WO2024028785A1 - Nanoparticules lipidiques multicomposants ayant une fusogénicité cellulaire élevée pour l'administration d'acides nucléiques et procédé de préparation associé - Google Patents

Nanoparticules lipidiques multicomposants ayant une fusogénicité cellulaire élevée pour l'administration d'acides nucléiques et procédé de préparation associé Download PDF

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WO2024028785A1
WO2024028785A1 PCT/IB2023/057827 IB2023057827W WO2024028785A1 WO 2024028785 A1 WO2024028785 A1 WO 2024028785A1 IB 2023057827 W IB2023057827 W IB 2023057827W WO 2024028785 A1 WO2024028785 A1 WO 2024028785A1
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dna
lipidic
nucleic acid
nanoparticles
vaccine
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PCT/IB2023/057827
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Giulio CARACCIOLO
Daniela POZZI
Erica QUAGLIARINI
Serena RENZI
Luca DIGIACOMO
Augusto Amici
Cristina Marchini
Lishan CUI
Junbiao WANG
Francesco CARDARELLI
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Università Degli Studi Di Roma - La Sapienza
Unicam - Università Degli Studi Di Camerino
Scuola Normale Superiore
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    • 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
    • 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
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • 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
    • A61K9/5192—Processes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics

Definitions

  • the present invention relates to the process of preparing multicomponent lipidic nanoparticles (LNP) with high cellular fusogenicity for the delivery of nucleic acids and in particular for DNA vaccination.
  • the invention also relates to the thus obtained nanoparticles.
  • the lipidic nanoparticles of the invention are obtained by combining cationic lipids and ionizable lipids in a specific ratio to each other to be administered both subcutaneously (intradermally or intramuscularly) and systemically with fewer side effects than the lipidic nanoparticles known in the art.
  • TAP 1 ,2-Dioleoyl-3-trimethylammonium propane is a cationic surfactant capable of forming stable cationic liposomes in solution which adsorb negatively charged organic compounds, such as e.g. the DNA. CAS Registry Number 113669-21 -9
  • - DPPC dipalmitoylphosphatidylcholine is a phospholipid (and a lecithin) consisting of two Ci6 palmitic acid groups attached to a phosphatidylcholine head group CAS Registry Number® 63-89-8
  • DOPE dioleoylphosphatidylethanolamine is a glycerophospholipid, CAS Registry Number® 2462-63-7
  • DOPG 1 ,2-Dioleoyl-sn-glycero-3-phosphoglycerol is a glycerophospholipid
  • DOPC 1 ,2-dioleoyl-sn-glycero-3-phosphocholine is a phospholipid
  • PEG or polyethylene glycol is a polymer prepared by polymerization of ethylene oxide and PEGylation is the process of covalent bonding of the PEG polymer chain to another molecule, usually a drug or a therapeutic protein.
  • Vaccination represents one of the most effective strategies to prevent large-scale mortality caused by infectious diseases (Rappuoli, Rino, et al. "Vaccines for the twenty-first century society.” Nature reviews immunology 1 1.12 (201 1 ): 865-872). In the 1970s, the World Health Organization (WHO), thanks to an effective vaccine, was able to declare the complete eradication of smallpox worldwide. Similarly, civilization is now making extensive use of vaccination as the main tool to deal with SARS-CoV-2 infection.
  • WHO World Health Organization
  • vaccines have been made from attenuated or inactivated bacteria or viruses.
  • these types of vaccines have been shown to be effective in inducing a robust immune response, but sometimes they can cause adverse reactions due to their toxicity.
  • the development of a live attenuated vaccine can be problematic when the pathogen replicates poorly in cell culture or when there is a risk of potential reversion to a virulent strain (e.g., HIV, rabies) (Baba, Timothy W., et al "Live attenuated, multiply deleted simian immunodeficiency virus causes AIDS in infant and adult macaques.” Nature medicine 5.2 (1999): 194- 203.
  • a specific pathogen e.g., hepatitis B surface antigen.
  • new generation vaccines such as recombinant proteinbased vaccines, synthetic peptide-based vaccines, lipid-based vaccines/antigens and polysaccharide-based vaccines, are potentially safer than traditional vaccines.
  • vaccines are often poorly immunogenic, in part because components with adjuvant activity are very often removed in the purification or synthesis process.
  • a further limitation is due to the reduced or lack of ability of some new generation vaccines to induce a cell-mediated immune response (CMI), following a response of cytotoxic T lymphocytes (CTL).
  • CRI cell-mediated immune response
  • CTL cytotoxic T lymphocytes
  • a recombinant protein-based vaccine elicits primarily a humoral immune response (i.e., IgG and IgE production) and should be administered with appropriate adjuvants. Therefore, the discovery made in the early 1990s that a vaccine based on plasmid DNA (pDNA) can induce, in the absence of adjuvants, both humoral and cellular immune responses was greeted with great enthusiasm by the scientific community operating in the field of vaccinology and immunology (Wolff, Jon A., et al. "Direct gene transfer into mouse muscle in vivo.” Science 247.4949 (1990): 1465-1468.).
  • pDNA plasmid DNA
  • DNA vaccine including 'genetic vaccine', 'polynucleotide vaccine' and 'nucleic acid vaccine'.
  • WHO chose the term 'nucleic acid vaccine' to comprise both DNA vaccines and RNA vaccines.
  • the DNA vaccine was used starting from the discovery that the administration of pDNA to an animal caused the expression of a foreign protein encoded by the plasmid.
  • this type of vaccine is based on pDNA of bacterial origin wherein the gene coding for the antigen capable of eliciting humoral and cell-mediated immunity is inserted, by means of recombinant DNA methods, by stimulating the production of neutralizing antibodies and cytotoxic T lymphocytes.
  • DNA vaccines reserve several advantages over current vaccines based on pathogens (i.e. attenuated and inactivated viral vaccines) or proteic vaccines (Khan, Kishwar Hayat. "DNA vaccines: roles against diseases. "Germs” 3.1 (2013): 26.). For instance, they require simple and rapid manufacturing processes, can be easily handled safely and modified through engineering of the pDNA which contains the information about the antigen that will be produced. Furthermore, the thermostability of DNA vaccines solves the complications related to the maintenance of the cold chain which instead is essential for less stable vaccines (e.g., mRNA vaccines) to avoid their inactivation during supply (Fuller, Deborah H., and Peter Berglund.
  • non-viral vectors are based on chemical or physical methods.
  • electroporation is based on the application of a controlled electric field capable of creating transient pores in cell membranes and allowing the entry of DNA into the cytoplasm of the target cell.
  • electroporation can induce nonspecific targeting, cause cell damage or lysis, and even lead to pDNA degradation (Liu, Feng, and Leaf Huang. "A syringe electrode device for simultaneous injection of DNA and electrotransfer.” Molecular Therapy 5.3 (2002): 323-328.).
  • nanocarriers protect the load of nucleic acids; (ii) they are highly versatile in terms of surface properties and therefore can be designed to cross cell barriers; (iii) in contact with the acidic pH of the endosomes they destabilize and release the genetic load into the cytoplasm of the cell.
  • microfluidically prepared Lipidic nanoparticles or LNPs are one of the most advanced delivery platforms.
  • microfluidic preparation increases the reproducibility of the experimental data and allows the comparison between experimental data obtained from different research groups.
  • the physico-chemical properties of microfluidically produced LNPs can be controlled by finely modulating the microfluidic operating parameters including the flow ratio, and total flow rate of the lipid solution in the organic step and of the aqueous solution containing the pDNA.
  • This technology is known (Lee et al., 201 1 - Microfluidic Mixing: A Review - Int. J. Mol. Sci. 12(5): pg. 3263-3287) and has recently been used for the synthesis of LNPs, loaded with short interfering RNA (siRNA) and messenger RNA (mRNA), which have received regulatory approval.
  • siRNA short interfering RNA
  • mRNA messenger RNA
  • Onpattro® an "orphan drug” based on LNPs approved in 2018 by the Food and Drug Administration (FDA) for the treatment of polyneuropathy caused by hereditary transthyretin amyloidosis (hATTR).
  • FDA Food and Drug Administration
  • hATTR hereditary transthyretin amyloidosis
  • GivlaariTM was approved for the treatment of acute hepatic porphyria (AHP).
  • LNPs LNPs mRNA vaccines developed to deal with the SARS-Cov2 emergency and which received conditional approval from the FDA and the European Medicines Agency (EMA) in December 2020: mRNA -1273/ SpikeVax (Moderna) and BNT162b2/Comirnaty (BioNTech/Pfizer).
  • EMA European Medicines Agency
  • Lipid-based nanoparticles consisting of a mixture of 4 lipids - a cationic or ionizable lipid, a phospholipid, a sterol and a PEGylated lipid - have been developed for the non-immunogenic delivery of siRNA and mRNA in the liver after systemic administration. Yet, despite the clinical successes of lipid-based gene delivery systems for mRNA and siRNA transport, to date there are no clinically approved pDNA encapsulating LNPs. Historically, LNPs technology was designed to encapsulate small RNAs (siRNAs).
  • siRNAs ensures that in the microfluidic channels where mixing with lipids occurs, they are completely coated with lipid and that many lipid-coated siRNA molecules form small (100-150 nm) and homogeneous nanoparticles. These characteristics, small and homogeneous dimensions, are not very dependent on the lipid composition and make the technique versatile and easy to apply. Only later the technique was extended to the encapsulation of mRNAs whose size is about 100 times greater than that of siRNAs. This makes the encapsulation less "precise” and, consequently, the particle size less controllable and more dependent on the lipid species involved.
  • An efficient vector for DNA vaccination must exhibit the following distinguishing characteristics: (i) controlled size and surface charge; (ii) high DNA encapsulation efficiency; (iii) effective cellular internalization; (iv) cytoplasmic release of nucleic acid content; (v) ability to stimulate antibody production in vivo.
  • LNPs represent the system of choice. Compared to liposomes, which have represented (for a long time) the most widespread class of nanocarriers worldwide, LNPs have peculiar chemical-physical properties such as the nanometric size ( ⁇ 150 nm), the homogeneity of the colloidal dispersion, the cationic zeta potential and a high encapsulating efficiency. These properties represent optimal conditions for the cellular internalization of the complexes and the delivery of the nucleotide load. However, it should be noted that the efficiency and cytotoxicity of LNPs are modulated by several factors. Among these, the synthesis parameters and their modulation (Roces, Carla B., et al.
  • the invention aims to solve the technical problems highlighted by the prior art.
  • LNP lipidic nanoparticle
  • nucleic acid preferably DNA
  • phospholipids preferably selected from dipalmitoyl phosphatidylcholine (DPPC) and dioleoyl phosphatidylethanolamine (DOPE) and mixtures thereof;
  • DPPC dipalmitoyl phosphatidylcholine
  • DOPE dioleoyl phosphatidylethanolamine
  • sterols preferably cholesterol, DC-Chol-3B-[N-(N',N'- dimethylaminoethane)-carbamoyl]cholesterol and its salts, preferably hydrochloride, and mixtures thereof;
  • PEG lipids preferably DOPE functionalized with polyethylene glycol (DOPE-PEG2k); and characterized by comprising: one or more cationic surfactants, preferably 1 ,2-Dioleoyl-3- trimethylammonium propane (DOTAP or 18:1TAP).
  • DOPE-PEG2k DOPE functionalized with polyethylene glycol
  • cationic surfactants preferably 1 ,2-Dioleoyl-3- trimethylammonium propane (DOTAP or 18:1TAP).
  • Another object of the invention is to provide the lipidic nanoparticles as defined herein, obtained by microfluidics.
  • Another object of the invention is to provide lipidic nanoparticles (LNP) comprising one or more nucleic acid molecules wherein one or more nucleic acid molecules are encapsulated in a lipidic structure comprising:
  • DOTAP 1,2-Dioleoyl-3-trimethylammonium propane
  • the nucleic acid is preferably a nucleic acid encoding the extracellular and transmembrane domains of the human tyrosine kinase receptor HER2.
  • Still another object of the invention is to provide a method for the preparation of an LNP according to the present invention, comprising:
  • a first alcoholic composition comprising said phospholipids, sterols, PEG lipids and cationic surfactants in a suitable alcoholic solvent, preferably ethanol;
  • Still another object of the invention is to provide a method for the preparation of a lipidic nanoparticle containing one or more molecules of a nucleic acid wherein the one or more molecules of nucleic acid is encapsulated in a lipidic structure. This method comprises the steps of:
  • a further object of the invention is to provide a pharmaceutical composition or a vaccine comprising one or more lipidic nanoparticles as defined herein and an acceptable pharmaceutical carrier.
  • Still another object of the invention are lipidic nanoparticles, pharmaceutical compositions or vaccines as defined herein for use in human or veterinary medicine
  • Another object of the present invention is a composition comprising said nanoparticles for use in the treatment of cancer or infectious diseases caused by viruses or bacteria.
  • Figure 1 Scheme of the preparation process of lipidic nanoparticles. Microfluidic mixing is performed using the Ignite® platform (Precision NanoSystems Inc., Vancouver, BC, Canada).
  • Figure 2. (a) HEK-293 cells were transiently transfected with the pVAX-hECTM- loaded LNP15 nanoparticle formulation and analyzed under a fluorescence microscope 48 hours post-transfection. Trastuzumab was used as primary antibody and a secondary fluorescent antibody (Alexa-fluor 488) to detect membrane expression of HER2 antigen. Magnification 40x (10 pm bar), (b) FACS analysis of the anti-HER2 antibody response induced by LNP15 hECTM vaccine compared to LNP pVAX control and LNP13 hECTM vaccine. Serums from immunized mice were tested on SK-BR-3 target cells. Data are shown as MFI ⁇ SEM; t test **** P ⁇ 0.0001 LNP hECTM vs. LNP pVAX (control).
  • Figure 3 Confocal image of the LNP15 formulation (green channel) and lysosomes (red channel) after treatment of HEK-293 cells at 37°C (a,b) and 4°C (c,d). Quantification of the degree of colocalization in terms of Pearson coefficient (e) and Manders coefficient (f). Boxplots represent the distribution of measured values on a data set consisting of 10 images per class.
  • Figure 4 Percentage of release of pDNA content from LNP15 incubated with increasing amounts of DOPG.
  • FIG. 1 x, 2x, 5x refer to the dose, i.e. 1 , 2 or 5 micrograms DNA per well.
  • the d-LNP condition is the same LNP15 formulation but surface decorated with DNA (in a molar ratio of lipid molecules to DNA bases between 0.33 and 1 ). Thus, d-LNP internally encapsulates the same amount of DNA as LNP15, and is then surface decorated with additional DNA.
  • nucleic acid delivery systems e.g., small non coding RNA, mRNA, pDNA, CRISPR/CAS,
  • nucleic acid in the context of the invention means a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA), more preferably mRNA.
  • Nucleic acids comprise according to the invention genomic DNA, plasmid DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules.
  • DNA and RNA comprise double-stranded DNA and RNA, single-stranded DNA and RNA, isolated DNA and RNA as partially purified DNA and RNA, essentially pure DNA and RNA, synthetic DNA and RNA, recombinant DNA and RNA, as well as modified DNA and RNA that differs from naturally occurring DNA and RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides.
  • the invention relates to delivery systems and plasmid DNA to be used for making vaccines.
  • the delivery systems of the invention are not liposomes, which are pre-constituted lipid particles wherein the active ingredients (such as, for instance, nucleic acids) are encapsulated.
  • the invention is aimed at creating delivery systems that are not obtained with a process wherein the lipids aggregate into vesicles and then encapsulate the Nucleic acid.
  • the process of the invention provides for the lipids to be dissolved in an organic phase, typically in ethanol. In this way the lipids remain single molecules in the organic phase and do not assemble to form vesicular structures.
  • fusogenicity refers to the ability to produce fusion between lipid membranes, i.e. the process by which two separate lipid bilayers fuse to become one.
  • fuseogen refers to the peculiar ability to induce membrane fusion.
  • the inventors have therefore designed and prepared by microfluidics a library of different formulations of LNPs encapsulating nucleic acids, preferably plasmid DNA, which differ in the number and species of lipidic molecules, for pegylation (i.e., the surface coating with PEG), for the total flow rate of the microfluidic mixing and for the weight ratio (Rw) of lipids to DNA, size and polydispersity index (pdl) or polydispersity.
  • pegylation i.e., the surface coating with PEG
  • Rw weight ratio
  • pdl polydispersity index
  • microfluidic mixing is to achieve rapid and thorough mixing of multiple samples (i.e., lipid phase and nucleic acid phase) in a microscale device.
  • microfluidic mixing devices can be used, such as those described in Lee et al., 2011 .
  • a particularly suitable microfluidic mixing device according to the present invention is that of the Ignite® platform, by Precision NanoSystems Inc., Vancouver, BC, Canada.
  • Table 1 shows a library of 16 different formulations of plasmid DNA encapsulating LNPs.
  • Pegylated formulations have a 1.5% pegylated lipidic molar fraction. Of the 16 formulations, only 8, those in gray, had the size and polydispersity index (pdl) adequate to move on to the next stage of in vitro validation.
  • This formulation i.e., LNP15 in Table 1
  • lipidic structure consisting of 1 ,2-dioleoyl-3-trimethylammonium- propane (DOTAP) (about 14.9%), 3B-[N-(N',N'-dimethylaminoethane)- carbamoyl]cholesterol hydrochloride (DC-Chol) (about 32%), cholesterol (about 8.2), dioleoylphosphatidylethanolamine (DOPE) (about 38.1%) and polyethylene glycol functionalized DOPE 2k (DOPE-PEG 2K, commercial product 1 ,2-dioleoyl- sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000, molecular weight 2785 a.m., purchased from Avanti Polar Lipids, Alabaste, AL, USA), (about 6.8%), DO PC (0%).
  • DOTAP 1,2-dioleoyl-3-trimethylammonium- propane
  • DC-Chol 3B-[N-(N',
  • DOTAP dioleoyl-3-trimethylammonium- propane
  • DC-Chol DC-Chol
  • DOPE dioleoylphosphatid
  • lipid concentration 12.5 mM lipid concentration.
  • pVAX-hECTM (about 5000 bp), which encodes for the extracellular and transmembrane domain of the human receptor with tyrosine kinase activity HER2, was inserted as a plasmid in the LNP object of the present invention.
  • the pVAX-hECTM is an anticancer DNA vaccine developed against the HER2 oncoprotein (as described in Quaglino E. et al. “A better immune reaction to Erbb-2 tumors is elicited in mice by DNA vaccines encoding rat/human chimeric proteins” Cancer Res.70(7) (2010) 2604-12).
  • the plasmid DNA of interest can be replaced with plasmid DNA encoding for luciferase, or for proteins (viral or oncogenic antigens) suitable for the induction of specific immune responses for use in vaccines against pathogenic microorganisms or tumours.
  • a per se known microfluidic mixing aid Ignite®, Precision NanoSystems Inc., Vancouver, BC, Canada
  • Y-junction Ignite® NxGen cartridge
  • the LNPs remain cationic and this allows for better interaction with the keratinocytes.
  • step iv) is to mix the nanoparticles loaded with the plasmid with an additional amount of plasmid. This occurs through an incubation of between 30 minutes and 3 hours between the nanoparticles and a solution of plasmid DNA dissolved in water at an even concentration (between 0.05 mg/ml and 0.15 mg/ml), double (between 0.15 mg/ml and 0.25 mg/ml), or triple (between 0.25 mg/ml and 0.35 mg/ml) compared to the concentration of encapsulated DNA. Nanoparticles and coating DNA are incubated in equal volumes.
  • the DNA can impart a less cationic or even anionic charge to the nanoparticles obtained in step iv. Since most plasma proteins are negatively charged at physiological pH, a weakly cationic or anionic particle becomes much less abundantly covered with plasma proteins in general. In particular, it is covered much less by opsonins with the advantage that the nanoparticles become invisible to the immune system. This is a distinct advantage when the application of the particles requires that they be administered systemically. The expert in the field, by combining his knowledge with the indications provided in the present description, is able to obtain the results in accordance with the invention.
  • the LNPs of the invention are obtained with cationic lipids which allow them to better interact with keratinocytes, resulting particularly suitable for subcutaneous/intramuscular administration as they show fewer side effects than similar nanoparticles obtained with ionizable lipids (such as for instance 1 ,2- dilinoleyl-N,N-dimethyl-3-aminopropane (DLinDMA), (4-hydroxybutyl)azanediyl- bis(hexane-6,1 -diyl)bis(2-hexyldecanoate) (ALC-0315), heptadecan-9-yl- 8-((2- hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102).
  • ionizable lipids such as for instance 1 ,2- dilinoleyl-N,N-dimethyl-3-aminopropane (
  • LNP15 was identified as the best system for in vitro transfection and was further validated at three different doses (i.e., 1 , 2, and 5pg DNA/well) in HEK-293 (CRL- 1573), HaCaT (PCS -200-01 1 ) and CaSki (CRM-CRL-1550) cell lines.
  • the transfection efficiency of LNP15 in HEK-293, HaCaT and CaSki cell lines is shown in Fig. 5a-c, while the corresponding cell viability is shown in Fig. 5d-f.
  • the same figure also shows the transfection efficiency and cell viability of the negative controls (i.e. the untreated cells) and the positive controls (i.e. the treatments with Lipofectamine TM 3000).
  • the LNP15 formulation has a size of about 120 nm, a zeta potential of about 20 mV, and an encapsulation efficiency of about 85% (Table 3).
  • HEK-293 cells were transiently transfected with the pVAX-hECTM-loaded nanoparticle formulation LNP15, which encodes the extracellular and transmembrane domain of the HER2 receptor, and analyzed under a fluorescence microscope 48 hours after transfection.
  • HER2 is a tumor antigen overexpressed in several cancers, including breast cancer, and is an excellent target for immunotherapies.
  • T rastuzumab as primary antibody and a secondary fluorescent antibody were used to detect membrane expression of HER2 antigen.
  • the membrane of HEK-293 cells transfected with pVAX-hECTM appears green labeled, indicating that the HER2 antigen has been correctly expressed and is localized, as expected, in the plasma membrane.
  • the obtained results demonstrate that LNP15 nanoparticles efficiently delivered the pVAX-hECTM DNA vaccine in HEK-293 cells.
  • Efficacy was then evaluated in vivo through the quantification of antibodies induced by vaccination of C57BL/6 mice with pVAX-hECTM encapsulated in the LNP15 formulation (LNP15 hECTM) and in the LNP13 formulation compared with those induced by the empty pVAX vector (without insert - Invitrogen by ThermoFisher Scientific) encapsulated in the same two formulations (control, LNP-pVAX).
  • the C57BL/6 strain was chosen among the other inbred mouse strains since it is the most commonly used in immunological research (Rosshart SP et al. "Laboratory mice born to wild mice have natural microbiota and model human immune responses”. Science.
  • mice/experimental group were vaccinated by intramuscular injection (tibial muscle) of 100 microliters/mouse of a suspension of LNP-pDNA (1 mg/mL), 50 microliters/tibial muscle.
  • the vaccination protocol prescribed a first injection, followed by a booster after 21 days. Blood samples at baseline, the day before the first vaccination, and 15 days after the last vaccination were taken to allow the evaluation of the antibody titre.
  • Quantitative analysis of antibody response against HER2 protein was analyzed by flow cytometry (BD FACSCalibur) using serums from vaccinated mice and targeting human HER2-positive breast cancer cells, SK-BR-3 (HTB-30). The results are summarized in Fig. 2b.
  • the graph shows the mean fluorescence intensity of SK-BR-3 cells after incubation with serums from vaccinated animals, followed by incubation with an anti-mouse secondary antibody conjugated with Alexa-Fluor-488 fluorochrome detected by FACS. Data were acquired and analyzed with BD cells quest pro software (6.0.2) and Flowjo 8.7, respectively.
  • the in vivo experiment clearly demonstrates that the pVAX-hECTM DNA vaccine is strongly immunogenic when it is administered encapsulated in the LNP15 lipidic formulation object of the present invention.
  • the effective production of antibodies in vivo can be attributed to the fusogenicity of the LNP15 object of the present invention with cell membranes. This peculiar property of the developed formulation was demonstrated by confocal fluorescence microscopy experiments.
  • a fluorescent variant of the formulation was prepared by incorporating a fluorescent lipid (Texas RedTM 1 ,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine Triethylammonium Salt) (Texas RedTM DHPE) in the proportion of 1 fluorescent lipid molecule into the LNPs per 1000 non-fluorescent lipid molecules.
  • the fluorescent LNPs were administered to HEK-293 cells.
  • the intracellular distribution of the nanoparticles was evaluated after 3 hours of incubation at 37°C.
  • Lystracker DeepRed a commercial fluorescent marker specific for these compartments at acidic pH.
  • the two fluorescent markers can be distinguished by laser excitation at suitable wavelengths (e.g.: 543 nm for lipid-TexasRed and 633 nm for Lysotracker Deep Red) and collection of the fluorescence in 2 distinct channels (detector).
  • suitable wavelengths e.g.: 543 nm for lipid-TexasRed and 633 nm for Lysotracker Deep Red
  • collection of the fluorescence in 2 distinct channels detector.
  • the Pearson coefficient measures the intensity correlation between the green and red channels, while the Manders coefficient quantifies the portion of the green pixel that colocalizes with the red ones. Both are commonly used statistical parameters to measure the correlation of fluorescence signals in confocal fluorescence images and, therefore, quantify the colocalization of fluorescent structures. Both colocalization parameters indicate that the cellular degradation process is largely inhibited at 4°C, i.e. when plasma membrane fusion mechanisms are the dominant internalization processes of LNP15.
  • LNP 1 x, LNP 2x, LNP 5x refer to dosage, i.e. increasing amounts of LNP incorporating 1 , 2 or 5 micrograms DNA per well.
  • the d-LNP condition is the same LNP15 formulation but also superficially decorated with DNA (therefore the DNA is distributed between the inside and the surface of the nanoparticles).
  • the DNA plasmid-containing nanoparticles exhibit a biomolecular coating obtained by exposure to biological fluids such as plasma, dermal interstitial fluid, and lung lining fluid.
  • biological fluids such as plasma, dermal interstitial fluid, and lung lining fluid.
  • the present invention provides a pharmaceutical composition comprising one or more LNPs as defined above.
  • compositions containing the LNPs of the invention are obtained by combining the LNPs with a pharmaceutically acceptable carrier and are particularly suitable as a vaccine.
  • the invention also provides a vaccine comprising one or more LNPs according to the present invention.
  • the vaccine of the invention can be used to induce an immune response, in particular an immune response against an antigen or disease-associated cells expressing an antigen associated with a disease, such as an immune response against a cancer or virus.
  • the vaccine may be used for the prophylactic and/or therapeutic treatment of a disease involving a disease-associated antigen or cells expressing a disease-associated antigen, such as cancer, viral or bacterial diseases.
  • compositions and vaccines and LNPs of the present invention are specifically intended for both subcutaneous (intradermal or intramuscular) and systemic (intravenous) administration, nevertheless the easier subcutaneous route of administration is preferred and the pharmaceutical and vaccine compositions of the invention are particularly suitable for this type of administration, including pediatric ones.
  • the present invention also provides the pharmaceutical compositions and LNP vaccines according to the present invention for use in human or veterinary medicine.
  • the human being can be of any age, as the compositions and vaccines are suitable for the administration of adults, the elderly, children and infants.
  • compositions and LNP vaccines according to the present invention for human or veterinary medicine as mentioned above.
  • the invention provides a method for the prophylaxis and treatment of human and veterinary diseases by administering the LNPs, pharmaceutical compositions and vaccines according to the present invention to a subject in need thereof.
  • the present invention further provides the use of a LNP, a pharmaceutical composition or a vaccine according to the present invention for the immunogenic delivery of said one or more nucleic acid molecules.
  • compositions and vaccine of LNP of the present invention are highly useful in the treatment of various human and veterinary diseases.
  • the present invention provides the pharmaceutical compositions and vaccines of LNPs of the present invention for use in the treatment of cancer or infectious diseases and bacterial conditions.
  • the plasmid DNA (pmirGLO) was purchased from Promega (USA).
  • the 16 formulations of lipidic nanoparticles encapsulating plasmid DNA were produced via the microfluidic mixing technique using the NanoAssemblr® Benchtop instrument from Precision NanoSystems Inc. (Vancouver, BC, Canada).
  • the microfluidic chip consists of a channel with a Y-junction and staggered herringbone grooves.
  • the lipids were individually dissolved in 100% ethanol and subsequently mixed to obtain the following weight ratios reported in Table 1 and a final lipid concentration equal to 12.5 mM.
  • the respective mother solutions i.e. the ethanolic lipidic solution and the acetate buffer solution containing pDNA
  • the respective mother solutions i.e. the ethanolic lipidic solution and the acetate buffer solution containing pDNA
  • the synthesis took place at room temperature.
  • the eluted solution was collected in a test tube and subjected to a dialysis process to completely remove the residual ethanol (25% vol).
  • the dialysis process involved the use of Slide-A-Lazer dialysis cassettes (0.5-3 mL, MWCO 3 kDa, Thermo Scientific, Rockford, USA).
  • the solution obtained by microfluidic mixing was taken with a syringe and injected into the dialysis cassette.
  • the solution containing the pDNA encapsulating lipidic nanoparticles was taken with a syringe from the dialysis cassette, collected in a vial and placed at 4°C.
  • the pDNA encapsulating lipidic nanoparticles can be kept at 4°C for a period ranging from a few hours up to 15 days.
  • lipidic nanoparticles were incubated with the pDNA at room temperature for 30 min to 3 hours.
  • a fixed volume of lipidic nanoparticle solution (generally between 100 pl and 2 ml) was incubated with an equal volume of aqueous solution containing pDNA, respectively at the same concentration (between 0.05 mg/ml and 0.15 mg/ml), double (between 0.1 mg/ml and 0.30 mg/ml), or triple (between 0.15 mg/ml and 0.45 mg/ml), compared to the lipidic nanoparticle solution, or concentration between 0.15 and 0.25 mg/ml or between 0.25 and 0.35 mg/ml.
  • the obtained pDNA-coated lipidic nanoparticles were placed at 4°C.
  • the storage of the nanoparticles at 4°C can last for a period ranging from a few minutes up to 2-3 hours.
  • the Quant-iT Pico-Green dsDNA assay kit (ThermoFisher Scientific, Waltham, Massachusetts) was used to measure the encapsulation efficiency of lipidic nanoparticles.
  • the lipidic nanoparticles were diluted 150-fold in a 10 mM Tris-HCI, 1 mM EDTA buffer (TE buffer) at pH 7.5 for a total volume of 200 pl. From this batch of sample, a 100 pL volume was incubated with an equal volume of TE buffer solution containing Quant-iTTM PicoGreenTM fluorophore selective for double-stranded DNA, to quantify pDNA not encapsulated in lipidic nanoparticles.
  • the remaining 100 pl of sample was incubated with 100 pl of Quant-iTTM PicoGreenTM and 2 pl of Triton X-100 surfactant (i.e. 1 % of the total volume).
  • the Triton X-100 lyses the lipid membranes and induces the release of the pDNA encapsulated in the nanoparticles allowing the detection of the total pDNA or the sum of the encapsulated and non-encapsulated ones.
  • incubation took place in the wells of Sigma-Aldrich Corning® 96 Well Solid Polystyrene Microplate cell plates for between 5 and 10 min, at room temperature and protected from light.
  • the samples were excited at 475 nm and the fluorescence emission intensity was measured in the wavelength range 500 to 550 nm using the Glomax Discover System spectrofluorimeter (Promega, Madison, Wl, USA). The intensity of the fluorescence emission was then plotted against the pDNA concentration.
  • the pDNA concentration was estimated using a calibration curve obtained from pDNA measurements at several known concentrations.
  • the encapsulation efficiency (EE%) was determined according to the following equation:
  • the size and surface charge of the lipidic nanoparticles were measured by elastic light scattering experiments (with Zetasizer Nano ZS90, Malvern, UK), and microelectrophoresis (with Zetasizer Nano ZS90, Malvern, UK), respectively. The measurements were made by diluting the sample 1 :100 with distilled water and the results were reported as the average ⁇ standard deviation of three experimental replicates.
  • lipidic nanoparticles The biological validation of lipidic nanoparticles was studied through in vitro cell transfection experiments on human embryonic kidney cell line (HEK-293), human cervical cancer cell line (Caski), and immortalized human keratinocytes (HaCaT).
  • HEK-293 human embryonic kidney cell line
  • Caski human cervical cancer cell line
  • HaCaT immortalized human keratinocytes
  • the cell lines were treated with lipidic nanoparticles encapsulating plasmid DNA (pmirGLO) coding for luciferase (in the figure indicated as LNP) with a concentration of pmirGLO equal to 0.1 mg/ml and with the lipidic nanoparticles covered by a pmirGLO envelope at the incubation condition ratio Encapsulated pmirGLO : External pmirGLO equal to 1 :1 w/w (shown as d-LNP). Untreated cells were used as a negative control.
  • pmirGLO lipidic nanoparticles encapsulating plasmid DNA
  • LNP lipidic nanoparticles coding for luciferase
  • HEK-293 and HaCaT were cultured with DMEM cell medium, while CaSki with RPMI cell medium, both mediums were supplemented with 10% fetal bovine serum (FBS) and 1 % penicillin-streptomycin.
  • FBS fetal bovine serum
  • 40'000 cells per well were seeded in 24-well cell plates.
  • three 400 pl solutions of Optimem (Life Technologies, USA) containing respectively 10, 20 and 50 pl of LNP were separately added to each well (i.e. the conditions in the figure indicated as LNP 1 x, LNP 2x and 5x) and a solution with 400 pl of Optimem containing 20 pl of d-LNP (indicated in the figure as d-LNP 1 x).
  • the supernatant (with a volume between 70 and 80 pl) was collected in a new tube and a volume equal to 30 pl was collected and placed in three separate wells (10 pl per well) of a clear cell culture plate with 96 wells. This operation was repeated on a 96-well white cell plate.
  • the white cell culture plate containing the samples was inserted into the Glomax luminescence detection instrument and luciferase expression was measured with the luciferase assay, the Luciferase Assay System (Promega, USA).
  • 100 pl of luciferase substrate was automatically added by the instrument to each well containing 10 pl of sample to measure the relative light unit (RLU).
  • the assay based on bicinchoninic acid (BCA), i.e.
  • the BCA Assay Protein Kit (Thermo Fisher Scientific, USA) was used to evaluate the protein content of the samples. To each well of the clear cell culture plate were added 200 pl of a solution of BCA and left to incubate for 30 min at 37°C. After 30 min, the absorbance of each sample, directly proportional to the protein concentration, was measured at 560 nm by means of the Glomax. The amount of protein in milligrams for each sample was calculated from a calibration curve obtained from a known concentration of bovine serum albumin (BSA). In conclusion, the transfection efficiency (TE) was expressed as relative light units (RLU), per milligram of protein.
  • RLU relative light units
  • Human embryonic kidney-293 (HEK-293) and SK-BR-3 (HER2-positive human breast cancer cell line) cell lines were obtained from the American Type Culture Collection (Rockville, MD) and maintained in Dulbecco's Modified Essential Medium (DMEM, Gibco, Life Technologies) with 10% fetal bovine serum (FBS, Gibco, Life T echnologies) and 1 % penicillin-streptomycin (Gibco, Life T echnologies), at 37°C in a humidified atmosphere and with 5% CO2.
  • DMEM Dulbecco's Modified Essential Medium
  • FBS fetal bovine serum
  • penicillin-streptomycin Gibco, Life T echnologies
  • Plasmid preparation (DNA vaccine)
  • the DH5alpha bacterial strain was transformed with the plasmids pVAX or pVAX- hECTM (encoding the extracellular and transmembrane domain of the HER2 oncoprotein) and was grown in Luria-Bertani medium supplemented with kanamycin.
  • HEK-293 cells were seeded in a 24-well plate (2x10 5 cells/well). The next day, when they had reached 70-90% confluency, they were transiently transfected with 1 microgram of the plasmid pVAX-hECTM encapsulated in NLP15. Two days after transfection, cells were fixed for 10 minutes with 4% paraformaldehyde in phosphate buffered saline (PBS) (Sigma, St. Louis, MO), left in blocking buffer (PBS-10% bovine serum albumin (BSA; Sigma)) for 20 minutes and incubated for one hour at 37°C with trastuzumab (primary anti-human HER2 antibody; 1 :50).
  • PBS phosphate buffered saline
  • BSA bovine serum albumin
  • the inbred C57BL/6 mouse strain (mus musculus) was chosen to carry out the vaccination experiments, since it is the most commonly used in immunological research.
  • mice 8-10 weeks were divided into 4 groups (4 animals/group), according to the experimental scheme:
  • Vaccination was performed by intramuscular injection (tibial muscle), bilaterally inoculating 50 micrograms of N PL-encapsulated plasmid DNA (100 microliter/mouse of a suspension of LNP-pDNA (1 mg/mL), 50 microliter/tibial muscle). Two doses of vaccine were administered 21 days apart. From all experimental groups, both controls and treated, blood samples were taken at baseline, the day before the first vaccination, and 15 days after the last vaccination to allow the evaluation of the antibody titre. The blood sampling method is retro- orbital, performed during isoflurane anesthesia.
  • Quantitative analysis of the antibody response against human HER2 protein was analyzed by flow cytometry using SK-BR-3 cells as a target.
  • a suspension of 1 x10 6 SK-BR-3 cells was dispensed into single tubes, washed in staining buffer (0.05% NaNa, 2% FBS in PBS) and incubated for one hour at 4°C with the immuno serums (1 :20). After three washes, the cells were incubated for one hour at 4°C with the anti-mouse IgG Alexa Fluor 488 secondary antibody. Finally, after three washes, the cells were resuspended in PBS and analyzed by FACS (BD FACSCalibur). The Cells Quest pro version (6.0.2) program was used for data acquisition, while the Flowjo software version 8.7 was used for their analysis. Confocal microscopy experiments
  • lysosomes were labeled with Lystracker DeepRed (a commercial fluorescent marker specific for these compartments at acidic pH) while the nanoparticles were labeled with fluorescent lipid (Texas RedTM).
  • Lystracker DeepRed a commercial fluorescent marker specific for these compartments at acidic pH
  • fluorescent lipid Texas RedTM
  • the intracellular localization was studied by fluorescence confocal microscopy, using a Zeiss Airyscan 800 inverted confocal microscope equipped with a 60x objective and a GASP detector.
  • the two fluorescent markers can be distinguished by laser excitation at suitable wavelengths (e.g.: 543 nm for lipid-TexasRed and 633 nm for Lysotracker Deep Red) and collection of the fluorescence in 2 distinct channels, 550-600 nm the first and above 650 nm the second.
  • suitable wavelengths e.g.: 543 nm for lipid-TexasRed and 633 nm for Lysotracker Deep Red

Abstract

La présente invention concerne le procédé de préparation de nanoparticules lipidiques (NPL) multicomposants ayant une fusogénicité cellulaire élevée pour l'administration d'acides nucléiques et en particulier pour la vaccination par ADN. L'invention concerne également les nanoparticules ainsi obtenues. En particulier, les nanoparticules lipidiques de l'invention sont obtenues par combinaison de lipides cationiques et de lipides ionisables dans un rapport spécifique les uns par rapport aux autres pour être administrés à la fois par voie sous-cutanée (intradermique ou intramusculaire) et systémiquement avec moins d'effets secondaires que les nanoparticules lipidiques connues dans l'état de la technique.
PCT/IB2023/057827 2022-08-02 2023-08-02 Nanoparticules lipidiques multicomposants ayant une fusogénicité cellulaire élevée pour l'administration d'acides nucléiques et procédé de préparation associé WO2024028785A1 (fr)

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Citations (2)

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Publication number Priority date Publication date Assignee Title
US20190307689A1 (en) * 2013-03-15 2019-10-10 The University Of British Columbia Lipid nanoparticles for transfection and related methods
US20210403950A1 (en) * 2018-11-13 2021-12-30 Oncorus, Inc. Encapsulated polynucleotides and methods of use

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190307689A1 (en) * 2013-03-15 2019-10-10 The University Of British Columbia Lipid nanoparticles for transfection and related methods
US20210403950A1 (en) * 2018-11-13 2021-12-30 Oncorus, Inc. Encapsulated polynucleotides and methods of use

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Title
DUGUAY BRETT A ET AL: "Lipofection of plasmid DNA into human mast cell lines using lipid nanoparticles generated by microfluidic mixing", vol. 104, no. 3, 28 August 2018 (2018-08-28), GB, pages 587 - 596, XP093025471, ISSN: 0741-5400, Retrieved from the Internet <URL:https://academic.oup.com/jleukbio/article-pdf/104/3/587/48171484/jlb10145.pdf> DOI: 10.1002/JLB.3TA0517-192R *
ROCES CARLA B. ET AL: "Manufacturing Considerations for the Development of Lipid Nanoparticles Using Microfluidics", PHARMACEUTICS, vol. 12, no. 11, 15 November 2020 (2020-11-15), pages 1 - 19, XP055972635, DOI: 10.3390/pharmaceutics12111095 *

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