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WO2025137252A1 - Procédé à haut rendement pour cribler l'efficacité et la cytotoxicité de nanoparticules lipidiques - Google Patents

Procédé à haut rendement pour cribler l'efficacité et la cytotoxicité de nanoparticules lipidiques Download PDF

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Publication number
WO2025137252A1
WO2025137252A1 PCT/US2024/060999 US2024060999W WO2025137252A1 WO 2025137252 A1 WO2025137252 A1 WO 2025137252A1 US 2024060999 W US2024060999 W US 2024060999W WO 2025137252 A1 WO2025137252 A1 WO 2025137252A1
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lnps
synthetic
lnp
synthetic cells
mixture
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Markita Landry
Alison LUI
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University of California Berkeley
University of California San Diego UCSD
Chan Zuckerberg Biohub San Francisco
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University of California Berkeley
University of California San Diego UCSD
Chan Zuckerberg Biohub San Francisco
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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
    • 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

Definitions

  • the present disclosure relates to synthetic cell membranes and their use in screening the toxicity and efficacy of lipid nanoparticle formulations in vitro, in a high throughput format.
  • Lipid nanoparticles have demonstrated great promise in recent years in the delivery of nucleic acid-based therapeutics.
  • FDA United States Food and Drug Administration
  • RNAi first-ever RNA interference
  • ONPATTRO® RNA interference
  • LNPs Cellular uptake of LNPs begins with endocytosis followed by endosomal escape, LNP degradation, and cargo release into the cytosol (Zhang et al. (2014) J Control Release. 174:7-14). LNPs however face several delivery pharmacokinetic and biodistribution barriers, including nonspecific serum protein interactions, rapid clearance, off-target localization (Zhang et al. (2014) J Control Release. 174:7-14). Moreover, the ability of LNP encapsulated cargo to escape early endosomes is critical for efficacy, failure results in residence in late-stage endosomes or lysosomes, where mRNA degradation occurs (Zhang et al. (2014) J Control Release.
  • lipid-based nucleic acid delivery platforms that are undergoing clinical studies or on the market consist of four components: an ionizable lipid, cholesterol, a PEGylated lipid, and a helper phospholipid (e.g., 1 ,2- distearoyl-sn-glycero-3-phosphocholine (DSPC)) (Zhu et al. (2022) Nat Common.
  • a helper phospholipid e.g., 1 ,2- distearoyl-sn-glycero-3-phosphocholine (DSPC)
  • the selection of lead compounds relies on an extensive characterization of the LNP library using cellular models for the in vitro screening followed by the selection of a limited number of formulations for in vivo testing(Akinc et al. (2008) Nature biotechnology. 26, 561-569, Jayaraman, M. et al. (2012) Angewandte Chemie. 51 , 8259-8533, Wang, M. et al (2012) ACS Synthetic Biology 1 (9): 403-407). Yet this paradigm is changing since recent studies have adopted to test the LNP formulations after their synthesis immediately in animal models (Tome et al. (2021) APL Bioeng. 5 (3): 031511).
  • HTS high throughput
  • the present disclosure provides first-pass high throughput screening methods for determining LNP cytotoxicity or efficacious biomolecular cargo endosomal release into the cytosol, thus allowing the prioritization of the most promising LNP formulations for further development or in vivo testing.
  • the disclosure features two complementary fluorescencebased methods and compositions for measuring LNP-induced membrane permeabilization and LNP- membrane fusion, which are proxies for LNP toxicity and efficacy respectively.
  • the disclosure provides a method of measuring membrane permeabilization of a synthetic cell by a lipid nanoparticle (LNP), the method comprising: a) preparing a mixture of LNPs and synthetic cells under conditions that allow contact between the LNPs and the synthetic cells; b) measuring the difference in fluorescence signal intensity between (i) the mixture of LNPs and synthetic cells and (ii) a composition comprising only synthetic cells; wherein the synthetic cells comprise a water-soluble fluorescent dye; and wherein an increase in fluorescence correlates with membrane permeabilization.
  • LNP lipid nanoparticle
  • the synthetic cell is a large unilamellar vesicle (LUV).
  • LUV large unilamellar vesicle
  • the LUV is approximately from 30-400 nm in diameter.
  • the water-soluble fluorescent dye is present in the synthetic cell at a self-quenching concentration.
  • the dye is included in the assay at or above its self-quenching concentration.
  • the increase in fluorescence intensity results from a decrease in self-quenching of the water-soluble fluorescent dye.
  • the water-soluble fluorescent dye is selected from the group consisting of sulforhodamine B, FITC, 5(6)-carboxyfluorescein, and calcein.
  • the water-soluble fluorescent dye is 5(6)-carboxyfluorescein (CF).
  • the fluorescence signal intensity is measured using fluorescence spectroscopy.
  • the preparing in step (a) and/or the measuring in step (b) occurs in a multi-well plate.
  • the LNP comprises a cargo selected from the group consisting of a peptide, a nucleic acid, a small molecule, and a protein or combinations of the same.
  • the LNP comprises a ribonucleic acid, protein, or drug cargo.
  • the ribonucleic acid is selected from the group consisting of a small interfering RNA (siRNA), an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a Dicer- substrate RNA (dsRNA), an antisense oligonucleotide (ASO), an RNA aptamer, a small hairpin RNA (shRNA), a messenger RNA (mRNA), and a long non-coding RNA (IncRNA), a guide RNA (gRNA).
  • siRNA small interfering RNA
  • aiRNA asymmetrical interfering RNA
  • miRNA microRNA
  • dsRNA Dicer- substrate RNA
  • ASO antisense oligonucleotide
  • RNA aptamer an RNA aptamer
  • shRNA small hairpin RNA
  • mRNA messenger RNA
  • IncRNA long non-coding RNA
  • gRNA guide RNA
  • the nucleic acid is a messenger RNA (mRNA).
  • mRNA messenger RNA
  • the conditions in step (a) optionally include a range of incubation parameters selected from the group consisting of temperature range, salinity range, ionic strength range, and pH range.
  • the conditions in step (a) include a pH of approximately pH 7.4.
  • the conditions in step (a) include a pH of approximately pH 5.5.
  • the disclosure provides a method measuring fusion of a LNP and a synthetic cell, the method comprising: a.) preparing a mixture of LNPs and synthetic cells under conditions that allow fusion between the LNPs and the synthetic cells; b.) measuring the difference in fluorescence signal intensity between (i) the mixture of LNPs and synthetic cells and (ii) a composition comprising only synthetic cells; wherein the synthetic cells comprise a lipophilic fluorescent dye; and wherein an increase in fluorescence correlates with fusion.
  • the synthetic cell is a large unilamellar vesicle (LLIV).
  • the LLIV is approximately from 30-400 nm in diameter.
  • the lipophilic fluorescent dye is present in the synthetic cell at a self-quenching concentration.
  • the dye is included in the assay at or above its self-quenching concentration.
  • the lipophilic fluorescent dye is R18.
  • the fluorescence signal intensity is measured using fluorescence spectroscopy.
  • the preparing in step (a) and/or the measuring in step (b) occurs in a multi-well plate.
  • the LNP comprises a cargo selected from the group consisting of a peptide, a nucleic acid, a small molecule, and a protein or combinations of the same.
  • the LNP comprises a ribonucleic acid, protein, or drug cargo.
  • the ribonucleic acid is selected from the group consisting of a small interfering RNA (siRNA), an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a Dicer- substrate RNA (dsRNA), an antisense oligonucleotide (ASO), an RNA aptamer, a small hairpin RNA (shRNA), a messenger RNA (mRNA), and a long non-coding RNA (IncRNA), a guide RNA (gRNA).
  • the nucleic acid is a messenger RNA (mRNA).
  • the conditions in step (a) optionally include a range of incubation parameters selected from the group consisting of temperature range, salinity range, ionic strength range, and pH range.
  • the conditions in step (a) include a pH of approximately pH
  • the conditions in step (a) include a pH of approximately pH
  • the disclosure also provides a method of concurrently measuring permeabilization of synthetic cell membranes by an LNP and fusion of synthetic cell membranes with LNPs, the method comprising: a.) preparing a first mixture of LNPs and synthetic cells under conditions that allow contact between the LNPs and the synthetic cells, and preparing a second mixture of LNPs and synthetic cells under conditions that allow fusion between the LNPs and the synthetic cells; b.) measuring the difference in fluorescence signal intensity between (i) the mixture of LNPs and synthetic cells and (ii) a composition comprising only synthetic cells; wherein the synthetic cells in the first mixture comprise a water-soluble fluorescent dye; wherein the synthetic cells in the second mixture comprise a lipophilic fluorescent dye; wherein an increase in fluorescence measured in the first mixture correlates with membrane permeabilization; and wherein an increase in fluorescence measured in the second mixture correlates with membrane fusion.
  • the disclosure provides a method of concurrently measuring (i) membrane permeabilization and (ii) fusion of a synthetic cell by a lipid nanoparticle (LNP), the method comprising: a.) preparing a first mixture of LNPs and synthetic cells under conditions that allow contact between the LNPs and the synthetic cells, and preparing a second mixture of LNPs and synthetic cells under conditions that allow fusion between the LNPs and the synthetic cells; b.) measuring the difference in fluorescence signal intensity between (i) the mixture of LNPs and synthetic cells and (ii) a composition comprising only synthetic cells; wherein the synthetic cells in the first mixture are LUVs and comprise 5(6)-carboxyfluorescein; wherein the synthetic cells in the second mixture are LUVs and comprise R18; wherein an increase in fluorescence measured in the first mixture correlates with membrane permeabilization; wherein an increase in fluorescence measured in the second mixture correlates with fusion.
  • LNP lipid nanoparticle
  • Figure 1 is a simplified, schematic representation of outcomes of the interaction between LNPs encapsulating cargo and target cells;
  • Figure 2 provides schematic representations of a fluorescence-based assay measuring LNP interactions with synthetic cell membranes.
  • Figure 2A is a schematic drawing illustrating the dye leakage assay of the present disclosure, LNP interaction results in LUV membrane permeabilization and leakage of the aqueous CF dye whose fluorescence increases from the dye dequenching. In this assay, the extent of dye leakage (increase in fluorescence) is a proxy for LNP toxicity.
  • Figure 2B is a mock graph exemplifying three possible traces of fluorescence intensity depending on the release rate of CF from LUVs; fast, slow or minimal, after mixing with LNPs.
  • FIG. 2C is a schematic drawing illustrating the membrane lipid-mixing assay of the present disclosure, the assay is based on increased R18 fluorescence due to dye dilution (dequenching) upon fusion of LUVs with fluorescently labeled membranes with non-labeled LNPs, the extent of membrane fusion (fluorescence increase) is a proxy for LNP efficacy.
  • Figure 2D is a mock graph exemplifying three possible R18 fluorescence profiles after mixing R18 labeled LUVs with unlabeled LNPs depending on whether the membranes fuse fast and completely, interact and mix slowly, or they do not fuse at all.
  • FIG 3 is a schematic illustrating an embodiment of a workflow of the fluorescence -based assays of the disclosure.
  • a multi well plate of the user’s preferred format can be loaded with the fluorescent synthetic cells of the present disclosure.
  • An automated fluorescence well plate reader can measure the time-dependent interactions between the LNP drug carrier or vaccine candidates and said synthetic cells. Curves unique to each LNP under a range of conditions (temperature, pH etc.) can be analyzed for their kinetic data and compared to identify LNP formulations with optimal efficacy vs toxicity profiles.
  • Figure 4 shows characteristics of the disclosed LLIVs and LNPs by Dynamic light scattering (DLS).
  • DLS Dynamic light scattering
  • Figure 4A is a schematic drawing illustrating one embodiment of the LUVs (synthetic cells) of the present disclosure.
  • Figure 4B shows chemical structures of POPC and POPG lipids.
  • Figure 4D is a graph of DLS measurements demonstrating that both POPC or POPG liposomes can be extruded in such a way that their size distributions are indistinguishable.
  • Figure 4E is a bar graph showing size change of LUVs by DLS before and after complete solubilization with Triton detergent.
  • Figure 4F is schematic drawing of a cargo loaded LNP.
  • Figure 4G is a bar graph showing the z-average diameter of LNP formulated with 35 mol% of 200oi10, 304oi10, 306o10, or 306oi10 lipidoids in either pH 7.4 or pH 5.5 1X PBS buffer as measured by DLS.
  • Figure 5 provides graphs showing differences in the rate and extent of CF dye leakage and R18 dilution depending on the LUV composition and LNP formulation and concentration.
  • Figure 5A is a graph of CF leakage from POPC LUVs at pH 7.4 after the addition of different LNP formulations (LNPs A-D) at the same concentration.
  • Figure 5B and Figure 5C are graphs of R18 dilution of POPC LUVs by different LNP formulations (LNPs A- D) at the same concentration, at pH 7.4 and pH 5.5 respectively.
  • Figure 5D provides graphs of CF leakage from POPC and POPG LUVs after the addition of different concentrations of LNP made with 35 mol% 200oi10 lipidoid at pH 7.4 and 35°C.
  • Figure 5E provides graphs of R18 dilution from POPC and POPG LUVs after the addition of different concentrations of the same LNP made with 35 mol% 200oi10 lipidoid at 35 C at pH 7.4 (top panel) or pH 5.5 (bottom panel).
  • Figure 6 provides Arrhenius plots for calculating the activation energy for membrane permeabilization at pH 7.4 (Figure 6A) or membrane fusion at pH 5.5 ( Figure 6B) or pH 7.4 ( Figure 6C). Curves are fit to a linear regression by relative LNP concentration (0.01-0.5X). Collapse of these curves to a single line demonstrates high confidence that the fluorescence dequenching has linear dependence on LNP concentration and Arrhenius dependence on temperature.
  • Figure 6A Prominent examples of curve collapse can be seen in Figure 6A for POPC liposomes and LNP with 306o10 lipidoid (3 rd row, 2 nd column), in Figure 6B for POPC and POPG liposomes mixed with LNP with 200oi10 lipidoid (3 rd row), and in Figure 6C for POPC liposomes mixed with LNP with 306oi10, 306o10, and 200oi10 lipidoids (3 rd -5 th row, 2 nd column).
  • Figure 7 provides a comparison of predicted values for each mRNA-carrying LNP and LUV pair.
  • the toxicity score on the vertical axis is derived from results of the neutral pH CF-LUV membrane leakage assay, with lower values implying faster membrane permeation and higher toxicity.
  • the efficacy score on the horizontal axis is derived from results of the endosomal pH R18-LUV membrane fusion assay, with lower values implying faster membrane fusion and higher delivery efficiency. Error bars describe a 95% confidence interval around the predicted value.
  • the present disclosure addresses the aforementioned needs in the art and provides, in various embodiments, a high throughput dual dye-dequenching screening method where increases in the fluorescence signal results from LNP-mediated cell membrane permeabilization or LNP-membrane fusion, which are read outs for LNP cytotoxicity and efficacy respectively.
  • the disclosed methods further enable the acquisition of large quantities of predictive structure-activity data of different LNP formulations.
  • the term “dequenching,” refers to an increase in the fluorescence intensity of fluorophores due to their dilution.
  • the present disclosure also contemplates methods for measuring vaccine efficacy and toxicity profiles in vitro and in a high-throughput manner.
  • LNP components capable of permeabilizing a cell membrane (e.g., causing cellular toxicity) / fusing with a cell membrane (enabling endosomal escape, which thus determines vaccine efficiency) are also provided herein.
  • LNP particle size is one of the most important physical characteristics that affects biodistribution, cellular uptake, efficacy, toxicity, aggregation, and stability of LNP formulations.
  • Light scattering methods such as dynamic light scattering (DLS) and Nanoparticle Tracking Analysis (NTA), are typically used to characterize some physical properties of LNPs.
  • DLS and NTA are label-free techniques that provide information about the size of LNPs, including the mean particle size, shape, diffusion coefficient, and the polydispersity index (PDI) of LNP formulations.
  • DLS measures intensity changes of scattered light which can be converted to a relative particle size distribution
  • NTA calculates the diffusivity of particles from optical videos which can be converted to particle size and concentration. Both techniques can be used to monitor the stability of LNPs over time. By measuring the changes in size distribution, they can indicate potential aggregation, particle growth, or degradation of LNPs. Stability studies using either instrument can assess the effects of storage conditions, temperature, pH, and other factors on the LNP formulation.
  • DLS and NTA are popular and useful screening tools, they do not provide any information on dynamics of LNP interaction, such as membrane permeability or fusion (toxicity or efficacy). Complementary information using different approaches is therefore necessary to completely characterize any LNP sample to support process development and optimization.
  • the present disclosure presents a method of measuring membrane permeabilization of a synthetic cell by a lipid nanoparticle (LNP).
  • LNP lipid nanoparticle
  • Liposomes remain the most widely employed type of synthetic cells because of their close structural similarity to cell membranes. Like cells, liposomes allow functional macromolecules (e.g., proteins, electron carriers) to be incorporated into their selfassembled membranes (Guindani et al. (2022) Angew Chem Int Ed Engl.
  • hydrophobic layer of these cell-like compartments may contain functional biomolecules, such as pore-forming proteins, or be synthetically tuned to be endowed with the desired permeability properties (Guindani et al. (2022) Angew Chem Int Ed Engl. 61 (16):e202110855).
  • a common feature of liposomes is that they can be formed with a wide range of sizes (10 nm-100 pm), depending on how they are prepared (Guindani et al. (2022) Angew Chem Int Ed Engl. 61 (16):e202110855; Walde et al. (2010) Chembiochem. 11 (7):848-65.).
  • small unilamellar vesicles are 10-100 nm in diameter
  • large unilamellar vesicles are 100-1000 nm in diameter
  • giant unilamellar vesicles are 1000nm or larger in diameter.
  • polymers and liposomes can be used to create synthetic nanocompartments with sizes comparable to that of cellular organelles, as well as microcompartments with sizes comparable to whole biological cells (Guindani et al. (2022) Angew Chem Int Ed Engl.
  • Liposomes can be used to characterize LNP-induced membrane permeabilization and the subsequent content leakage. There are various options to characterize induced membrane permeabilization. Most commonly, a fluorescent dye or a dye-quencher pair is entrapped in large unilamellar vesicles (LUVs), a perturbant molecule (such as a peptide) is added, and the rate and extent of release of the entrapped probe is measured (Guha et al.
  • LUVs large unilamellar vesicles
  • Endosomal escape of LNPs relies on endosomal-LNP membrane fusion followed by translocation of nucleic acid cargo into the cytosol (Patel et al. (2020) Nat Common. 11 (1 ):983). Although the mechanism for LNP endosomal escape has not yet been fully understood, it has been suggested that protonation of the ionizable lipid within the LNPs at low endosomal pH promotes electrostatic interactions with the anionic endosomal membrane, which triggers the release of nucleic acid to the cytosol (Patel et al. (2020) Nat Commun. 11 (1 ):983).
  • nanoformulation means a formulation containing nanoparticles as an active ingredient (U.S. Patent Application Publication No. 20090104269).
  • nanoparticle refers to a particle having a diameter, such as an average diameter, from about 10 nm up to but not including about 1 micron, preferably from 100 nm to about 1 micron (International Patent Publication No. WO 2021/184010).
  • the particles can have any shape. Nanoparticles having a spherical shape are generally referred to as "nanospheres" (International Patent Publication No. WO 2021/184010).
  • Lipid nanoparticles may comprise one or more lipid species (International Patent Publication No. WO 2021/184010).
  • a membrane fusion or lipid mixing assay two sets of membranes are mixed - one membrane that is fluorescently-labelled and one membrane that is not fluorescently- labelled - under controlled experimental conditions (Gnopo YMD, Putnam D. Methods. 177:74-79).
  • Exemplary fluorescent probes that are used are either self-quenching (R18) or are a pair of fluorescent acceptor and donor probes that undergo a process known as FRET whereby the acceptor probe quenches the fluorescence of the donor probe when they are in close proximity ( ⁇ 10 nm) (Gnopo YMD, Putnam D. Methods. 177:74-79).
  • synthetic cells or “artificial cells”, or “protocells” mean chemical or biochemical systems based on micro-compartments that enclose a set of reacting molecules, mimicking cell structure and behavior (Stano P. (2018) Life (Basel, Switzerland) 9(1 ):3). Synthetic cells encompass vesicles, membrane-free compartments and colloidosomes. Vesicle models include liposomes, polymersomes, and hybrid lipopolymersomes.
  • the synthetic cell is a liposome.
  • liposome refers to an artificially prepared vesicle composed of a lipid bilayer. A liposome may be classified as a unilamellar vesicle or a multivesicular vesicle (U.S. Patent No. 9457082).
  • the synthetic cell is a large unilamellar vesicle (LUV).
  • LUV large unilamellar vesicle
  • the bilayer membrane includes two layers (or “leaflets”) of lipids; an inner layer and an outer layer (U.S. Patent Application Publication No. 20140079773).
  • the outer leaflet of lipid molecules is oriented with the hydrophilic head portions toward the external aqueous environment and the hydrophobic tails pointed downward toward the interior of the liposome (U.S. Patent Application Publication No. 20140079773).
  • Multilamellar liposomes also referred to as “multilamellar vesicles” or “multiple lamellar vesicles,” include more than one lipid bilayer membrane, which membranes define more than one closed aqueous compartment (U.S. Patent Application Publication No. 20140079773).
  • the membranes are typically concentrically arranged so that the different membranes are separated by aqueous compartments, much like an onion (U.S. Patent Application Publication No. 20140079773).
  • lipid bilayer refers to a membrane made of two layers of lipid molecules (U.S. Patent No. 9457082).
  • the lipid bilayer may have a similar thickness as that of a naturally existing bilayer, such as a cell membrane, a nuclear membrane, and endocytic membranes (U.S. Patent No. 9457082).
  • the lipid bilayer may have a thickness of about 10 nm or less, for example, in a range of about 1 nm to about 9 nm, about 2 nm to about 8 nm, about 2 nm to about 6 nm, about 2 nm to about 4 nm, or about 2.5 nm to about 3.5 nm (U.S.
  • the lipid bilayer is a barrier that keeps ions, proteins, and other molecules in an area, and/or prevents them from diffusing into other areas (U.S. Patent No. 9457082).
  • the “lipid molecules” forming the lipid bilayer may be a molecule including a hydrophilic head and hydrophobic tails (U.S. Patent No. 9457082).
  • the lipid molecule may have 14 to 50 carbon atoms (U.S. Patent No. 9457082). In some embodiments, the lipid molecules have more than 50 carbon atoms.
  • the lipid bilayer may comprise, functionalized cholesterol, synthetic ionizable lipid- like amphiphilic molecules, lipid conjugated to polyethylene glycol (PEG), cholesterol, or any combination thereof (U.S. Patent No. 9457082).
  • the liposomes that are used in the present invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol (U.S. Patent Application Publication No. 20140079773).
  • the selection of lipids is generally guided by consideration of, e.g., liposome size and stability of the liposomes in the bloodstream (U.S. Patent Application Publication No. 20140079773).
  • amphipathic lipids that find use are zwitterionic, anionic, cationic, and neutrally charged lipids (U.S. Patent Application Publication No. 20140079773).
  • zwitterionic amphipathic lipids are phosphatidylcholines, phosphatidylethanolamines, sphingomyelins, etc (U.S. Patent Application Publication No. 20140079773).
  • anionic amphipathic lipids are phosphatidylglycerols, phosphatidylserines, phosphatidylinositols, phosphatidic acids, etc (U.S.
  • cationic amphipathic lipids are diacyl trimethylammonium propanes (DOTAP), N1-[2-((1S)-1-[(3- aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]- benzamide (MVL5), dimethyldioctadecylammonium bromide (DDAB), etc (U.S. Patent Application Publication No. 20140079773).
  • DOTAP diacyl trimethylammonium propanes
  • DDAB dimethyldioctadecylammonium bromide
  • neutral lipids examples include diglycerides, such as diolein, dipalmitolein, and mixed caprylin-caprin; triglycerides, such as triolein, tripalmitolein, trilinolein, tricaprylin, and trilaurin; and combinations thereof (U.S. Patent Application Publication No. 20140079773). Additionally, cholesterol or plant sterols are used in some embodiments, e.g., to make multivesicular liposomes (U.S. Patent Application Publication No. 20140079773).
  • the cationic lipids can be DMTAP, DPTAP, DOTAP, DC-Chol, MoChol or HisChol, or combinations thereof
  • the anionic lipids can be CHEMS5 DGSucc, Cet-P, DMGSucc, DOGSucc, POGSucc, DPGSucc, DG Succ, DMPS, DPPS, DOPS, POPS5 DMPG, DPPG5 DOPG, POPG, DMPA, DPPA5 DOPA, POPA or combinations thereof (International Patent Publication No. WO 2007/064857).
  • the liposomes also include neutral lipids.
  • the neutral lipids include sterols and derivatives thereof (International Patent Publication No. WO 2007/064857).
  • the neutral lipids may also include neutral phospholipids.
  • the phospholipids include phosphatidylcholines and phosphoethanolamines (International Patent Publication No. WO 2007/064857).
  • the phosphatidylcholines are POPC, OPPC, natural or hydrogenated soybean PC, natural or hydrogenated egg PC, DMPC, DPPC, or DOPC and derivatives thereof and the phosphatidylethanolamines are DOPE, DMPE, DPPE or derivatives and combinations thereof (International Patent Publication No. WO 2007/064857).
  • the phosphatidylcholine is POPC, OPPC, soybean PC or egg PC and the phosphatidylethanolamines is DOPE (International Patent Publication No. WO 2007/064857).
  • the CF-Liposomes of the present disclosure comprise 100 mol% POPC. In another embodiment, the CF-Liposomes described herein comprise 100 mol% POPG.
  • the R18-Liposomes described herein comprise 10 mol% R18 and 90 mol% of either POPC or POPG.
  • Sonicating a liposome suspension either by bath or probe sonication produces a progressive size reduction down to small unilamellar vesicles (SUV) less than about 0.05 microns in size (U.S. Patent Application Publication No. 20140079773).
  • Homogenization is another method that relies on shearing energy to fragment large liposomes into smaller ones (U.S. Patent Application Publication No. 20140079773).
  • multilamellar vesicles are recirculated through a standard emulsion homogenizer until selected liposome sizes, typically between about 0.1 and 0.5 microns, are observed (U.S. Patent Application Publication No. 20140079773).
  • the liposome may be a stimulus-sensitive liposome (i.e., sensitive to one or more stimuli), and the stimulus-sensitive liposome may control release of materials that are encapsulated therein (U.S. Patent No. 9457082).
  • “sensitive” to stimuli refers to the ability of a liposome to release its contents in response to exposure to one or more stimuli or the like, or to disintegrate in response to one or more stimuli or the like (U.S. Patent No. 9457082).
  • Suitable cationic lipids include, but are not limited to: 3p-(N-(N',N'- dimethylaminoethane)-carbamoyl)cholesterol hydrochloride (DC-Chol); 1 ,2-dioleoyl-3- trimethylammonium-propane (DOTAP); 1 ,2-dioleoyl-3-dimethylammonium-propane (DODAP); dimethyldioctadecylammonium bromide salt (DDAB); 1 ,2-dilauroyl-sn-glycero-3- ethylphosphocholine chloride (DL-EPC); N-(1-(2,3-dioleyloyx) propyl)-N-N-N-trimethyl ammonium chloride (DOTMA); N-(1-(2,3-dioleyloyx) propyl)-N-N-N-dimethyl ammonium chloride (DOD
  • cationic lipids in available preparations could be used, such as LIPOFECTIN® (from GIBCO/BRL), LIPOFECTAMINE® (from GIBCO/MRL), siPORT NEOFX® (from Applied Biosystems), TRANSFECTAM® (from Promega), and TRANSFECTIN® (from Bio-Rad Laboratories, Inc.) (U.S. Patent No. 10555910).
  • LIPOFECTIN® from GIBCO/BRL
  • LIPOFECTAMINE® from GIBCO/MRL
  • siPORT NEOFX® from Applied Biosystems
  • TRANSFECTAM® from Promega
  • TRANSFECTIN® from Bio-Rad Laboratories, Inc.
  • lipid nanoparticle formulations presently disclosed may include lipidoids.
  • lipidoids refers to lipid-like structures containing multiple secondary and tertiary amine functionalities, prepared from the conjugate addition of alkylamines to acrylates, and which confer highly efficient interaction with anionic nucleic acids, for example siRNA molecules (de Groot et al. (2016) Mol Ther Nucleic Acids. 11 : 159- 169; Akinc et al. (2008) Nat Biotechnol. 26(5) :561 -9; Whitehead et al. (2014) Nat Commun. 27;5:4277).
  • Non-limiting examples of lipidoids include the 200oi10, 304oi10, 306o10, and 306oi10 lipidoids of the present disclosure.
  • Reducible or hydrolysable linkages may be applied to prevent accumulation of the formulation in vivo and subsequent cytotoxicity (U.S. Patent No. 10555910).
  • lipid nanoparticle preparation are suitable to synthesize the lipid nanoparticles of the present disclosure (U.S. Patent No. 10555910).
  • ethanol dilution, freeze-thaw, thin film hydration, sonication, extrusion, high pressure homogenization, detergent dialysis, microfluidization, tangential flow diafiltration, sterile filtration, and/or lyophilization may be utilized (U.S. Patent No. 10555910).
  • several methods may be employed to decrease the size of the lipid nanoparticles (U.S. Patent No. 10555910).
  • homogenization may be conducted on any devices suitable for lipid homogenization such as an Avestin Emulsiflex C5® device (U.S. Patent No. 10555910).
  • Extrusion may be conducted on an Avanti Mini-extruder using a polycarbonate membrane of appropriate pore size (0.05 to 0.4 pm). Multiple particle size reduction cycles may be conducted to minimize size variation within the sample (U.S. Patent No. 10555910).
  • the resultant lipid nanoparticles may then be passed through a size exclusion column containing a Sephadex® G-25 resin or processed by tangential flow diafiltration to purify the lipid nanoparticles.
  • any embodiment of the lipid nanoparticles described herein may further include ethanol in the preparation process (U.S. Patent No. 10555910).
  • ethanol in the preparation process
  • the incorporation of about 30-50% ethanol in lipid nanoparticle formulations destabilizes the lipid bilayer and promotes electrostatic interactions among charged moieties such as cationic lipids with anionic oligonucleotides, such as ASO and siRNA (U.S. Patent No. 10555910).
  • Lipid nanoparticles prepared in high ethanol solution are diluted before administration.
  • ethanol may be removed by dialysis, or diafiltration, which also removes non-encapsulated NA (U.S. Patent No. 10555910).
  • the lipid nanoparticles be sterilized. This may be achieved by passing of the lipid nanoparticles through a 0.2 or 0.22 pm sterile filter with or without pre-filtration (U.S. Patent No. 10555910).
  • lipid nanoparticles Physical characterization of the lipid nanoparticles can be carried through many methods (U.S. Patent No. 10555910). Dynamic light scattering (DLS), atomic force microscopy (AFM), or Nanoparticle Tracking Analysis (NTA) can be used to determine the average diameter and its standard deviation (U.S. Patent No. 10555910). In certain embodiments, it is especially desirable that the lipid nanoparticles have about a 200 nm diameter (U.S. Patent No. 10555910). Zeta potential measurement via zeta potentiometer is useful in determining the relative stability of particles (U.S. Patent No. 10555910).
  • Both dynamic light scattering analysis and zeta potential analysis may be conducted with diluted samples in deionized water or appropriate buffer solution (U.S. Patent No. 10555910).
  • Cryogenic transmission electron microscopy (Cryo-TEM), Transmission electron microscopes (TEM), and scanning electron microscopy (SEM) may be used to determine the detailed morphology of lipid nanoparticles (U.S. Patent No. 10555910).
  • the LNPs typically have a mean diameter of from about 100 nm to about 150 nm, from about 100 nm to about 200 nm, from about 100 nm to about 250 nm, from about 150 nm to about 250 nm, and are substantially non-toxic.
  • nucleic acids when present in the lipid particles of the invention, are resistant in aqueous solution to degradation with a nuclease (International Patent Publication No. WO 2020/219941). Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Patent Publication Nos. 20040142025 and 20070042031 , the disclosures of which are herein incorporated by reference in their entirety for all purposes (International Patent Publication No. WO 2020/219941 ).
  • lipid nanoparticles described herein can be used as platforms for therapeutic delivery of oligonucleotide (ON) therapeutics, such as siRNA, shRNA, miRNA, anti-miR, and antisense ODN.
  • ON oligonucleotide
  • Nucleic acid-based therapeutic agents are highly applicable to the lipid nanoparticle formulations of the present disclosure.
  • nucleic acid-based therapeutic agents include, but are not limited to: antisense DNA or RNA compositions, chimeric DNA:RNA compositions, allozymes, aptamers, ribozyme, decoys and analogs thereof, plasmids and other types of expression vectors, and small nucleic acid molecules, RNAi agents, short interfering nucleic acid (siNA), messenger ribonucleic acid (messenger RNA, mRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules, peptide nucleic acid (PNA), a locked nucleic acid ribonucleotide (LNA), morpholino nucleotide, threose nucleic acid (TNA), glycol nucleic acid (GNA), sisiRNA (small internally segmente
  • modifications to the substituent nucleic acids and/or phosphodiester linker can be made. Such modifications include, but are not limited to: backbone modifications (e.g., phosphothioate linkages); 2' modifications (e.g., 2'-O-methyl substituted bases); zwitterionic modifications (6'-aminohexy modified ODNs); the addition of a lipophilic moiety (e.g., fatty acids, cholesterol, or cholesterol derivatives); and combinations thereof (U.S. Patent No. 10555910).
  • the modified sequences synergize with the lipid nanoparticle formulations disclosed herein (U.S. Patent No. 10555910).
  • addition of a 3'-cholesterol to an ODN supplies stability to a lipid nanoparticle complex by adding lipophilic interaction in a system otherwise solely held together by electrostatic interaction (U.S. Patent No. 10555910).
  • this lipophilic addition promotes cell permeation by localizing the ODN to the outer leaflet of the cell membrane (U.S. Patent No. 10555910).
  • Applying a peptide such as gramicidin or JTS-1 further promotes cell permeation of the formulation due to its fusogenic properties (U.S. Patent No. 10555910).
  • addition of an enzyme such as proteinase K could further aid the ODN in resisting degradation (U.S. Patent No. 10555910).
  • the method comprises preparing a mixture of LNPs and synthetic cells loaded with a water-soluble fluorescent dye under conditions that allow contact between the LNPs and the synthetic cells and determining membrane permeability by assaying for fluorescent dye release from the synthetic cells.
  • the synthetic cells have mean diameters of about 50-100 nm, about 100-170 nm, about 150-170 nm, or about 150-220 nm.
  • contacting means establishing a physical connection between two or more entities.
  • contacting a synthetic cell with a nanoparticle means that the synthetic cell and a nanoparticle are made to share a physical connection.
  • the contacting may involve varied amounts of nanoparticle compositions.
  • one synthetic cell may be contacted by more than one nanoparticle composition.
  • the water-soluble fluorescent dye is selected from the group consisting of sulforhodamine B (SRB), FITC, calcein, 5(6)-carboxyfluorescein (CF) and other fluorescein and rhodamine derivatives.
  • the water-soluble fluorescent dye is CF.
  • CF-LUVs have been described, for example, to demonstrate fluorescent marker uptake into cultured frog retinal cells (Weinstein et al. (1977) Science. 1977; 195(4277) :489- 492.), to study lipoprotein induced permeability (Weinstein JN, Blumenthal R, Klausner RD. (1986) Methods Enzymol.128:657-668) and, in determining cell permeability changes due to inorganic environmental nanoparticles (Moghadam et al. (2012) Langmuir. 28(47):16318- 26).
  • the water-soluble fluorescent dyes are loaded into the synthetic cells at self-quenching concentrations.
  • the self-quenching concentration range of the water-soluble fluorescent dye is between 30-50 mM or between 35-100 mM.
  • membrane permeability is evaluated by following dye leakage from synthetic cells with time.
  • dye release reduces self-quenching (dequenching) and leads to an increase in fluorescence, the magnitude of which is proportional to the extent of leakage of the dye from the lipid vesicle.
  • fluorescence spectroscopy is used to monitor dye leakage.
  • the present disclosure further provides compositions and methods for measuring membrane fusion between a synthetic cell and an LNP.
  • membrane fusion refers to the mechanism by which two apposed (docked) membrane bilayers coalesce in rapid, transient steps that enable the successive merging of the outer and inner leaflets allowing lipid intermixing and subsequent mixing of the two previously separate compartments (Dabral D, Coorssen JR. Int J Biochem Cell Biol. 85:1 -5). “Membrane fusion,” as used herein can also mean mixing of only outer leaflets of two docked bilayers.
  • Endosomal-membrane fusion is required for LNP cargo release. Described herein is a lipid mixing assay for quantifying endosomal-membrane LNP fusions resulting from interactions between synthetic cells and LNPs.
  • the method comprises preparing a mixture of LNPs and synthetic cells with fluorescently labeled membranes under conditions that allow fusion between the LNPs and the labeled synthetic cells.
  • fluorescent tagging refers to the attachment of a reactive derivative of a fluorescent molecule known as a fluorophore to aid in the detection of a biomolecule e.g., membrane lipids.
  • the membranes of the synthetic cells are labeled with a lipophilic fluorescent dye.
  • lipophilic fluorescent dyes include, but are not limited to, Octadecyl Rhodamine B Chloride (R18), Diphenylhexatriene (DPH), N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (Rh-PE), 2-(4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s- indacene-3-dodecanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (p-BODIPY- C12HPC), N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)-1 ,2- dihexadecanoyl-sn-glycero-3-phosphoethanolamine (triethylammonium salt) (BODIPY FL
  • the lipophilic fluorescent dye is R18.
  • fusion of the unlabeled LNPs membranes, with the labeled membranes of the synthetic cells dilutes the probe, with a concomitant increase in fluorescence that allows for the measurement of lipid mixing.
  • the maximal fluorescence was achieved by the addition of 0.2% (v/v) Triton X-100.
  • the fluorescence intensity is measured using a spectrofluorometer using a multi-well plate.
  • the LNP comprises a cargo selected from the group consisting of a peptide, a nucleic acid, a small molecule, and a protein or combinations of the same.
  • the LNP comprises a ribonucleic acid, protein, plasmid, or a small molecule drug cargo.
  • ribonucleic acid cargo examples include but are not limited to small interfering RNA (siRNA), an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a Dicersubstrate RNA (dsRNA), an antisense oligonucleotide (ASO), an RNA aptamer, a small hairpin RNA (shRNA), a messenger RNA (mRNA), trans-activating clustered regularly interspaced short palindromic repeat RNA (tracrRNA), clustered regularly interspaced short palindromic repeat RNA (crRNA), and a long non-coding RNA (IncRNA), a guide RNA (gRNA).
  • siRNA small interfering RNA
  • aiRNA asymmetrical interfering RNA
  • miRNA microRNA
  • dsRNA Dicersubstrate RNA
  • ASO antisense oligonucleotide
  • RNA aptamer examples include but are not limited to small inter
  • the method comprises, in some embodiments, preparing a first mixture of LNPs and synthetic cells encapsulating a water-soluble fluorescent dye under conditions that allow contact between the LNPs and the synthetic cells, and preparing a second mixture of LNPs and synthetic cells with fluorescently labeled membranes under conditions that allow fusion between the LNPs and the synthetic cells.
  • the method further comprises measuring the difference in fluorescence signal intensity between (i) the mixture of LNPs and synthetic cells and (ii) a composition comprising only synthetic cells after normalization to maximum fluorescence values from full homogenization by Triton X-100 or other surfactants.
  • the extent of membrane permeabilization at each time point is calculated by normalizing the respective fluorescence values of the mixture comprising the synthetic cells encapsulating a water-soluble fluorescent dye to the maximal fluorescence dequenching seen upon detergent lysis of said synthetic cells.
  • the extent of membrane fusion at each time point is calculated by normalizing the respective fluorescence values of the mixture comprising the synthetic cells with fluorescently labeled membranes to maximal fluorescence dequenching seen upon detergent lysis of the synthetic cells.
  • the fluorescently labeled membranes are labeled with R18 dye.
  • a 100 nm polycarbonate filter and Mini-Extruder Set was purchased from Avanti Polar Lipids.
  • LUVs large unilamellar vesicles
  • Pure lipids POPC, POPG
  • Octadecyl Rhodamine B chloride R18 was purchased from Sigma Aldrich, dissolved in chloroform, and stored at -80 C.
  • lipids in chloroform were added to a glass cuvette and dried under a N 2 stream, then dried under vacuum in the dark at room temperature for at least 12 hours.
  • lipid and R18 in chloroform were added to a glass cuvette and mixed by swirling before being dried under N 2 and then dried under vacuum in the dark at room temperature for at least 12 hours.
  • Lipids for the R18 assay were rehydrated with 20 mM HEPES buffer at either pH 7.4 or pH 5.5 and vortexed to disperse.
  • Lipids for the CF assay were rehydrated with ⁇ 36 mM CF in 20 mM HEPES buffer at pH 7.4. Hydrated lipid underwent 5 freeze-thaw cycles and was extruded through a 100 nm polycarbonate filter (purchased from Avanti Polar Lipids) 29 times to produce unilamellar liposomes of a consistent size. LUV were stored at 4C in the dark.
  • CF-LUV were filtered through a column packed with Sephadex® G-25 Medium media, washed through with 20 mM pH 7.4 HEPES buffer. After filtration, CF absorbance was measured by UV-VIS spectrophotometer, size was measured using DLS, size was also measured by Transmission Electron Microscopy (TEM).
  • LNP components Lipophilic LNP components (lipidoid, helper lipid, PEG lipid, and cholesterol) were dissolved in ethanol and stored at -80 °C. Aliquots of mRNA were stored at -80 C and thawed slowly on ice before use. LNP components were mixed at room temperature and diluted with ethanol and 10 mM sodium citrate buffer pH 4. In parallel, mRNA was diluted with 10 mM sodium citrate buffer at pH 4 at room temperature. Lipid solution was quickly added to mRNA solution, pipetting to mix, to spontaneously form conjugates. LNPs were then diluted with 1 X PBS at pH 7.4 or pH 5.5. Ethanol was removed by dialysis using 3.5k MWCO filters (Thermo Scientific) into the same PBS buffer at room temperature. LNP were stored at 4C in the dark for up to one week. Size characterization was performed by DLS and TEM.
  • Liposomes were diluted in 20 mM HEPES at pH 7.4 or pH 5.5 to approximately 0.1 mg/mL before measurement. LNP were diluted to a concentration of approximately 5 zg/mL mRNA in 1X PBS at pH 7.4 or pH 5.5 before measurement.

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Abstract

La présente divulgation concerne des compositions et des procédés de criblage à haut rendement de formulations de nanoparticules lipidiques (LNP) in vitro pour identifier les meilleurs candidats sur la base de la cytotoxicité et de l'échappement endosomal effectif. Les LNP sont des agents actifs au niveau membranaire, susceptibles de compromettre la barrière de perméabilité constituée de membranes plasmatiques phospholipidiques conduisant à la cytotoxicité. Suite à une absorption cellulaire effective, les LNP efficaces doivent s'échapper avec succès de l'endosome, par fusion membranaire LNP-endosomique pour administrer leur charge au cytosol. Les procédés décrits ici sont basés sur des cellules synthétiques qui encapsulent un colorant fluorescent hydrosoluble ou dont les membranes sont marquées avec un colorant fluorescent lipophile à des concentrations de colorant présentant une autoextinction.
PCT/US2024/060999 2023-12-20 2024-12-19 Procédé à haut rendement pour cribler l'efficacité et la cytotoxicité de nanoparticules lipidiques Pending WO2025137252A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200129445A1 (en) * 2017-03-15 2020-04-30 Modernatx, Inc. Lipid nanoparticle formulation
US20200222324A1 (en) * 2014-01-21 2020-07-16 Anjarium Biosciences Ag Hybridosomes, compositions comprising the same, processes for their production and uses thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200222324A1 (en) * 2014-01-21 2020-07-16 Anjarium Biosciences Ag Hybridosomes, compositions comprising the same, processes for their production and uses thereof
US20200129445A1 (en) * 2017-03-15 2020-04-30 Modernatx, Inc. Lipid nanoparticle formulation

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