[go: up one dir, main page]

WO2025171027A1 - Npl phlip® pour la délivrance intracellulaire ciblée d'agents thérapeutiques à base d'acides nucléiques - Google Patents

Npl phlip® pour la délivrance intracellulaire ciblée d'agents thérapeutiques à base d'acides nucléiques

Info

Publication number
WO2025171027A1
WO2025171027A1 PCT/US2025/014619 US2025014619W WO2025171027A1 WO 2025171027 A1 WO2025171027 A1 WO 2025171027A1 US 2025014619 W US2025014619 W US 2025014619W WO 2025171027 A1 WO2025171027 A1 WO 2025171027A1
Authority
WO
WIPO (PCT)
Prior art keywords
bis
amino
ethyl
phlip
composition
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/014619
Other languages
English (en)
Inventor
Yana K. Reshetnyak
Oleg A. Andreev
Donald M. Engelman
Umberto ROMEO
Eleonora FRATUS
Mathieu Giraud
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Phlip Inc
Yale University
Rhode Island University
Original Assignee
Phlip Inc
Yale University
Rhode Island University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Phlip Inc, Yale University, Rhode Island University filed Critical Phlip Inc
Publication of WO2025171027A1 publication Critical patent/WO2025171027A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • 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
    • 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
    • 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
    • A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • PHLIP®-LNP FOR TARGETED INTRACELLULAR DELIVERY OF NUCLEIC ACID THERAPEUTICS CROSS REFERENCE TO A RELATED APPLICATION
  • LNP lipid nanoparticle
  • pHLIP ® -LNP nucleic acids
  • RNA- and gene-based therapeutics are rapidly emerging due to progress in the synthesis of stable nucleic acids.
  • the progress in lipid formulations e.g., lipid nanoparticle (“LNP”)
  • LNP lipid nanoparticle
  • key challenges remain, including, but not limited to, i) targeted delivery of nucleic acid therapeutics to diseased tissues other than the liver, and ii) enhanced cytoplasmic delivery of nucleic acid payloads into specific cells.
  • the one or more nucleic acid payloads are encapsulated within the LNP to which the pHLIP ® peptide is covalently bonded or linked with a non-cleavable linker.
  • the one or more pHLIP ® peptides comprise the following sequence: X n Y m ; Y m X n ; X n Y m X j ; Y m X n Y i ; Y m X n Y i X j ; X n Y m X j Y i ; Y m X n Y i X j Y l ; XnYmXjYiXl; YmXnYiXjYlXh; XnYmXjYiXhYg; YmXnYiXjYlXhYg; XnYmXjYiXhYgXf; (XY)n; (YX)n; (XY)nYm; (YX)nYm; (XY)nXm; (YX)nXm; (YX)nXm; (YX)nX
  • the one or more pHLIP ® peptides of Formula (I) or pHLIP ® - LNPs described herein comprises a sequence comprising the sequence AXDNDNDNPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO.226), wherein “X” is a functional group for conjugation purposes, selected from lysine (Lys), cysteine (Cys), Azido-containing amino acid or other modified amino acids.
  • the naturally occurring analogs of cholesterol are selected from vitamin D3, vitamin D2, calcipotriol, stigmasterol, ⁇ - sitosterol, sitostanol, betulin, lupeol, ursolic acid, oleanolic acid, or chemical alkyl derivatives (stigmastanol, campesterol, fucosterol, brassicasterol, ergosterol, dehydroergosterol), and combinations thereof.
  • the LNP of the pHLIP ® -LNPs described herein additionally comprises about 5% to about 25% of other lipid ingredients.
  • the LNP additionally comprises PEG-lipids. In other aspects, the LNP additionally comprises PEG-free polymers (e.g. pSar-lipids). [0017] In some aspects, the compositions of Formula (I) described herein further encompasses one or more nucleic acid payloads (pHLIP ® -LNPs). In the pHLIP ® -LNPs, the one or more nucleic acid payloads are encapsulated within the LNP to which the pHLIP ® peptide is covalently bonded or linked with a non-cleavable linker.
  • pHLIP ® -LNPs the one or more nucleic acid payloads are encapsulated within the LNP to which the pHLIP ® peptide is covalently bonded or linked with a non-cleavable linker.
  • the nucleic acid payloads of the pHLIP ® -LNPs are RNA and/or DNA molecules.
  • the nucleic acid payloads of the pHLIP ® -LNPs are selected mRNA (messenger RNA), miRNA (micro RNA), saRNA (self-Amplyfing RNA), saRNA (small activating RNA), siRNA (small interfering RNA), snRNA (small nuclear RNA), snoRNA (small nucleolar RNA), piRNA (piwi-interacting RNA), crRNA (CRISPR RNA), gRNA (guide RNA), tracrRNA (trans-activating CRISPR RNA), ncRNA (non-coding RNA), rRNA (ribosomal RNA), tRNA (transfer RNA), regions of lncRNA (long non-coding RNA), and other RNAs from protein-nucleic acid complexes such as signal recognition particles, aptamers
  • the pHLIP ® peptide and LNP of Formula (I) are linked by a covalent bond or a non-cleavable linker. In some aspects, the pHLIP ® peptide and LNP of Formula (I) described herein are linked by a covalent bond. [0020] In some aspects, the N-terminus of the pHLIP ® peptide is covalently bonded to the LNP of Formula (I). [0021] In some aspects, pharmaceutical compositions comprising any one of the compositions described herein and one or more pharmaceutically acceptable excipients are provided herein.
  • provided herein are methods of delivering a nucleic acid payload to a cell and/or a diseased tissue comprising administering to the cell and/or diseased tissue a composition or a pharmaceutical composition described herein.
  • methods of treating diseases in a subject in need thereof comprising administering a composition or a pharmaceutical composition described herein to the subject are provided herein.
  • methods of prevention diseases by vaccination in a subject in need thereof comprising administering a composition or a pharmaceutical composition described herein to the subject are provided.
  • Figure 1 shows a schematic presentation of the pHLIP ® -LNP structure.
  • Figure 2 shows a DSPE-PEG-pHLIP ® structure using a pHLIP ® peptide having the sequence ACDNDNDNPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO.227).
  • Figure 3 shows an exemplary reaction pathway for the synthesis of DMG- PEG2000.
  • Figure 4 shows a HPLC chromatogram indicating the presence of the pHLIP ® - DSPE-PEG2000 conjugate after the completion of coupling reaction.
  • Figure 5 shows a HPLC chromatogram indicating the presence of the pHLIP ® - DSPE-PEG2000 conjugate in a final sample after purification.
  • compositions comprising multiple pHLIP ® peptides linked to an LNP described herein.
  • the LNP comprises one or more nucleic acids.
  • an advantage of the disclosed compositions comprising pHLIP ® peptides linked to an LNP and pHLIP ® -LNPs is targeted delivery of, e.g., nucleic acid therapeutics to diseased tissue(s) and enhanced delivery of the nucleic acid payloads to the cells of the diseased tissue(s).
  • compositions comprising pHLIP ® peptides linked to an LNP, as described herein, provide stable and homogeneous formulations without diminishing encapsulation efficiency of nucleic acid payloads.
  • a lipid nanoparticle (“LNP”) is a liposome-like structure composed primarily of cationic lipids, along with other lipid ingredients. The LNP can encapsulate nucleic acids.
  • a pHLIP ® peptide (also known as a pH Low Insertion Peptide) is a linear, water- soluble membrane peptide that interacts weakly with a cell membrane at neutral pH, without insertion into the lipid bilayer, but inserts into the cell membrane and forms a stable transmembrane alpha-helix at a slightly acidic pH ( ⁇ 7.0).
  • a pHLIP ® peptide senses acidity or low pH and targets it to the surfaces of cancer and stromal cells, activated macrophages, and some other immune cells in tumors, inflamed tissues, or any naturally or artificially created acidic tissues.
  • pHLIP ® peptides When pHLIP ® peptides are exposed to a low pH environment at the surfaces of acidic cells, its negatively charged residues are protonated to become neutral, which enhances the pHLIP ® hydrophobicity and triggers insertion of the peptides into the plasma membranes of targeted cells. Multiple insertion events of multiple pHLIP ® peptides into cellular membrane promotes membrane fusion and release of an encapsulated payload in the cytoplasm.
  • pHLIP ® peptides By binding pHLIP ® peptides, or a pHLIP ® equivalent, to an LNP encapsulated with nucleic acids, pHLIP ® peptides will target the LNP(s) to acidic diseased tissues or artificially created acidic tissues and promote intracellular delivery of nucleic acid payloads into the cytosols of targeted cells. Delivering therapeutic nucleic acid payloads encapsulated within the pHLIP ® -LNPs described herein activates and/or blocks synthesis of specific proteins for treatment and/or prevention of diseases. [0033] As used herein, “effective” when referring to an amount of a composition described herein, refers to the quantity of the composition that is sufficient to yield a desired response.
  • the amount of cargo, e.g., nucleic acid in the LNP yields a desired response, e.g., therapeutic effect in targeted tissue, without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this disclosure.
  • a subject administered the composition of Formula (I) or pHLIP ® -LNPs described herein is a mammal. In certain aspects, the subject is a mammal.
  • the mammal is a rodent (e.g., a mouse or a rat), a primate (e.g., a chimpanzee, a gorilla, a monkey, a gibbon, a baboon), a cow, a camel, a dog, a cat, a horse, a llama, a sheep, a goat, or a pig.
  • the subject is a human.
  • the transitional term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.
  • transitional phrase “consisting of” excludes any element, step, or ingredient not specified in the claim.
  • the transitional phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention.
  • the singular forms “a,” “an,” and “the” include the plural reference unless the context clearly dictates otherwise.
  • a reference to “a disease,” “a disease state”, or “a nucleic acid” is a reference to one or more such embodiments, and includes equivalents thereof known to those skilled in the art and so forth.
  • treating encompasses, e.g., inhibition, regression, or stasis of the progression of a disorder. Treating also encompasses the prevention or amelioration of any symptom or symptoms of the disorder.
  • inhibition of disease progression or a disease complication in a subject means preventing or reducing the disease progression and/or disease complication in the subject.
  • a “pharmaceutically acceptable” carrier or excipient refers to a carrier or excipient that is suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio.
  • a “pHLIP ® -LNP” refers to one or more nucleic acid payloads encapsulated within a lipid nanoparticle to which one or more peptides (e.g., pHLIP ® peptide) is covalently bonded or linked with a non-cleavable linker.
  • the pHLIP ® peptide can be linked (i.e., covalently bonded or linked with a non-cleavable linker) to the LNP either before or after the one or more nucleic acid payloads is encapsulated.
  • the one or more nucleic acid payloads is encompassed in the LNP prior to linking the pHLIP ® peptide to the LNP.
  • Each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiments. Thus, all combinations of the various elements described herein are within the scope of the invention. [0041] Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below.
  • the LNP of the pHLIP ® -LNPs described herein comprises from about 10% to about 60% of one or more cationic or ionizable lipids.
  • the ionizable lipids have a pK of about 5.5 to about 9.5.
  • the ionizable lipids are propargylamine-based lipids.
  • the LNP of Formula (I) or the pHLIP ® -LNPs described herein comprises from about 10% to about 60% of cholesterol and/or its naturally occurring analogs.
  • the LNP of Formula (I) or the pHLIP ® -LNPs described herein comprises cholesterol.
  • the cholesterol is from plant origin (e.g., BotaniChol ® ).
  • the LNP of Formula (I) or pHLIP ® -LNPs described herein comprise at least one naturally occurring analog of cholesterol.
  • one or more pHLIP ® peptides of Formula (I) or pHLIP ® -LNPs described herein comprises a sequence consisting of the sequence AXDNDNDNPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO.226), wherein “X” is a functional group for conjugation purposes, selected from lysine (Lys), cysteine (Cys), Azido-containing amino acid or other modified amino acids.
  • one or more pHLIP ® peptides of Formula (I) or pHLIP ® -LNPs described herein comprises a sequence comprising the sequence ACDNDNDNPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO.227).
  • one or more pHLIP ® peptides of Formula (I) or pHLIP ® -LNPs described herein comprises a sequence consisting of the sequence ACDNDNDNPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO.227).
  • Other exemplary pHLIP ® peptides that can be included in the pHLIP ® -LNPs described herein are shown in the Tables below. [0051] The sequences are provided N-terminus to C-terminus. Table 1 Table 2 * Ac means Acetylated N-terminus; and Am means Amidated C-terminus Table 3.
  • Coded and exemplary non-coded amino acids including L-isomers, D-isomers, alpha-isomers, beta-isomers, glycol-, and methyl-modifications.
  • Table 4. Non-limiting examples of protonatable residues and their substitutions including L-isomers, D- isomers, alpha-isomers, and beta-isomers. Table 5. Examples of coded amino acid substitutions Table 6.
  • Each non-polar residue could be replaced by its coded amino acid substitution from Table 5, and/or non-coded amino acid substitutions from Table 3.
  • pHLIP ® sequences A cysteine, a lysine, an azido- modified amino acid, or an alkynyl modified amino acid can be incorporated at the N- terminal (first 6 residues) or C-terminal (last 6 residues) parts of the peptides for conjugation with a cargo, and a linker.
  • one or more pHLIP ® peptides of Formula (I) or pHLIP ® -LNPs described herein comprises a sequence selected from the group consisting of any one of SEQ ID NOs.1-225.
  • 1 to 10,000 pHLIP ® peptides are independently covalently bonded and/or linked with a non-cleavable linker to a single LNP.
  • Methods of Treatment [0057] Methods of using the compositions described herein for treating a disease or disorder in a subject in need thereof comprising administering an effective amount of a composition or a pharmaceutical composition described herein are also provided.
  • the subject is a human.
  • the disease or disorder comprises one or more of liver disease, cancer, heart disease, ocular disease, and muscular dystrophy.
  • administering comprises contacting a cell with a composition comprising one or more pHLIP ® peptides of Formula (I) or pHLIP ® -LNPs described herein or a pharmaceutical composition comprising one or more pHLIP ® peptides of Formula (I) or pHLIP ® -LNPs described herein.
  • the contacting occurs in vitro.
  • the contacting occurs in vivo.
  • the contacting occurs ex vivo.
  • Step 5 Direct coupling of the alcohol DMG-PEG2000-OH with an unprotected maleimide was challenging. To overcome the issues of instability the maleimide function was protected by a Diels-Alder reaction with furane. This furane protected maleimide was very stable and was isolated in high purity by standard chromatographic purification.
  • Step 6 To deprotect the furane protected product a retro-Diels-Alder reaction was performed in toluene at 90°C and the product was obtained in very high yield without degradation.
  • the mol% lipid composition of pHLIP ® -LNP was the following: 50 mol% of ionizable lipids SM-102, 10 mol% of DSPC natural phospholipids, 38.5 mol% of Botanichol (cholesterol) and 1.5 mol% of pegylated lipids among them were 1.35 mol% of DSPE-PEG2000 and 0.15 mol% of DSPE-PEG2000-Mal for conjugation with pHLIP ® .
  • LNP formulation was prepared using the Automated Nanoparticle System by Dolomite Microfluidics. All lipids were dissolved in ethanol as an organic phase, with a final lipid concentration of 12.5 mM.
  • composition of lipids is shown in Table 8.
  • Table 8 Composition of pHLIP ® -LNP formulation [0061] mRNA was solubilized in 100 mM acetate buffer (pH 4) to reach a final concentration of 0.12 mg/mL. The aqueous phase and organic phase were injected into sample loops within the system later combined on a microfluidic chip.
  • the Flow Rate Ratio (FRR) was 3:1 and the Total Flow Rate (TFR) was 21 mL/min including an in-line dilution step with 1X PBS pH 7.4. After preparation of the LNP formulations an-offline dilution step (1:4) with Tris buffer 100 mM pH 8.0 was immediately performed to reduce the percentage of ethanol.
  • Reaction parameters are presented in Table 9. Table 9. Reaction parameters [0062] For coating of LNPs with pHLIP ® we used the following sequence: pHLIP ® peptide: ACDNDNDNPWRAYLDLLFPTDTLLLDLLWA (Seq. ID NO 227) [0063] pHLIP ® peptide (1.2 eq) was dissolved in DMSO for conjugation with the surface of LNPs through the coupling of SH group of single Cys residue at the pHLIP ® peptide with the maleimide group at the distal end of DSPE-PEG2000-Mal. Reaction was complete in 5 min.
  • pHLIP ® -LNP was purified by ultracentrifugation in Amicon® Ultra Centrifugal Filter devices (100 kDa MWCO, Millipore at 2000 RCF 4°C) following diafiltration (3 diavolumes) against Tris buffer 100 mM (pH 8.0). The presence of pHLIP ® in pHLIP ® - LNP formulation was confirmed by HPLC ( Figure 5). At the final step, sucrose was added to cryo-preserve samples (the final sucrose concentration in the sample was 10%). [0066] pHLIP ® -LNP formulation was characterized by measuring the amount of encapsulated mRNA, particle size and polydispersity index (PDI).
  • PDI polydispersity index
  • the particle size and PDI were determined by dynamic light scattering (DLS) using a Zetasizer Ultra (Malvern), the data are presented in Table 10.
  • Encapsulated mRNA was measured by a fluorescence-based Quant-iTTM RiboGreen kit (Thermofisher) according to the manufacturer’s protocol. The efficiency of mRNA encapsulation was 77% after off-line dilution and 74% after addition of sucrose.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Nanotechnology (AREA)
  • Immunology (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Dispersion Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Cell Biology (AREA)
  • Optics & Photonics (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne des compositions comprenant plusieurs peptides pHLIP® liés à une nanoparticule lipidique encapsulant un ou plusieurs acides nucléiques (NPL pHLIP®) et des procédés de préparation des NPL pHLIP®.
PCT/US2025/014619 2024-02-05 2025-02-05 Npl phlip® pour la délivrance intracellulaire ciblée d'agents thérapeutiques à base d'acides nucléiques Pending WO2025171027A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202463549959P 2024-02-05 2024-02-05
US63/549,959 2024-02-05

Publications (1)

Publication Number Publication Date
WO2025171027A1 true WO2025171027A1 (fr) 2025-08-14

Family

ID=96700657

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2025/014619 Pending WO2025171027A1 (fr) 2024-02-05 2025-02-05 Npl phlip® pour la délivrance intracellulaire ciblée d'agents thérapeutiques à base d'acides nucléiques

Country Status (1)

Country Link
WO (1) WO2025171027A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180021447A1 (en) * 2012-05-23 2018-01-25 Ohio State Innovation Foundation Lipid Nanoparticle Compositions and Methods of Making and Methods of Using the Same
US20180344655A1 (en) * 2015-05-11 2018-12-06 Yale University Functionalized polymeric particles for treatment of gliomas
US20200253872A1 (en) * 2010-08-13 2020-08-13 Rhode Island Council On Postsecondary Education Liposome compositions and methods of use thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200253872A1 (en) * 2010-08-13 2020-08-13 Rhode Island Council On Postsecondary Education Liposome compositions and methods of use thereof
US20180021447A1 (en) * 2012-05-23 2018-01-25 Ohio State Innovation Foundation Lipid Nanoparticle Compositions and Methods of Making and Methods of Using the Same
US20180344655A1 (en) * 2015-05-11 2018-12-06 Yale University Functionalized polymeric particles for treatment of gliomas

Similar Documents

Publication Publication Date Title
JP3907662B2 (ja) 自己構築ポリヌクレオチド送達システム
JP6126072B2 (ja) 遺伝子発現を抑制する治療におけるリポソームによる効率的な送達のプロセスおよび組成物
AU738083B2 (en) Cationic amphiphile/ DNA complexes
US6172049B1 (en) Phosphonic acid-based cationic lipids
US20100240731A1 (en) Lipopeptides for delivery of nucleic acids
US20120015865A1 (en) Novel class of cationic lipids for transporting active agents into cells
US20250213718A1 (en) Lipids for nucleic acid delivery
EP2157096B1 (fr) Molécule amphipathique, agrégat moléculaire comprenant la molécule amphipathique, et utilisation de l'agrégat moléculaire
US20160243246A1 (en) Hyaluronic acid-nucleic acid conjugate and composition for nucleic acid delivery containing the same
PL211710B1 (pl) Zastosowanie lipozomów zawierających związek o wzorze (II) ,jako nośnika do transportu leków lub substancji posiadających aktywność kosmetyczną
US20220378702A1 (en) Peptide-lipid conjugates
US20230355539A1 (en) Lipid-polymer compositions and methods of use
JP2001515060A (ja) 新リポポリアミン、その調製および適用
WO2025171027A1 (fr) Npl phlip® pour la délivrance intracellulaire ciblée d'agents thérapeutiques à base d'acides nucléiques
WO2025034764A1 (fr) Lipides pour l'administration d'acides nucléiques à des cellules eucaryotes
US6331524B1 (en) Organ-specific targeting of cationic amphiphile / DNA complexes for gene therapy
TW202315601A (zh) ApoE胜肽之使用方法
KR20230088412A (ko) 단일 치환된 동종 2가 링커를 포함하는 다중접합체
US20240000966A1 (en) Lipid Compositions For In Vivo Delivery
CN119604312A (zh) 用于体内递送的脂质组合物
WO2025217110A1 (fr) Nanoparticules lipidiques pour l'administration de médicament et leurs procédés de fabrication et d'utilisation
AU2024255972A1 (en) Modified trems, compositions, and related methods thereof
WO2025034802A2 (fr) Agents pour l'administration d'acides nucléiques
KR20240076588A (ko) 핵산 전달용 아미노산-지질 접합 화합물 및 이를 포함하는 지질 나노입자
WO2025200610A1 (fr) Lipide cationique contenant une liaison disulfure et un groupe latéral ionisable et son utilisation

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 25752759

Country of ref document: EP

Kind code of ref document: A1