormones such as luteinizing hormone releasing hormone (LHRH) and analogues and
  • TGF-.beta. fibroblast growth factor
  • FGF nerve growth factor
  • GRF growth hormone releasing factor
  • EGF epidermal growth factor
  • FGFHF fibroblast growth factor homologous factor
  • HGF hepatocyte growth factor
  • IGF insulin growth factor
  • tumor necrosis factor-.alpha. & .beta. tumor necrosis factor-.alpha. & .beta.
  • IIF-2 invasion inhibiting factor-2
  • BMP 1-7 bone morphogenetic proteins 1-7
  • somatostatin thymosin-. alpha.- 1 ; . gamma. -globulin; superoxide dismutase (SOD); complement factors; anti-angiogenesis factors; antigenic materials; and pro-drugs.
  • Chemotherapeutic agents for use with the compositions and methods of treatment described herein include, but are not limited to alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine ; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly crypto
  • compositions and methods of the invention can comprise or include the use of other biologically active substances, including therapeutic drugs or pro-drugs, for example, other chemotherapeutic agents or antigens useful for cancer vaccine applications.
  • chemotherapeutic agents and/or additional active agents may be used. These include, without limitation, such forms as uncharged molecules, molecular complexes, salts, ethers, esters, amides, and the like, which are biologically active.
  • the agents and substances described herein can be delivered to the subject in a pharmaceutically suitable, or acceptable or biologically compatible carrier.
  • pharmaceutically suitable/ acceptable or biologically compatible mean suitable for pharmaceutical use (for example, sufficient safety margin and if appropriate, sufficient efficacy for the stated purpose), particularly as used in the compositions and methods of this invention.
  • compositions described herein may be delivered by any suitable route of administration for treating the cancer, including orally, nasally, transmucosally, ocularly, rectally, intravaginally, parenterally, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intra-articular, intra- stemal, intra- synovial, intra-hepatic, through an inhalation spray, or other modes of delivery known in the art.
  • VEGFR1 The nucleic acid sequence for VEGFR1 (FLT1) can be found at “fins related receptor tyrosine kinase 1 [ Homo sapiens (human) ]” Gene ID: 2321, www.ncbi.nlm.nih.gov/gene, updated on 7-Dec-2019
  • the nucleic acid sequence for VEGFR2 (KDR) can be found at “kinase insert domain receptor [ Homo sapiens (human)]” Gene ID: 3791, www.ncbi.nlm.nih.gov/gene, updated on 7-Dec-2019
  • the nucleic acid sequence for VEGFR3 (FLT4) can be found at “fins related receptor tyrosine kinase 4 [ Homo sapiens (human) ]” Gene ID: 2324, www.ncbi.nlm.nih.gov/gene, updated on 7- Dec-2019, the nucleic acid sequence for HER2/
  • T Cell Beta Chain [ Homo sapiens (human) ]
  • a gene editing technique to alter genomic DNA sequences within tumors can be used so that the protein product is targeted to the cell surface membrane as described in this invention (see e.g., US Patent 8,697, 359 for a description of CRISPR techniques).
  • Delivery of CRISPR/CAS9 with a sgRNAs to a tumor cell and other sequences necessary to effect the desired change following cleavage of the targeted DNA can be provided by use of Reagent 1.
  • a number of vectors have been used in humans and these can be used to express the genetic material in different cell types. Such methods are known to those of skill in the art. Means to target expression of the receptors that targetmers have affinity for are also known to those of skill.
  • genetically engineered vectors exist that direct the expression of receptors that facilitate targetmer delivery into a particular cell type. An example is given in Figure 3. This construct also includes a reporter gene that allows efficiency of transduction of the virus into the tumor to be quantitated.
  • the above approaches can be combined with other cancer therapies including immune-modulators such as checkpoint inhibitor ligands for PD-1 CTLA-4, ICOS, OX40;; lymphokines, cytokines and their receptors and strategies designed to increase major and minor histocompatibility antigens. Additionally, the methods of the present invention can be combined with other standard cancer therapies such as radiotherapy and chemotherapy. [ 0088 ] The above approaches can be used to deliver nucleic acids for the repair genetic defects underlying Mendelian disease and others that are the product of a DNA mutation or a change in genomic sequence that occurs only in somatic cells.
  • the targeted therapeutic composition described herein comprises three parts:
  • Reagent 1 that carries nucleic acid sequences necessary to produce the therapeutic effect
  • Reagent 2 that is a molecule that binds to the minor groove of a double-stranded nucleic acid
  • Reagent 3 that is a ligand specific for a receptor(s) on the targeted cell.
  • the composition comprises a means of attaching Reagent 2 to Reagent 3 — either through a covalent chemical bond or by a non-covalent association.
  • the attachment may or may -not involve a linker.
  • the targetmer is synthesized using solid state peptide synthesis to create distamycin and related minor groove binders derived from pyrrole— imidazole (Py— Im) polyamides by solid phase synthesis, most commonly using Fmoc chemistry to create compounds of different length and composition [23].
  • heteroaryl ring compounds that are comprised of 5 to 14 atoms, including thienyl, furyl, pyrrolyl, indolyl, pyrimidinyl, isoxazolyl, purinyl, imidazolyl, pyridyl, pyrazolyl, quinolyl, pyrazinyl and their derivatives in order to optimize the properties of the minor-groove binding compound for nucleic acid delivery.
  • the incorporation of histidine may make certain properties of the compound pH dependent to enable release of reagent 1 from endosomes [42] while the incorporation of tryptophan may increase its hydrophobicity to promote incorporation into membranes [43].
  • Reactive groups for covalent coupling of Reagent 2 to Reagent 3 are introduced during chemical synthesis of reagent 2.
  • scheme 1 they are introduced to the N-terminus at the end of synthesis using standard FMOC chemistry that enables the introduction of azido-homoalanine, propargyl glycine, formyl glycine, thiol [44] as well as a primary amine (Fig. 4).
  • scheme 2 they can be introduced by attachment of a modified amino acid bearing 3 -amino-propylazide or propargyl functional groups as first described by Barany and modified by Ten Brink et al. [45, 46] (Fig. 6).
  • Reagent 3 bearing the appropriate reactive group can then be coupled to the modified peptide. Most commonly the coupling will be through an amino group using a NHS ester or by reductive amidation of an aldehyde group. Alternatively coupling via click chemistry involving either an azide or alkyne group will be the preferred embodiment (Fig. 7 A and B).
  • FIG. 8 An example of a folate receptor a receptor targetmer with distamycin A linked through a polyethylene glycol linker to folate is shown in FIG. 8.
  • Example 5 Reagent 3 : Antibody
  • Reagent 3 may be an antigen-specific antibody, a lectin specific for abnormal glycoproteins on a cancer cell (for example N -gly colylneuraminic acid by the B submit of the subtilase cytotoxin) antibodies specific for viral proteins (for example, antibodies derived from individuals immune to a particular virus) a nucleic acid, modified or not, that can bind sequence specifically to another nucleic acid.
  • a lectin specific for abnormal glycoproteins on a cancer cell for example N -gly colylneuraminic acid by the B submit of the subtilase cytotoxin
  • viral proteins for example, antibodies derived from individuals immune to a particular virus
  • Reagent 1 can be combined with different targetmers.
  • the targetmer specific for a cell surface receptor may be used in conjunction with other targetmers composed of a nucleic sequence or drug that inhibits Toll-like Receptor (TLR) activation of immune responses.
  • TLR Toll-like Receptor
  • one of the targetmer may incorporate telomere sequences to inhibit TLR9, or GpC sequences to inhibit TLR9 [57], or small molecules to inhibit TLR4 [58, 59],
  • RNA nanoparticles harboring annexin A2 aptamer can target ovarian cancer for tumor-specific doxorubicin delivery, Nanomedicine 13(3) (2017) 1183-1193.
  • RNA aptamer blockade of osteopontin inhibits growth and metastasis of MDA-MB231 breast cancer cells, Mol Ther 17(1) (2009) 153-61.

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Abstract

Sont ici décrits des procédés et des compositions pour l'administration spécifique d'agents thérapeutiques codés par acide nucléique à l'aide de polymères-cibles Les polymères-cibles comprennent une sonde de ligand du petit sillon (MGB) liée à un ligand pour un récepteur qui est exprimé sur la cellule cible.
PCT/US2020/063610 2019-12-13 2020-12-07 Méthodes et compositions pour l'administration ciblée d'agents thérapeutiques par l'acide nucléique Ceased WO2021118927A1 (fr)

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