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WO2025210592A1 - Dosages de contrôle de qualité de lnp-arnm de cellules musculaires - Google Patents

Dosages de contrôle de qualité de lnp-arnm de cellules musculaires

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Publication number
WO2025210592A1
WO2025210592A1 PCT/IB2025/053600 IB2025053600W WO2025210592A1 WO 2025210592 A1 WO2025210592 A1 WO 2025210592A1 IB 2025053600 W IB2025053600 W IB 2025053600W WO 2025210592 A1 WO2025210592 A1 WO 2025210592A1
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WO
WIPO (PCT)
Prior art keywords
lnp
mrna
muscle cells
lipid
antigen
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
PCT/IB2025/053600
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English (en)
Inventor
Carla Jane ASQUITH
Sudha CHIVUKULA
Hillary DANZ
Adrien NOUGARÉDE
Shraddha SHARMA
Heesik YOON
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Sanofi Pasteur Inc
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Sanofi Pasteur Inc
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Publication of WO2025210592A1 publication Critical patent/WO2025210592A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0658—Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5061—Muscle cells
    • 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
    • C12N2510/00—Genetically modified cells
    • 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
    • C12N2510/00—Genetically modified cells
    • C12N2510/04—Immortalised cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the antigen is membrane localized on the plurality of isolated human muscle cells.
  • the in vitro system further comprises an antigen binding protein that specifically binds to the antigen.
  • the antigen binding protein comprises a detectable moiety.
  • the antigen is linked to a detectable protein.
  • the detectable protein comprises a fluorescent protein.
  • the fluorescent protein is GFP, RFP, YFP, or dsRed.
  • the antigen is secreted from the plurality of isolated human muscle cells.
  • the plurality of isolated human muscle cells are skeletal muscle cells.
  • the skeletal muscle cells are myocytes, myosatellite cells, or myoblasts.
  • the skeletal muscle cells are primary skeletal muscle cells.
  • the immortalized skeletal muscle cells exhibit SV40 large T antigen expression, hTERT expression, HPV16 E6/E7 expression, adenovirus E1A/E1 B expression, tumor suppressor gene inactivation, or fusion with one or more immortalized cell line.
  • the immortalized skeletal muscle cells express one or more of cyclin D1 (CCND1 ), cyclin-dependent kinase 4 (CDK4), telomerase (TERT), HPV16 E6/E7, or adenovirus E1A/E1 B.
  • CCND1 cyclin D1
  • CDK4 cyclin-dependent kinase 4
  • TERT telomerase
  • HPV16 E6/E7 adenovirus E1A/E1 B.
  • the LNP comprises at least one cationic lipid.
  • the LNP further comprises at least one polyethylene glycol (PEG) conjugated (PEGylated) lipid, at least one cholesterol-based lipid, and at least one helper lipid.
  • PEG polyethylene glycol
  • the disclosure provides a multi-vessel in vitro system comprising a plurality of isolated human muscle cells contained within at least two separate vessels, wherein each of the at least two separate vessels comprises a plurality of lipid-nanoparticle (LNP)-encapsulated messenger RNA (mRNA), the plurality of LNP-encapsulated mRNA comprising an LNP, and wherein the LNP comprises a unique composition of lipids in each vessel.
  • LNP lipid-nanoparticle
  • mRNA messenger RNA
  • the LNP comprises at least one cationic lipid and wherein each of the at least two separate vessels comprises an LNP with a different cationic lipid.
  • the LNP further comprises at least one polyethylene glycol (PEG) conjugated (PEGylated) lipid, at least one cholesterol-based lipid, and at least one helper lipid.
  • PEG polyethylene glycol
  • the LNP comprises at least one PEGylated lipid and wherein each of the at least two separate vessels comprises an LNP with a different PEGylated lipid.
  • the LNP further comprises at least one cationic lipid, at least one a cholesterol-based lipid, and at least one helper lipid.
  • the LNP comprises at least one cholesterol-based lipid and wherein each of the at least two separate vessels comprises an LNP with a different cholesterol-based lipid.
  • the LNP further comprises at least one cationic lipid, at least one PEGylated lipid, and at least one helper lipid.
  • the LNP comprises at least one helper lipid and wherein each of the at least two separate vessels comprises an LNP with a different helper lipid.
  • the LNP further comprises at least one cationic lipid, at least one PEGylated lipid, and at least one cholesterol-based lipid.
  • the plurality of isolated human muscle cells are skeletal muscle cells.
  • the disclosure provides a method of screening LNP-encapsulated mRNA for polypeptide or protein expression, the method comprising: a) contacting a plurality of isolated human muscle cells with a plurality of lipid-nanoparticle (LNP)-encapsulated messenger RNA (mRNA), wherein the plurality of LNP- encapsulated mRNA contact the plurality of isolated human muscle cells in vitro and transfect the plurality of isolated human muscle cells; and b) detecting polypeptide or protein expression.
  • LNP lipid-nanoparticle
  • mRNA messenger RNA
  • each of the plurality of LNP-encapsulated mRNA comprises mRNA, wherein the mRNA encodes an antigen.
  • the antigen is membrane localized on the plurality of isolated human muscle cells.
  • the method further comprises an antigen binding protein that specifically binds to the antigen.
  • the antigen binding protein comprises a detectable moiety.
  • step b) comprises detecting the antigen binding protein comprising the detectable moiety.
  • the antigen is linked to a detectable protein.
  • the detectable protein comprises a fluorescent protein.
  • the fluorescent protein is GFP, RFP, YFP, or dsRed.
  • step b) comprises detecting the antigen linked to the detectable protein.
  • step b) is performed by fluorescence-activated cell sorting (FACS).
  • FACS fluorescence-activated cell sorting
  • the antigen is secreted from the isolated human muscle cells into cell culture media.
  • step b) comprises detecting the antigen in the cell culture media.
  • the plurality of isolated human muscle cells are skeletal muscle cells.
  • the disclosure provides a method of producing a validated lipid- nanoparticle (LNP)-encapsulated messenger RNA (mRNA), the method comprising: a) mixing LNPs or LNP-forming lipids with mRNAs, thereby forming the LNP-encapsulated mRNA; b) contacting a plurality of isolated human muscle cells with the LNP-encapsulated mRNA; and c) detecting polypeptide or protein expression from the plurality of isolated human muscle cells to validate quality of the LNP-encapsulated mRNA, wherein the quality is validated if expression of the mRNA is detected above a baseline value.
  • the mRNA encodes an antigen.
  • the antigen is membrane localized on the plurality of isolated human muscle cells.
  • the method further comprises an antigen binding protein that specifically binds to the antigen.
  • the antigen binding protein comprises a detectable moiety.
  • step b) comprises detecting the antigen binding protein comprising the detectable moiety.
  • the antigen is linked to a detectable protein.
  • the detectable protein comprises a fluorescent protein.
  • the fluorescent protein is GFP, RFP, YFP, or dsRed.
  • step b) comprises detecting the antigen linked to the detectable protein.
  • step b) is performed by fluorescence-activated cell sorting (FACS).
  • FACS fluorescence-activated cell sorting
  • the antigen is secreted from the isolated human muscle cells into cell culture media.
  • step b) comprises detecting the antigen in the cell culture media.
  • the plurality of isolated human muscle cells are skeletal muscle cells.
  • the baseline value is a value from a plurality of isolated human muscle cells transfected with an LNP encapsulated with an mRNA encoding a polypeptide that is not detected.
  • the disclosure provides an LNP-encapsulated mRNA produced by the method of producing an lipid-nanoparticle (LNP)-encapsulated messenger RNA (mRNA) described herein.
  • LNP lipid-nanoparticle
  • mRNA messenger RNA
  • FIG. 1 depicts a bar graph demonstrating that human skeletal muscle cells (HSkMC) display a lower autofluorescence background compared to HeLa cells.
  • FIG. 2 depicts HSkMCs displaying a high reproducibility for expressing proteinencoding mRNA.
  • FIGs. 5A-5C depict the potency of various LNP formulated mRNA evaluated in HSkMC (FIG. 5A) and compared to NHP (FIGs. 5B and 5C).
  • FIGs. 7A-7B depict an example of a potency assay in HSkMC used to evaluate conformational correctness.
  • FIG. 7A depicts the conversion of the prefusion form of RSV F to post-fusion F.
  • FIG. 7B depicts the ability to distinguish the presence or absence of key protein epitopes in mRNA constructs (RSV F) in HSkMC.
  • RSV F mRNA constructs
  • FIGs. 8A-8C depict three different methods of protein detection in HSkMCs transfected with mRNA LNPs: cytosolic antigen eGFP (FIG. 8A) analyzed by Countess 3 FL imaging and/or flow cytometry, secreted antigen hEPO (FIG. 8B) analyzed by ELISA, and membrane-bound antigen HA (FIG. 8C) analyzed by Countess 3 FL and/or flow cytometry.
  • FIG. 9 depicts the reproducibility of a potency assay performed in three separate experiments. HSkMCs were treated with 1 pg mRNA LNP, and eGFP expression was detected. Similar expression profiles were observed between experiments with small increases in the percentage of cells expressing eGFP in later experiments.
  • FIG. 10 depicts a plot correlating in vitro HA expression in human myoblasts against in vivo HAI titers.
  • the present disclosure is directed to, inter alia, an in vitro system comprising a plurality of isolated human muscle cells and a plurality of lipid-nanoparticle (LNP)- encapsulated messenger RNA (mRNA), wherein the plurality of LNP-encapsulated mRNA contacts the plurality of isolated human muscle cells in vitro and transfect the isolated human muscle cells.
  • LNP lipid-nanoparticle
  • mRNA messenger RNA
  • an “adjuvant” refers to a substance or vehicle that enhances the immune response to an antigen.
  • Adjuvants can include, without limitation, a suspension of minerals (e.g., alum, aluminum hydroxide, or phosphate) on which antigen is adsorbed; a water-in-oil or oil-in-water emulsion in which antigen solution is emulsified in mineral oil or in water (e.g., Freund’s incomplete adjuvant).
  • killed mycobacteria is included (e.g., Freund’s complete adjuvant) to further enhance antigenicity.
  • a “subject” refers to any member of the animal kingdom. In some embodiments, “subject” refers to humans. In some embodiments, “subject” refers to non-human animals. In some embodiments, subjects include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms. In certain embodiments, the non-human subject is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig).
  • a mammal e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig.
  • the disclosure provides an in vitro system comprising: (a) a plurality of isolated human muscle cells; and (b) a plurality of lipid-nanoparticle (LNP)- encapsulated messenger RNA (mRNA), wherein the plurality of isolated LNP- encapsulated mRNA contact the plurality of isolated human muscle cells in vitro and transfect the plurality of isolated human muscle cells.
  • LNP lipid-nanoparticle
  • mRNA messenger RNA
  • the isolated human muscle cells include human skeletal muscle cells (HSkMCs).
  • HSkMCs human skeletal muscle cells
  • the HSkMCs described herein can replicate the environment that LNP-encapsulated mRNA are exposed to upon intramuscular administration of LNP-encapsulated mRNA to humans, a common administration route for LNP-encapsulated mRNA-based vaccination.
  • the isolated human muscle cells described herein can be maintained under cell culture conditions appropriate for their maintenance and propagation, for example, under the conditions described in Example 4.
  • the HSkMCs may comprise any one or more of myocytes, myosatellite cells, and myoblasts (e.g., fetal myoblasts).
  • myocyte is a general term for a muscle cell, including a cardiac muscle cell, a smooth muscle cell, and a skeletal muscle cell.
  • a myocyte corresponds to a skeletal muscle cell (e.g., a human skeletal muscle cell).
  • myoblast refers to an embryonic precursor cell that can differentiate into any one of a skeletal muscle cell, a cardiac muscle cell, or a smooth muscle cell.
  • myosatellite cell refers to a myoblast in skeletal muscle that does not form muscle fibers and dedifferentiates into the myosatellite cell. Myosatellite cells remain adjacent to a skeletal muscle fiber, situated between the sarcolemma and the basement membrane of the endomysium (the connective tissue that divides the muscle fascicles (bundle of muscle fibers) into individual fibers) (see Zammit et al. 2006. J Histochem Cytochem. 54(11 ): 1177-91 , incorporated herein by reference).
  • the HSkMCs are primary HSkMCs.
  • Primary HSkMCs include HSkMCs that are directly isolated from skeletal muscle tissue.
  • the HSkMCs are differentiated primary cells (e.g., myoblasts) isolated from a human donor.
  • the HSkMCs are immortalized HSkMCs.
  • Immortalized HSkMCs include cells that have been manipulated to proliferate indefinitely and can thus be cultured for long periods of time. Suitable methods of immortalizing cells can be used.
  • the immortalized skeletal muscle cells express SV40 large T antigen. Expression of the SV40 large T antigen can create immortalized cells, in part, by overcoming p53- and pRB-dependent cell cycle arrest (see May et al. Nucleic Acids Res. 2004. 32(18): 5529-5538, incorporated herein by reference).
  • the immortalized skeletal muscle cells express one or more of cyclin D1 (CCND1 ), cyclin-dependent kinase 4 (CDK4), and telomerase (TERT). Each of CCND1 , CDK4, and TERT have several activities that promote cell division.
  • the immortalized skeletal muscle cells express CCND1 and CDK4.
  • the immortalized skeletal muscle cells express CCND1 and TERT.
  • the immortalized skeletal muscle cells express CDK4 and TERT.
  • the immortalized skeletal muscle cells express CCND1 , CDK4, and TERT.
  • CCND1 , CDK4, and TERT to create immortalized skeletal muscle cells is described in Pantic et al. (Exp Cell Res. 2016. 342(1 ): 39-51 ), Arandel et al. (Disease Models & Mechanisms. 2017. 10(4): 487-497), and Thorley et al. (Skeletal Muscle, volume 6, Article number: 43. 2016), each of which is incorporated herein by reference.
  • the immortalized skeletal muscle cells express one or more of HPV16 E6/E7 or adenovirus E1A/E1 B.
  • HPV16 E6/E7 or adenovirus E1A/E1 B to immortalize cells is described in Halbert et al. (J Virol. 1992 Apr; 66(4): 2125-2134) and Douglas et al. (J Virol. 1995 Dec;69(12):8061 -5), 2016), each of which is incorporated herein by reference.
  • the HSkMCs are transfected with an expression vector encoding one or more of SV40 large T antigen, CCND1 , CDK4, TERT, HPV16 E6/E7, and adenovirus E1A/E1 B.
  • one or more of SV40 large T antigen, CCND1 , CDK4, TERT, HPV16 E6/E7, and adenovirus E1A/E1 B are expressed constitutively from the expression vector.
  • one or more of SV40 large T antigen, CCND1 , CDK4, TERT, HPV16 E6/E7, and adenovirus E1 A/E1 B are expressed inducibly from the expression vector.
  • the HSkMCs are transduced with a viral vector that expresses one or more or SV40 large T antigen, CCND1 , CDK4, TERT, HPV16 E6/E7, and adenovirus E1A/E1 B.
  • the viral vector is an adeno-associated virus (AAV).
  • the viral vector is a lentivirus.
  • the HSkMCs are immortalized by inactivating a tumor suppressor gene, such as p16, p53, and pRb.
  • the HSkMCs are immortalized by fusing said HSkMCs with one or more immortalized cell lines.
  • each of the plurality of LNP-encapsulated mRNA in the in vitro system described herein comprises an mRNA, wherein the mRNA encodes a polypeptide.
  • the polypeptide is an antigen. The antigen may be capable of eliciting an immune response in a subject.
  • the mRNA or the polypeptide encoded by said mRNA may be detected.
  • Detection can be achieved intracellularly (i.e., the polypeptide expressed in the cell), on the surface of the isolated human muscle cells (i.e., the polypeptide is anchored to the cell membrane), and/or in the cell culture media (i.e., the polypeptide is secreted from the isolated human muscle cell).
  • the polypeptide (e.g., the antigen) is membrane localized on the plurality of isolated human muscle cells.
  • the polypeptide may be modified such that it is directed to the membrane.
  • the polypeptide may be modified to contain a membrane localization sequence and a transmembrane domain.
  • the polypeptide (e.g., the antigen) is secreted from the plurality of isolated human muscle cells.
  • the polypeptide may be modified such that it is secreted out of the cell.
  • the polypeptide may be modified to contain a secretion sequence.
  • the polypeptide when the polypeptide is secreted, the polypeptide may not comprise a transmembrane domain.
  • the polypeptide e.g., the antigen
  • an antigen binding protein e.g., an antibody
  • the antigen binding protein comprises a detectable moiety.
  • detectable moiety is a moiety that is linked to the antigen binding protein and is capable of being imaged or otherwise detected.
  • the detectable moiety is a small molecule label, e.g., a fluorophore, a chromophore, a spin resonance probe, an imaging agent, or a radiolabel.
  • exemplary fluorophores include fluorescent dyes (e.g., fluorescein, rhodamine, and the like) and other luminescent molecules (e.g., luminal).
  • a fluorophore may be environmentally- sensitive such that its fluorescence changes if it is located close to one or more residues in the modified antigen-binding protein that undergo structural changes upon binding a substrate (e.g., dansyl probes).
  • radiolabels include small molecules containing atoms with one or more low sensitivity nuclei.
  • the radionuclide can be, for example, a gamma, photon, or positron-emitting radionuclide with a half-life suitable to permit activity or detection after the elapsed time between administration and localization to the imaging site.
  • the detectable moiety is a polypeptide.
  • Exemplary detectable polypeptides include enzymes with fluorogenic or chromogenic activity, e.g., the ability to cleave a substrate which forms a fluorophore or chromophore as a product (i.e. , reporter proteins such as luciferase).
  • detectable proteins may have intrinsic fluorogenic or chromogenic activity (e.g., green, red, and yellow fluorescent bioluminescent aequorin proteins from bioluminescent marine organisms, i.e., green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), or Discosoma Red (dsRed)), or they may comprise a protein containing one or more low-energy radioactive nuclei ( 13 C, 15 N, 2H, 125 l, 124 l, 123 l, "Tc, 43 K, 52 Fe, 64 Cu, 68 Ga, 111 In, and the like).
  • GFP green fluorescent protein
  • RFP red fluorescent protein
  • YFP yellow fluorescent protein
  • dsRed Discosoma Red
  • the disclosure provides a multi-vessel in vitro system comprising a plurality of isolated human muscle cells contained within at least two separate vessels, wherein each of the at least two separate vessels comprises a plurality of lipid-nanoparticle (LNP)-encapsulated messenger RNA (mRNA), the plurality of LNP-encapsulated mRNA comprising an LNP, wherein the LNP comprises a unique composition of lipids in each vessel.
  • LNP lipid-nanoparticle
  • mRNA messenger RNA
  • each vessel may contain a unique composition of lipids in the LNP, thereby allowing for the determination of activity of each LNP.
  • the disclosure provides a method of producing a validated lipid- nanoparticle (LNP)-encapsulated messenger RNA (mRNA), the method comprising: a) mixing LNPs or LNP-forming lipids with mRNAs, thereby forming LNP-encapsulated mRNAs; b) contacting a plurality of isolated human muscle cells with the LNP-encapsulated mRNAs; and c) detecting polypeptide expression (i.e., from the LNP-encapsulated mRNAs) in the plurality of isolated human muscle cells to validate guality or integrity of the LNP-encapsulated mRNA.
  • LNP lipid- nanoparticle
  • mRNA messenger RNA
  • the disclosure provides methods of screening LNP- encapsulated mRNA to predict in vivo polypeptide expression (e.g., protein expression or antigen expression) or antibody response.
  • the methods may comprise: a) contacting a plurality of isolated human muscle cells or myoblasts with a plurality of LNP-encapsulated mRNA, wherein the plurality of LNP-encapsulated mRNA contact the plurality of isolated human muscle cells or myoblasts in vitro and transfect the plurality of isolated human muscle cells or myoblasts and b) detecting the presence of mRNA (e.g., the LNP-encapsulated mRNA) in the plurality of isolated human muscle cells or myoblasts.
  • mRNA e.g., the LNP-encapsulated mRNA
  • the in vitro mRNA accumulation and/or mRNA uptake and/or the in vitro polypeptide expression may be predictive of in vivo polypeptide expression and/or of an antibody response to in vivo polypeptide expression.
  • the antibody response may correspond to antibody titers.
  • the methods may further comprise studying a mechanism of action. That is, mechanistic studies such as, visualizing LNP/mRNA uptake and trafficking by live imaging, smFISH, or immunofluorescence (IF) and protein trafficking studies can be performed in the plurality of human muscle cells or myoblasts.
  • mechanistic studies such as, visualizing LNP/mRNA uptake and trafficking by live imaging, smFISH, or immunofluorescence (IF) and protein trafficking studies can be performed in the plurality of human muscle cells or myoblasts.
  • RNA expression may be predictive of an in vivo response (e.g., in vitro polypeptide expression may be correlative of in vivo polypeptide expression or antibody response/titers).
  • the mRNA component of the mRNA-LNP formulations of the present disclosure comprises at least one ribonucleic acid (RNA) comprising an ORF encoding a polypeptide.
  • the RNA is a messenger RNA (mRNA) comprising an open reading frame encoding a polypeptide.
  • the RNA e.g., mRNA
  • the RNA further comprises at least one 5’ UTR, 3’ UTR, poly(A) tail, and/or 5’ cap.
  • An mRNA 5’ cap can provide resistance to nucleases found in most eukaryotic cells and promote translation efficiency. Suitable types of 5’ caps can be used.
  • a 7-methylguanosine cap (also referred to as “m 7 G” or “Cap-0”) comprises a guanosine that is linked through a 5’ - 5’ - triphosphate bond to the first transcribed nucleotide.
  • a 5’ cap can be added as follows: first, an RNA terminal phosphatase can be used to remove one of the terminal phosphate groups from the 5’ nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) can then be added to the terminal phosphates via a guanylyl transferase, producing a 5 ‘5 ‘5 triphosphate linkage; and the 7-nitrogen of guanine can then be methylated by a methyltransferase.
  • Examples of cap structures include, but are not limited to, m7G(5’)ppp, (5’(A,G(5’)ppp(5’)A, and G(5’)ppp(5’)G. Additional cap structures are described in U.S. Publication No. US 2016/0032356 and U.S. Publication No. US 2018/0125989, which are incorporated herein by reference.
  • [0142]5’-capping of polynucleotides may be completed concomitantly during the in vitro- transcription reaction using the following chemical RNA cap analogs to generate the 5’-guanosine cap structure according to manufacturer protocols: 3’-0-Me- m7G(5’)ppp(5’)G (the ARCA cap); G(5’)ppp(5’)A; G(5’)ppp(5’)G; m7G(5’)ppp(5’)A; m7G(5’)ppp(5’)G; m7G(5’)ppp(5’)(2’OMeA)pG; m7G(5’)ppp(5’)(2’OMeA)pU; m7G(5’)ppp(5’)(2’OMeG)pG (New England BioLabs, Ipswich, MA; TriLink Biotechnologies).
  • 5’-capping of modified RNA may be completed post- transcriptionally using a vaccinia virus capping enzyme to generate the Cap-0 structure: m7G(5’)ppp(5’)G.
  • Cap-1 structure may be generated using both vaccinia virus capping enzyme and a 2’-0 methyl-transferase to generate m7G(5’)ppp(5’)G- 2’-0-methyl.
  • Cap 2 structure may be generated from the Cap-1 structure followed by the 2’-O-methylation of the 5’-antepenultimate nucleotide using a 2’-0 methyltransferase.
  • Cap 3 structure may be generated from the Cap 2 structure followed by the 2’-O-methylation of the 5’-preantepenultimate nucleotide using a 2’-0 methyl-transferase.
  • the mRNA of the disclosure comprises a 5’ cap of:
  • the mRNA of the disclosure includes a 5’ and/or 3’ untranslated region (UTR).
  • the 5’ UTR may start at the transcription start site and continue to the start codon but not include the start codon.
  • the 3’ UTR may start immediately following the stop codon and continue until the transcriptional termination signal.
  • the mRNA disclosed herein comprises a 5’ UTR that includes one or more elements that affect stability or translation of the mRNA.
  • a 5’ UTR may be about 10 to 5,000 nucleotides in length. In some embodiments, a 5’ UTR may be about 50 to 500 nucleotides in length.
  • the 5’ UTR is at least about 10 nucleotides in length, about 20 nucleotides in length, about 30 nucleotides in length, about 40 nucleotides in length, about 50 nucleotides in length, about 100 nucleotides in length, about 150 nucleotides in length, about 200 nucleotides in length, about 250 nucleotides in length, about 300 nucleotides in length, about 350 nucleotides in length, about 400 nucleotides in length, about 450 nucleotides in length, about 500 nucleotides in length, about 550 nucleotides in length, about 600 nucleotides in length, about 650 nucleotides in length, about 700 nucleotides in length, about 750 nucleotides in length, about 800 nucleotides in length, about 850 nucleotides in length, about 900 nucleotides in length, about 950 nucleotides in length, about 1 ,000
  • the mRNA disclosed herein comprise a 3’ UTR comprising one or more of a polyadenylation signal, a binding site for proteins that affect an mRNA’s stability of location in a cell, or one or more binding sites for miRNAs.
  • a 3’ UTR may be about 50 to 5,000 nucleotides in length or longer. In certain embodiments, a 3’ UTR may be about 50 to 1 ,000 nucleotides in length or longer.
  • the 3’ UTR is at least about 50 nucleotides in length, about 100 nucleotides in length, about 150 nucleotides in length, about 200 nucleotides in length, about 250 nucleotides in length, about 300 nucleotides in length, about 350 nucleotides in length, about 400 nucleotides in length, about 450 nucleotides in length, about 500 nucleotides in length, about 550 nucleotides in length, about 600 nucleotides in length, about 650 nucleotides in length, about 700 nucleotides in length, about 750 nucleotides in length, about 800 nucleotides in length, about 850 nucleotides in length, about 900 nucleotides in length, about 950 nucleotides in length, about 1 ,000 nucleotides in length, about 1 ,500 nucleotides in length, about 2,000 nucleotides in length, about 2,500 nucleotides
  • the 5’ and/or 3’ UTR sequences are derived from mRNA which are stable (e.g., globin, actin, GAPDH, tubulin, histone, or citric acid cycle enzymes) to increase the stability of the mRNA.
  • a 5’ UTR sequence may include a partial sequence of a CMV immediate-early 1 (IE1 ) gene, or a fragment thereof, to improve the nuclease resistance and/or improve the half-life of the mRNA.
  • IE1 CMV immediate-early 1
  • hGH human growth hormone
  • these modifications improve the stability and/or pharmacokinetic properties (e.g., half-life) of the mRNA relative to their unmodified counterparts, and include, for example, modifications made to improve such mRNA resistance to in vivo nuclease digestion.
  • Exemplary 5’ UTRs include a sequence derived from a CMV immediate-early 1 (IE1 ) gene (U.S. Publication Nos. 2014/0206753 and 2015/0157565, each of which is incorporated herein by reference), or the sequence GGGAUCCUACC (SEQ ID NO: 1 ) (U.S. Publication No. 2016/0151409, incorporated herein by reference).
  • IE1 CMV immediate-early 1
  • GGGAUCCUACC SEQ ID NO: 1
  • the 5’ UTR is derived from the 5’ UTR of a TOP gene.
  • TOP genes are typically characterized by the presence of a 5’-terminal oligopyrimidine (TOP) tract. Furthermore, most TOP genes are characterized by growth-associated translational regulation. However, TOP genes with a tissue specific translational regulation can also be used.
  • the 5’ UTR derived from the 5’ UTR of a TOP gene lacks the 5’ TOP motif (the oligopyrimidine tract) (e.g., U.S. Publication Nos. 2017/0029847, 2016/0304883, 2016/0235864, and 2016/0166710, each of which is incorporated herein by reference).
  • the 5’ UTR is derived from a ribosomal protein large 32 (L32) gene (U.S. Publication No. 2017/0029847, supra).
  • the 5’ UTR is derived from the 5’ UTR of an hydroxysteroid (17-b) dehydrogenase 4 gene (HSD17B4) (U.S. Publication No. 2016/0166710, supra).
  • the PEGylated lipid component may provide control over particle size and stability of the nanoparticle.
  • the addition of such components may prevent complex aggregation and provide a means for increasing circulation lifetime and increasing the delivery of the lipid-nucleic acid pharmaceutical composition to target tissues (Klibanov et al. FEBS Letters 268(1 ):235-7. 1990).
  • These components may be selected to rapidly exchange out of the pharmaceutical composition in vivo (see, e.g., U.S. Pat. No. 5,885,613).
  • imidazole cholesterol ester (“ICE”; WO 2011/068810), sitosterol (22,23-dihydrostigmasterol), [3- sitosterol, sitostanol, fucosterol, stigmasterol (stigmasta-5,22-dien-3-ol), ergosterol; desmosterol (3[3-hydroxy-5,24-cholestadiene); lanosterol (8,24- lanostadien-3b-ol); 7-dehydrocholesterol (A5,7-cholesterol); dihydrolanosterol (24,25-dihydrolanosterol); zymosterol (5a-cholesta-8,24-dien-3[3-ol); lathosterol (5a-cholest-7-en-3[3-ol); diosgenin ((3[3,25R)-spirost-5-en-3-ol); campesterol (campest-5-en-3[3-ol); campestanol (5
  • a helper lipid can enhance the structural stability of the LNP and help the LNP in endosome escape.
  • a helper lipid can improve uptake and release of the mRNA drug payload.
  • the helper lipid is a zwitterionic lipid, which has fusogenic properties for enhancing uptake and release of the drug payload.
  • helper lipids are 1 ,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE); 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); 1 ,2-dioleoyl-sn- glycero-3-phospho-L-serine (DOPS); 1 ,2-dielaidoyl-sn-glycero-3- phosphoethanolamine (DEPE); and 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DPOC), dipalmitoylphosphatidylcholine (DPPC), DMPC, 1 ,2-dilauroyl-sn-glycero- 3-phosphocholine (DLPC), 1 ,2-distearoylphosphatidylethanolamine (DSPE), and 1 ,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE).
  • DOPE 1,2-diste
  • helper lipids are dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), phosphatidylserine, sphingolipids, sphingomyelins, ceramides, cerebrosides, gangliosides, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1 -trans PE,
  • DOPC diole
  • a polyethylene glycol (PEG) conjugated (PEGylated) lipid at a molar ratio of 0.25% to 2.75% or 1.00% to 2.00% (e.g., a PEGylated lipid at a molar ratio of 0.25%, 0.50%, 0.75%, 1.00%, 1.25%, 1.50%, 1.75%, 2.00%, 2.25%, 2.50%, or 2.75%);
  • a cholesterol-based lipid at a molar ratio of 20% to 50%, 25% to 45%, or 28.5% to 43% e.g., a cholesterol-based lipid at a molar ratio of 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50%); and
  • a helper lipid at a molar ratio of 5% to 35%, 8% to 30%, or 10% to 30% e.g., a helper lipid at a molar ratio of 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, or 35%),
  • the LNP comprises: a cationic lipid at a molar ratio of 40%; a PEGylated lipid at a molar ratio of 1 .5%; a cholesterol-based lipid at a molar ratio of 28.5%; and a helper lipid at a molar ratio of 30%.
  • the PEGylated lipid is dimyristoyl-PEG2000 (DMG- PEG2000).
  • the cholesterol-based lipid is cholesterol
  • the helper lipid is 1 ,2-dioleoyl-SN-glycero-3- phosphoethanolamine (DOPE).
  • DOPE 1 ,2-dioleoyl-SN-glycero-3- phosphoethanolamine
  • the LNP comprises: GL-HEPES-E3-E12-DS-4-E10 at a molar ratio of 35% to 55%; DMG-PEG2000 at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DOPE at a molar ratio of 5% to 35%.
  • the LNP comprises: ALC-0315 at a molar ratio of 35% to 55%; ALC-0159 at a molar ratio of 0.25% to 2.75%; cholesterol at a molar ratio of 20% to 50%; and DSPC at a molar ratio of 5% to 35%.
  • the LNP comprises: 9-heptadecanyl 8- ⁇ (2- hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino ⁇ octanoate (SM-102) at a molar ratio of 50%; 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) at a molar ratio of 10%; cholesterol at a molar ratio of 38.5%; and 1 ,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (DMG-PEG2000) at a molar ratio of 1 .5%.
  • SM-102 9-heptadecanyl 8- ⁇ (2- hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino ⁇ octanoate
  • DSPC ,2-distearoyl-sn-glycero-3-
  • the LNP comprises: (4- hydroxybutyl)azanediyl]di(hexane-6,1 -diyl) bis(2-hexyldecanoate) (ALC-0315) at a molar ratio of 46.3%; 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) at a molar ratio of 9.4%; cholesterol at a molar ratio of 42.7%; and 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159) at a molar ratio of 1.6%.
  • the LNP comprises: (4-hydroxybutyl)azanediyl]di(hexane- 6,1 -diyl) bis(2-hexyldecanoate) (ALC-0315) at a molar ratio of 47.4%; 1 ,2- distearoyl-sn-glycero-3-phosphocholine (DSPC) at a molar ratio of 10%; cholesterol at a molar ratio of 40.9%; and 2-[(polyethylene glycol)-2000]-N,N- ditetradecylacetamide (ALC-0159) at a molar ratio of 1 .7%.
  • the molar amount of the cationic lipid is first determined based on a desired N/P ratio, where N is the number of nitrogen atoms in the cationic lipid and P is the number of phosphate groups in the mRNA to be transported by the LNP.
  • N is the number of nitrogen atoms in the cationic lipid
  • P is the number of phosphate groups in the mRNA to be transported by the LNP.
  • the molar amount of each of the other lipids is calculated based on the molar amount of the cationic lipid and the molar ratio selected. These molar amounts are then converted to weights using the molecular weight of each lipid.
  • the nucleic acid and/or LNP can be formulated in combination with one or more carriers, targeting ligands, stabilizing reagents (e.g., preservatives and antioxidants), and/or other pharmaceutically acceptable excipients.
  • excipients are parabens, thimerosal, thiomersal, chlorobutanol, bezalkonium chloride, and chelators (e.g., EDTA).
  • the LNP compositions of the present disclosure can be provided as a frozen liquid form or a lyophilized form.
  • cryoprotectants may be used, including, without limitation, sucrose, trehalose, glucose, mannitol, mannose, dextrose, and the like.
  • the cryoprotectant may constitute 5-30% (w/v) of the LNP composition.
  • the LNP composition may comprise trehalose, e.g., at 5- 30% (e.g., 10%) (w/v).
  • the LNP compositions may be frozen (or lyophilized and cryopreserved) at -20°C to -80°C.
  • the LNP compositions may be provided to a patient in an aqueous buffered solution - thawed if previously frozen, or if previously lyophilized, reconstituted in an aqueous buffered solution at bedside.
  • the buffered solution typically is isotonic and suitable for, e.g., intramuscular or intradermal injection.
  • the buffered solution is a phosphate-buffered saline (PBS).
  • the present LNPs can be prepared by various techniques.
  • multilamellar vesicles may be prepared according to conventional techniques, such as by depositing a selected lipid on the inside wall of a suitable container or vessel by dissolving the lipid in an appropriate solvent, and then evaporating the solvent to leave a thin film on the inside of the vessel or by spray drying. An aqueous phase may then be added to the vessel with a vortexing motion that results in the formation of MLVs.
  • Unilamellar vesicles (ULV) can then be formed by homogenization, sonication, or extrusion of the multilamellar vesicles.
  • unilamellar vesicles can be formed by detergent removal techniques.
  • the process of preparing mRNA-loaded LNPs includes a step of heating one or more of the solutions to a temperature greater than ambient temperature, the one or more solutions being the solution comprising the preformed lipid nanoparticles, the solution comprising the mRNA, and the mixed solution comprising the LNP-encapsulated mRNA.
  • the process includes the step of heating one or both of the mRNA solution and the preformed LNP solution prior to the mixing step.
  • the process includes heating one or more of the solutions comprising the pre-formed LNPs, the solution comprising the mRNA, and the solution comprising the LNP-encapsulated mRNA during the mixing step.
  • a suitable mRNA stock solution may contain mRNA in water or a buffer at a concentration at or greater than about 0.2 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.8 mg/ml, 1.0 mg/ml, 1.2 mg/ml, 1.4 mg/ml, 1.5 mg/ml, 1.6 mg/ml, 2.0 mg/ml, 2.5 mg/ml, 3.0 mg/ml, 3.5 mg/ml, 4.0 mg/ml, 4.5 mg/ml, or 5.0 mg/ml.
  • an mRNA stock solution is mixed with a buffer solution using a pump.
  • exemplary pumps include, but are not limited to, gear pumps, peristaltic pumps, and centrifugal pumps.
  • the buffer solution is mixed at a rate greater than that of the mRNA stock solution.
  • the buffer solution may be mixed at a rate at least 1X, 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X, 15X, or 20X greater than the rate of the mRNA stock solution.
  • a buffer solution is mixed at a flow rate ranging from about 60-6000 ml/minute (e.g., about 100-300 ml/minute, 300-600 ml/minute, 600-1200 ml/m inute, 1200-2400 ml/minute, 2400-3600 ml/minute, 3600-4800 ml/minute, 4800-6000 ml/minute, or 60-420 ml/minute).
  • a buffer solution is mixed at a flow rate of, or greater than, about 60 ml/minute, 100 ml/minute, 140 ml/minute, 180 ml/minute, 220 ml/minute, 260 ml/minute, 300 ml/minute, 340 ml/minute, 380 ml/minute, 420 ml/minute, 480 ml/minute, 540 ml/minute, 600 ml/minute, 1200 ml/minute, 2400 ml/minute, 3600 ml/minute, 4800 ml/minute, or 6000 ml/minute.
  • an mRNA stock solution is mixed at a flow rate ranging from about 10-600 ml/minute (e.g., about 5-50 ml/minute, about 10-30 ml/minute, about 30-60 ml/minute, about 60-120 ml/minute, about 120-240 ml/minute, about 240-360 ml/minute, about 360-480 ml/minute, or about 480-600 ml/minute).
  • a flow rate ranging from about 10-600 ml/minute (e.g., about 5-50 ml/minute, about 10-30 ml/minute, about 30-60 ml/minute, about 60-120 ml/minute, about 120-240 ml/minute, about 240-360 ml/minute, about 360-480 ml/minute, or about 480-600 ml/minute).
  • an mRNA stock solution is mixed at a flow rate of or greater than about 5 ml/minute, 10 ml/minute, 15 ml/minute, 20 ml/minute, 25 ml/minute, 30 ml/minute, 35 ml/minute, 40 ml/minute, 45 ml/minute, 50 ml/minute, 60 ml/minute, 80 ml/minute, 100 ml/minute, 200 ml/minute, 300 ml/minute, 400 ml/minute, 500 ml/minute, or 600 ml/minute.
  • the process of incorporation of a desired mRNA into a lipid nanoparticle is referred to as “loading.” Exemplary methods are described in Lasic et al. , FEBS Lett. (1992) 312:255-8.
  • the LNP-incorporated nucleic acids may be completely or partially located in the interior space of the lipid nanoparticle, within the bilayer membrane of the lipid nanoparticle, or associated with the exterior surface of the lipid nanoparticle membrane.
  • the incorporation of an mRNA into lipid nanoparticles is also referred to herein as “encapsulation,” wherein the nucleic acid is entirely or substantially contained within the interior space of the lipid nanoparticle.
  • Suitable LNPs may be made in various sizes. In some embodiments, decreased size of lipid nanoparticles is associated with more efficient delivery of an mRNA. Selection of an appropriate LNP size may take into consideration the site of the target cell or tissue and to some extent the application for which the lipid nanoparticle is being made.
  • Suitable methods for sizing of a population of lipid nanoparticles can be used.
  • Exemplary methods provided herein utilize Zetasizer Nano ZS (Malvern Panalytical) to measure LNP particle size.
  • 10 pl of an LNP sample are mixed with 990 pl of 10% trehalose. This solution is loaded into a cuvette and then put into the Zetasizer machine.
  • the z-average diameter (nm), or cumulants mean, is regarded as the average size for the LNPs in the sample.
  • the Zetasizer machine can also be used to measure the polydispersity index (PDI) by using dynamic light scattering (DLS) and cumulant analysis of the autocorrelation function.
  • Average LNP diameter may be reduced by sonication of formed LNP. Intermittent sonication cycles may be alternated with quasi-elastic light scattering (QELS) assessment to guide efficient lipid nanoparticle synthesis.
  • QELS quasi-elastic light scattering
  • the majority of purified LNPs i.e., greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the LNPs have a size of about 70-150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm).
  • nm e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm
  • substantially all (e.g., greater than 80% or 90%) of the purified lipid nanoparticles have a size of about 70-150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm).
  • the LNP has an average diameter of 30-200 nm. In various embodiments, the LNP has an average diameter of 80-150 nm. In some embodiments, the LNPs in the present composition have an average size of less than 150 nm, less than 120 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, less than 30 nm, or less than 20 nm.
  • greater than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the LNPs in the present composition have a size ranging from about 40-90 nm (e.g., about 45-85 nm, about 50-80 nm, about 55-75 nm, or about 60-70 nm), about 40-90 nm (e.g., about 45-85 nm, about 50-80 nm, about 55-75 nm, or about 60-70 nm), or about 50-70 nm (e.g., 55-65 nm).
  • the LNPs are suitable for pulmonary delivery via nebulization.
  • the dispersity, or measure of heterogeneity in size of molecules (PDI), of LNPs in a pharmaceutical composition provided by the present disclosure is less than about 0.5.
  • an LNP has a PDI of less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.28, less than about 0.25, less than about 0.23, less than about 0.20, less than about 0.18, less than about 0.16, less than about 0.14, less than about 0.12, less than about 0.10, or less than about 0.08.
  • the PDI may be measured by a Zetasizer machine as described herein.
  • lipid nanoparticles for use herein have an encapsulation efficiency of at least 90% (e.g., at least 91 %, 92%, 93%, 94%, or 95%).
  • an LNP has a N/P ratio of 1 to 10.
  • a lipid nanoparticle has a N/P ratio above 1 , about 1 , about 2, about 3, about 4, about 5, about 6, about 7, or about 8.
  • a typical LNP herein has an N/P ratio of 4.
  • a pharmaceutical composition according to the present disclosure contains at least about 0.5 pg, 1 pg, 5 pg, 10 pg, 100 pg, 500 pg, or 1000 pg of encapsulated mRNA. In some embodiments, a pharmaceutical composition contains about 0.1 pg to 1000 pg, at least about 0.5 pg, at least about 0.8 pg, at least about 1 pg, at least about 5 pg, at least about 8 pg, at least about 10 pg, at least about 50 pg, at least about 100 pg, at least about 500 pg, or at least about 1000 pg of encapsulated mRNA.
  • mRNA can be made by chemical synthesis or by in vitro transcription (IVT) of a DNA template.
  • IVT in vitro transcription
  • An exemplary process for making and purifying mRNA is described in Example 1.
  • a cDNA template is used to produce an mRNA transcript and the DNA template is degraded by a DNase.
  • the transcript is purified by depth filtration and tangential flow filtration (TFF).
  • TFF depth filtration and tangential flow filtration
  • the purified transcript is further modified by adding a cap and a tail, and the modified RNA is purified again by depth filtration and TFF.
  • the mRNA is then prepared in an aqueous buffer and mixed with an amphiphilic solution containing the lipid components of the LNPs.
  • An amphiphilic solution for dissolving the four lipid components of the LNPs may be an alcohol solution.
  • the alcohol is ethanol.
  • the aqueous buffer may be, for example, a citrate, phosphate, acetate, or succinate buffer and may have a pH of about 3.0-7.0, e.g., about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, or about 6.5.
  • the buffer may contain other components such as a salt (e.g., sodium, potassium, and/or calcium salts).
  • the aqueous buffer has 1 mM citrate and 150 mM NaCI at pH 4.5.
  • An exemplary, nonlimiting process for making an mRNA-LNP composition involves mixing of a buffered mRNA solution with a solution of lipids in ethanol in a controlled homogeneous manner, where the ratio of lipids:mRNA is maintained throughout the mixing process.
  • the mRNA is presented in an aqueous buffer containing citric acid monohydrate, tri-sodium citrate dihydrate, and sodium chloride.
  • the mRNA solution is added to the solution (1 mM citrate buffer, 150 mM NaCI, pH 4.5).
  • the lipid mixture of four lipids (e.g., a cationic lipid, a PEGylated lipid, a cholesterol-based lipid, and a helper lipid) is dissolved in ethanol.
  • the aqueous mRNA solution and the ethanol lipid solution are mixed at a volume ratio of 4:1 in a “T” mixer with a near “pulseless” pump system.
  • the resultant mixture is then subjected for downstream purification and buffer exchange.
  • the buffer exchange may be achieved using dialysis cassettes or a TFF system. TFF may be used to concentrate and buffer-exchange the resulting nascent LNP immediately after formation via the T-mix process.
  • the diafiltration process is a continuous operation, keeping the volume constant by adding appropriate buffer at the same rate as the permeate flow.
  • the mRNA-LNP vaccines can be formulated or packaged for parenteral (e.g., intramuscular, intradermal, or subcutaneous) administration or nasopharyngeal (e.g., intranasal) administration.
  • the vaccine compositions may be in the form of an extemporaneous formulation, where the LNP composition is lyophilized and reconstituted with a physiological buffer (e.g., PBS) just before use.
  • the vaccine compositions also may be shipped and provided in the form of an aqueous solution or a frozen aqueous solution and can be directly administered to subjects without reconstitution (after thawing, if previously frozen).
  • the present disclosure provides an article of manufacture, such as a kit, that provides the mRNA-LNP vaccine in a single container or provides the mRNA-LNP vaccine in one container (e.g., a first container) and a physiological buffer for reconstitution in another container (e.g., a second container).
  • the container(s) may contain a single-use dosage or multi-use dosage.
  • the containers may be pre-treated glass vials or ampules.
  • the article of manufacture may include instructions for use as well.
  • a single dose of the mRNA-LNP vaccine contains 1 -50 pg of mRNA (e.g., monovalent or multivalent).
  • a single dose may contain about 2.5 pg, about 5 pg, about 7.5 pg, about 10 pg, about 12.5 pg, or about 15 pg of the mRNA for intramuscular (IM) injection.
  • IM intramuscular
  • the vector can be used to express mRNA in a host cell.
  • the vector is used as a template for IVT.
  • the construction of optimally translated IVT mRNA suitable for therapeutic use is disclosed in detail in Sahin, et al. (2014). Nat. Rev. Drug Discov. 13, 759-780; Weissman (2015). Expert Rev. Vaccines 14, 265-281.
  • the vectors disclosed herein can comprise at least the following, from 5’ to 3’: an RNA polymerase promoter; a polynucleotide sequence encoding a 5’ UTR; a polynucleotide sequence encoding an ORF; a polynucleotide sequence encoding a 3’ UTR; and a polynucleotide sequence encoding at least one RNA aptamer.
  • the vectors disclosed herein may also comprise a polynucleotide sequence encoding a poly(A) sequence and/or a polyadenylation signal.
  • RNA polymerase promoters A variety of RNA polymerase promoters are known.
  • the promoter can be a T7 RNA polymerase promoter.
  • Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3, and SP6 promoters are known.
  • Also disclosed herein are host cells (e.g., mammalian cells, such as, e.g., human cells) comprising the vectors or RNA compositions disclosed herein.
  • host cells e.g., mammalian cells, such as, e.g., human cells
  • vectors or RNA compositions disclosed herein comprising the vectors or RNA compositions disclosed herein.
  • Polynucleotides can be introduced into target cells using any of a number of different methods, for instance, commercially available methods which include, but are not limited to, electroporation (Amaxa Nucleofector-ll (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendorf, Hamburg Germany), cationic liposome mediated transfection using lipofection, polymer encapsulation, peptide mediated transfection, biolistic particle delivery systems such as “gene guns” (see, for example, Nishikawa, et al. (2001 ). Hum Gene Then 12(8):861 -70, or the TransIT-RNA transfection Kit (Mirus, Madison Wl).
  • RNA purified according to this disclosure can be useful as a component in pharmaceutical compositions, for example, for use as a vaccine.
  • These compositions can include RNA and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition of the disclosure can also include one or more additional components such as small molecule immunopotentiators (e.g., TLR agonists).
  • a pharmaceutical composition of the disclosure can also include a delivery system for the RNA, such as a liposome, an oil-in-water emulsion, or a microparticle.
  • the pharmaceutical composition comprises a lipid nanoparticle (LNP).
  • the composition comprises an antigen-encoding nucleic acid molecule encapsulated within an LNP.
  • Embodiment 1 An in vitro system comprising: a plurality of isolated human muscle cells; and a plurality of lipid-nanoparticle (LNP)-encapsulated messenger RNA (mRNA), wherein the plurality of isolated LNP-encapsulated mRNA contact the plurality of isolated human muscle cells in vitro and transfect the plurality of isolated human muscle cells.
  • LNP lipid-nanoparticle
  • mRNA messenger RNA
  • Embodiment 2 The in vitro system of embodiment 1 , each of the plurality of LNP-encapsulated mRNA comprising an mRNA, wherein the mRNA encodes an antigen.
  • Embodiment 3 The in vitro system of embodiment 2, wherein the antigen is membrane localized on the plurality of isolated human muscle cells.
  • Embodiment 4 The in vitro system of embodiment 3, further comprising an antigen binding protein that specifically binds to the antigen.
  • Embodiment 5 The in vitro system of embodiment 4, wherein the antigen binding protein comprises a detectable moiety.
  • Embodiment 6 The in vitro system of embodiment 2, wherein the antigen is linked to a detectable protein.
  • Embodiment 7 The in vitro system of embodiment 6, wherein the detectable protein comprises a fluorescent protein.
  • Embodiment 8 The in vitro system of embodiment 7, wherein the fluorescent protein is GFP, RFP, YFP, or dsRed.
  • Embodiment 9 The in vitro system of embodiment 2, wherein the antigen is secreted from the plurality of isolated human muscle cells.
  • Embodiment 10 The in vitro system of any one of embodiments 1 -9, wherein the plurality of isolated human muscle cells are skeletal muscle cells.
  • Embodiment 11 The in vitro system of embodiment 10, wherein the skeletal muscle cells are myocytes, myosatellite cells, or myoblasts.
  • Embodiment 12 The in vitro system of embodiment 10 or 11 , wherein the skeletal muscle cells are primary skeletal muscle cells.
  • Embodiment 14 The in vitro system of embodiment 13, wherein the immortalized skeletal muscle cells exhibit SV40 large T antigen expression, hTERT expression, HPV16 E6/E7 expression, adenovirus E1A/E1 B expression, tumor suppressor gene inactivation, or fusion with one or more immortalized cell line.
  • Embodiment 15 The in vitro system of embodiment 13, wherein the immortalized skeletal muscle cells express one or more of cyclin D1 (CCND1 ), cyclin-dependent kinase 4 (CDK4), telomerase (TERT), HPV16 E6/E7, or adenovirus E1A/E1 B.
  • CCND1 cyclin D1
  • CDK4 cyclin-dependent kinase 4
  • TERT telomerase
  • HPV16 E6/E7 adenovirus E1A/E1 B.
  • Embodiment 16 The in vitro system of embodiment 13, wherein the immortalized skeletal muscle cells express cyclin D1 (CCND1 ), cyclin-dependent kinase 4 (CDK4), and telomerase (TERT).
  • CCND1 cyclin D1
  • CDK4 cyclin-dependent kinase 4
  • TERT telomerase
  • Embodiment 17 The in vitro system of any one of embodiments 1 -16, wherein the LNP comprises at least one cationic lipid.
  • Embodiment 18 The in vitro system of embodiment 17, wherein the LNP further comprises at least one polyethylene glycol (PEG) conjugated (PEGylated) lipid, at least one cholesterol-based lipid, and at least one helper lipid.
  • PEG polyethylene glycol
  • Embodiment 24 The multi-vessel in vitro system of embodiment 19, wherein the LNP comprises at least one cholesterol-based lipid and wherein each of the at least two separate vessels comprises an LNP with a different cholesterol-based lipid.
  • Embodiment 26 The multi-vessel in vitro system of embodiment 19, wherein the LNP comprises at least one helper lipid and wherein each of the at least two separate vessels comprises an LNP with a different helper lipid.
  • Embodiment 27 The multi-vessel in vitro system of embodiment 26, wherein the LNP further comprises at least one cationic lipid, at least one PEGylated lipid, and at least one cholesterol-based lipid.
  • Embodiment 28 The multi-vessel in vitro system of any one of embodiments 19-27, wherein the plurality of isolated human muscle cells are skeletal muscle cells.
  • Embodiment 29 A method of screening LNP-encapsulated mRNA for polypeptide expression, the method comprising: a) contacting a plurality of isolated human muscle cells with a plurality of lipid-nanoparticle (LNP)-encapsulated messenger RNA (mRNA), wherein the plurality of LNP-encapsulated mRNA contact the plurality of isolated human muscle cells in vitro and transfect the plurality of isolated human muscle cells; and b) detecting polypeptide expression.
  • LNP lipid-nanoparticle
  • mRNA messenger RNA
  • Embodiment 30 The method of embodiment 29, wherein each of the plurality of LNP-encapsulated mRNA comprises mRNA, wherein the mRNA encodes an antigen.
  • Embodiment 31 The method of embodiment 30, wherein the antigen is membrane localized on the plurality of isolated human muscle cells.
  • Embodiment 33 The method of embodiment 32, wherein the antigen binding protein comprises a detectable moiety.
  • Embodiment 34 The method of embodiment 33, wherein step b) comprises detecting the antigen binding protein comprising the detectable moiety.
  • Embodiment 36 The method of embodiment 35, wherein the detectable protein comprises a fluorescent protein.
  • Embodiment 37 The method of embodiment 36, wherein the fluorescent protein is GFP, RFP, YFP, or dsRed.
  • Embodiment 40 The method of embodiment 30, wherein the antigen is secreted from the isolated human muscle cells into cell culture media.
  • Embodiment 41 The method of embodiment 40, wherein step b) comprises detecting the antigen in the cell culture media.
  • Embodiment 42 The method of any one of embodiments 29-41 , wherein the plurality of isolated human muscle cells are skeletal muscle cells or skeletal muscle myoblasts.
  • Embodiment 43 A method of producing a validated lipid-nanoparticle (LNP)- encapsulated messenger RNA (mRNA), the method comprising: a) mixing LNPs with mRNAs, thereby forming the LNP-encapsulated mRNA; b) contacting a plurality of isolated human muscle cells with the LNP-encapsulated mRNA; andc) detecting polypeptide expression from the plurality of isolated human muscle cells to validate quality of the LNP-encapsulated mRNA, wherein the quality is validated if expression of the mRNA is detected above a baseline value.
  • LNP lipid-nanoparticle
  • mRNA messenger RNA
  • Embodiment 45 The method of embodiment 44, wherein the antigen is membrane localized on the plurality of isolated human muscle cells.
  • Embodiment 46 The method of embodiment 45, further comprising an antigen binding protein that specifically binds to the antigen.
  • Embodiment 47 The method of embodiment 46, wherein the antigen binding protein comprises a detectable moiety.
  • Embodiment 48 The method of embodiment 47, wherein step b) comprises detecting the antigen binding protein comprising the detectable moiety.
  • Embodiment 49 The method of embodiment 44, wherein the antigen is linked to a detectable protein.
  • Embodiment 50 The method of embodiment 49, wherein the detectable protein comprises a fluorescent protein.
  • Embodiment 51 The method of embodiment 50, wherein the fluorescent protein is GFP, RFP, YFP, or dsRed.
  • Embodiment 52 The method of any one of embodiments 49-51 , wherein step b) comprises detecting the antigen linked to the detectable protein.
  • Embodiment 53 The method of embodiment 52, wherein step b) is performed by fluorescence-activated cell sorting (FACS).
  • FACS fluorescence-activated cell sorting
  • Embodiment 54 The method of embodiment 44, wherein the antigen is secreted from the isolated human muscle cells into cell culture media.
  • Embodiment 55 The method of embodiment 54, wherein step b) comprises detecting the antigen in the cell culture media.
  • Embodiment 56 The method of any one of embodiments 43-55, wherein the plurality of isolated human muscle cells are skeletal muscle cells.
  • Embodiment 57 The method of any one of embodiments 43-56, wherein the baseline value is a value from a plurality of isolated human muscle cells transfected with an empty LNP.
  • Embodiment 58 The method of any one of embodiments 43-56, wherein the baseline value is a value from a plurality of isolated human muscle cells transfected with an LNP encapsulated with an mRNA encoding a polypeptide that is not detected.
  • Embodiment 59 An LNP-encapsulated mRNA produced by the method of any one of embodiments 43-58.
  • Embodiment 60 A method of screening lipid-nanoparticle (LNP)-encapsulated messenger mRNA (mRNA) to predict in vivo polypeptide expression or antibody response, the method comprising: a) contacting a plurality of isolated human muscle cells or myoblasts with a plurality of LNP-encapsulated mRNA, wherein the plurality of LNP-encapsulated mRNA contact the plurality of isolated human muscle cells or myoblasts in vitro and transfect the plurality of isolated human muscle cells or myoblasts; and one or both of: b) detecting the presence of the mRNA in the plurality of isolated human muscle cells or myoblasts; and c) detecting in vitro polypeptide expression.
  • LNP lipid-nanoparticle
  • mRNA messenger mRNA
  • Embodiment 61 The method of embodiment 60, comprising b) detecting the presence of the mRNA in the plurality of human muscle cells or myoblasts and d) correlating the in vitro mRNA accumulation and/or the mRNA uptake with in vivo polypeptide expression or antibody response.
  • Embodiment 62 The method of embodiment 60 or embodiment 61 , comprising c) detecting in vitro polypeptide expression and e) correlating the in vitro polypeptide expression with in vivo polypeptide expression or antibody response.
  • Embodiment 63 The method of any one of embodiments 60-62, wherein the antibody response corresponds to an antibody titer.
  • Example 1 Measurement of Autofluorescence Background of HSkMCs and HeLa Cells
  • Example 2 Measurements for Reproducibility of Expression of Proteinencoding mRNA in HSkMCs
  • HSkMCs were seeded at 6000 cells per well in a Greiner pCIear FlatBottom 96- well plate and incubated at 37°C with 5% CO2 for 24 hours. Cells were transfected 24 hours after seeding with 50 ng of GFP mRNA per well using Mirus TransIT lipofection reagents. Nuclei staining was performed for 30 minutes with H33342 nuclear stain at 1 pM just before image acquisition. Cells were then imaged on a Cytation 7 imaging system at 469 nm excitation and 525 nm emission bandwidth. After image segmentation based on nuclei staining, mean fluorescence intensity per cell was determined for each cell imaged.
  • HSkMCs displayed a high reproducibility for expressing protein-encoding mRNA (FIG. 2).
  • HSkMCs were seeded at 6000 cells per well in a Greiner pCIear FlatBottom 96- well plate and incubated at 37°C with 5% CO2 for 24 hours. Cells were incubated at various amounts of LNP-formulated HA-H3 mRNA (from 125 ng to 1000 ng per well) or not treated (NT) 24 hours after seeding. After 48 hours of incubation with LNP mRNA, cells were fixed for 20 minutes in 4% PFA solution.
  • HSkMCs can be used for high-throughput antigen expression assays, as only the HA-H3 mRNA-transfected cells produced detectable HA-H3 protein (FIG. 3).
  • Example 4 High-throughput Immunofluorescence Antigen Detection Method on hSKMCs Using Image Cytometry
  • the cells were centrifuged at 250 g for 3 minutes and the supernatant was discarded carefully without detaching the cell pellet.
  • the cell pellet was resuspended in 9 mL of M199 complete medium and diluted in a new Falcon 50 tube to 30,000 cells/mL for HSkMC.
  • the 30,000 cells/mL suspension (200 pL) was added to each empty well in the plate (60 wells). Manual orbital shaking of the plate was performed for 10 seconds, and the cells were left to stand on a bench (outside of the cell culture hood) for 20 to 30 minutes at room temperature and protected from light, then the flask was placed back in the incubator at 37°C and 5% CO2.
  • the liquid was aspirated from each well and 100 pL primary antibody dilution in PBS (0.1 % Triton X-100 and 5% BSA) or 100 pL of PBS (0.1 % Triton X-100 and 5% BSA; for the secondary antibody only control) was added. The plate was incubated for 60 minutes at room temperature and protected from light. The liquid was aspirated from each well and each well was washed three times with 200 pL of PBS (0.1 % Triton X-100) with the following protocol: a quick wash first then two 5-m inute washes.
  • Gen5 The following analysis parameters were used on Gen5: 1 ) Primary mask on nuclei (Hoechst 33342) staining; 2) Secondary mask on primary mask + 30 pm expansion for HSkMC; 3) Measurement of Mean fluorescence intensity for each ROI based on secondary mask; and 4) Export as a text file for subsequent analysis.
  • Threshold Mean(NT) + 3x SD(NT) and 2) Adaptative thresholding algorithm from NT (background) and positive control well.
  • HSkMCs were ordered from Lonza (SkMC, Catalog #: CC-2561 ).
  • 0.25% Trypsin-EDTA 1X and M199 were warmed in a water bath. Media were decanted from the T150 HSkMC culture flask, then 10 mL PBS was added to the flask, and the flask was tilted gently a couple of times. PBS was decanted, then another 10 mL PBS was added to the flask, and the flask was tilted gently a couple of times. PBS was decanted, and 5 mL 0.25% Trypsin-EDTA 1X was added to the flask. Cells were incubated in a 37°C incubator for up to 5 minutes (until the cells were detached).
  • Cells were washed off of the flask bottom with 10 mL warm M199 and were transferred to a 50 mL tube. Cells were spun at 1400 rpm for 7 minutes at 4°C. The supernatant was aspirated, and the cell pellet was resuspended in 5 mL M199 per flask harvested. A 10 pL sample was taken for counting at 1 :2 dilution, the number of cells were calculated, and the volume was brought up to 1 ,000,000 cells per mL. Typically, 450,000 cells were seeded weekly on T 150, and media were replaced once in the middle of the week. At the end of the week, the cells were harvested and seeded again. Cells were maintained until 10-12 passages.
  • Cells were washed with 15 mL warm M199 and were transferred to a 50 mL tube. The flask was washed again with 10 mL warm M199 and transferred to the 50 mL tube. The cells were spun at 90 g for 20 minutes at room temperature. The 50 mL tube was aspirated, and the cell pellet was resuspended in 10 mL PBS. A 10 pL sample was taken for counting at 1 :2 - 1 : 10 dilution with trypan blue. [0345] Cells were transferred (1 million cells/cuvette) and tubes were spun at 90 g for 20 minutes at room temperature. While the cells were spun, the Amaxa Nucleofector ll/llb Device was set to the D-033 program. The Nucleofector solution was prepared by mixing with supplement before adding to the cells.
  • the cuvettes were tapped to even out the layer before inserting into the Amaxa Nucleofector ll/llb Device.
  • 900 pL of warm M 199 was added to the cuvette and was mixed gently using a p1000 pipette. 1 mL was transferred to a well in a culture plate using a disposable pipette from the kit. Typically, all cells were added into 2 wells of a 6- well plate with 3 mL culture media each. A 12-, 24-, or 48-well plate may be used with adjustments to the number of cells and volume of media used.
  • the culture was incubated in a CO2 incubator at 37°C.
  • 0.25% Trypsin-EDTA 1X and M199 were warmed in a water bath. Media were decanted from the T150 HSkMC culture flask, then 10 mL PBS was added to the flask, and the flask was tilted gently a couple of times. PBS was decanted, then another 10 mL PBS was added to the flask, and the flask was tilted gently a couple of times. PBS was decanted, and 5 mL 0.25% Trypsin-EDTA 1X was added to the flask. Cells were incubated in a 37°C incubator for up to 5 minutes (until the cells are detached).
  • HSkMCs were added into each well (100 pL, 100,000 cells per well in a 24-well plate), 900 pL of culture media was added to each well, and the cells were incubated at 37°C. The cell number may be adjusted if other formats of cell culture plates are used.
  • LNP formulated mRNA was added to each well and the culture plate was swirled gently to evenly spread the LNP. Too much LNP can reduce transfectivity or cell survival (i.e. , 1 pg or 0.5 pg of MC3 or OF-02 is optimal for the transfection of 100,000 HSkMC in a 24-well plate), thus the dose of LNP is adjusted and/or optimized accordingly.
  • the cells were incubated in a CO2 incubator at 37°C.
  • the supernatant from the culture (50 pL - 100 pL) was harvested at desired time points and was stored at 4°C until analysis by ELISA.
  • ELISA was performed (Human Erythropoietin Quantikine IVD ELISA Kit, Cat. DEP00, www.rndsystems.com/products/human-erythropoietin-quantikine-ivd-elisa- kit_dep00) according to the manufacturer’s protocol.
  • a 1 :100 dilution was used for low expression samples and a 1 :5,000 dilution was used for high expression samples, but the dilution factors may be optimized and/or adjusted.
  • the media was aspirated from the cells, then 1 mL PBS was gently added to each well and the plate was tilted a couple of times. The PBS was aspirated and 0.5 mL TrypLE (12-well plate) was added to each well. Cells were incubated in a 37°C incubator for up to 5 minutes (until the cells are detached). Cells were resuspended with 0.5 mL PBS and 10 pL was transferred to a Countess slide. The Countess slide was inserted into the Countess 3 FL and the number of GFP positive cells was quantified - this was conducted for all wells.
  • the tubes were spun at 600 x g for 5 minutes at 4°C (all subsequent staining spins used these parameters). The supernatant was removed, and the tubes were lightly vortexed. 1 mL PBS was added to each tube, the tubes were spun, and the supernatant was removed.
  • the tubes or plates were spun down at 1400 rpm for 4 minutes (all subsequent staining spins used these parameters). The supernatant was removed, and the plates/tubes were lightly vortexed. 200 pL PBS was added to each well/tube, the plates/tubes spun, and the supernatant removed.
  • a solution of 1 :500 Live Dead Aqua (LDA) in PBS was prepared. 100 pL LDA solution was added to all of the sample wells and the LDA single color compensation control. The plates were placed on ice or at 4°C in the dark for 20 to 30 minutes or at room temperature for 10 minutes. 100 pL of PBS was added to the unstained wells. The cells were washed by adding 100 pL PBS to each well, then the plates were spun, PBS was removed, and the plates were vortexed. 200 pL FACS buffer was added to each well, then the plates were spun, FACS buffer was removed, and the plates were vortexed.
  • LDA Live Dead Aqua
  • the cells were fixed by adding 100 pL of 2% paraformaldehyde (PFA) to each well (all of the sample wells, the unstained wells, and the LDA wells) and the plates were incubated in the dark for 15 minutes on ice or at 4°C.
  • FACS buffer 100 pL was added to each well so that each well had a total volume of 200 pL.
  • the plates were spun, buffer/PFA were removed, the plates were vortexed, and FACS buffer (200 pL) was added to each well. The plates were centrifuged and the cells were stored as pellets in FACS buffer at 4°C overnight.
  • the plates were incubated on ice or at 4°C in the dark for 20 minutes.
  • the samples were washed twice in PermWash, and an appropriate secondary Ab conjugated with a fluorochrome was added (generally 0.2 - 0.5 pg/sample is used, but the amount of antibody should generally be titrated).
  • the plates were incubated on ice or at 4°C in the dark for 10 minutes, then the samples were washed twice with PermWash and once with FACS buffer.
  • the samples were resuspended in 200 pL FACS buffer (or a different volume depending on the experimental conditions).
  • the plates were wrapped in foil until the cells were ready to be acquired on the cytometer.
  • the fixed cells may be stored at 4°C for 2-3 days before acquisition on the cytometer; however, it is generally preferable to acquire them as quickly as possible. If the cells were sitting for more than 30 minutes before being placed on the cytometer, the cells were resuspended with a multi-channel pipet to decrease cell clumping.
  • other live, dead, or apoptotic markers can be used as a replacement for LDA.
  • the cells can be fixed and stained at a later time point without LDA. In some embodiments, the cells can be fixed and stained on the same day without LDA.
  • Cells were harvested and transferred to tubes or 96-well plates on ice to avoid further digestion for analysis by flow cytometry.
  • the tubes or plates were spun down at 1400 rpm for 4 minutes (all subsequent staining spins used these parameters). The supernatant was removed, and the plates/tubes were lightly vortexed. 200 pL PBS was added to each well/tube, the plates/tubes were spun, and the supernatant was removed.
  • a solution of 1 :500 Live Dead Aqua (LDA) in PBS was prepared. 100 pL LDA solution was added to all of the sample wells and the LDA single color compensation control. The plates were placed on ice or at 4°C in the dark for 20 to 30 minutes or at room temperature for 10 minutes. 100 pL of PBS was added to the unstained wells. The cells were washed by adding 100 pL PBS to each well, then the plates were spun, PBS was removed, and the plates were vortexed. 200 pL FACS buffer was added to each well, then the plates were spun, FACS buffer was removed, and the plates were vortexed.
  • LDA Live Dead Aqua
  • the primary antibody cocktail was diluted in FACS buffer, and 50 pL of this solution was added per sample well (generally 0.2 - 1 pg/sample is used, but the amount of antibody should generally be titrated).
  • the plates were incubated on ice or at 4°C in the dark for 20 minutes.
  • the samples were washed twice with FACS buffer, and an appropriate secondary Ab conjugated with a fluorochrome was added (generally 0.2 - 0.5 pg/sample is used, but the amount of antibody should generally be titrated).
  • the plates were incubated on ice or at 4°C in the dark for 10 minutes, then the samples were washed twice with FACS buffer.
  • HSkMCs were transfected with several hEPO-expressing mRNA-LNP formulations, each formulation containing a different cationic lipid. The same formulations were administered to NHPs via intramuscular injection. EPO protein levels were then measured by ELISA at 6, 24, and 48 hours post transection or injection. As shown in FIG. 4A and FIG. 4B, the results were comparable between the two model systems.
  • HSkMCs were used to evaluate expression of influenza antigens HA and NA, and RSV F protein.
  • Numerous mRNA-LNP formulations with mRNA expressing HA or NA were transfected into HSkMCs at a range of doses (6 pg, 1 .5 pg, 375 ng, 93.75 ng, 23.44 ng, and 5.86 ng).
  • FIG. 5A transfection of HSkMCs displayed a dose response effect, with decreasing HA expression levels with lower doses of mRNA. This result was comparable to NHP (FIGs. 5B and 5C).
  • a similar effect was shown with NA-expressing mRNA and was also comparable to NHP (FIG. 6A and FIG. 6B).
  • the data shows that mRNA-LNP formulation potency may be reliably tested in the HSkMC model.
  • HSkMCs may be transfected with an mRNA encoding a fluorescently labeled antigen (such as GFP) and detected in the cytoplasm of the cells. The number of cells positive for the antigen and fluorescence intensity can be calculated to assess the potency of the mRNA-LNP formulations.
  • the encoded antigen may be secreted into the cell culture media and detected there with antigen specific antibodies which allow direct protein quantitation.
  • the antigen may also be membrane bound and detected on the cell surface by cell imaging and flow cytometry.
  • HSkMCs were transfected with GFP-encoding mRNA-LNP formulations, with expression detected by FACS.
  • the LNP formulations for the mRNA-LNP were as follows:
  • cOrn-EE1 DMG-PEG:Cholesterol:DOPE:cOrn-EE1 at a ratio of 1 .5:38.5:20:40.
  • OF-02 DMG-PEG:Cholesterol:DOPE:OF-02 at a ratio of 1 .5:28.5:30:40.
  • MATE-Suc2-E12 DMG-PEG:Cholesterol:DOPE:MATE-Suc2-E12 at a ratio of 1.5:28.5:30:40.
  • SM 102 DMG-PEG:Cholesterol:DSPC:SM102 at a ratio of 1 .5:38.5:10:50.
  • MC3 DMG ⁇ EG: Cholesterol: DSPC:MC3 at a ratio of 1.5:38.5: 10:50.
  • cKK-E10 DMG-PEG:Cholesterol:DOPE: cKK-E10 at a ratio of 1 .5:40:28.5:30.
  • the HSkMCs yielded reproducible results across three separate experiments.
  • Example 7 Human Skeletal Muscle Myoblasts (HSMMs) as an In vitro Cell Model to Predict LNP Potency and Antibody Response In vivo
  • mice Female Balb/c mice (Mus musculus) per treatment group were immunized under isoflurane anesthesia with a dose of 0.05 mL of designated vaccine preparation or diluent via the IM route in the quadriceps, on day 0 in one hind leg and day 21 in the contralateral leg. Mice that lost more than 20% of their initial body weight and displayed severe clinical signs were euthanized after the veterinarian’s assessment of the animal’s health prior to the study termination.
  • HAI Assay - HAI assays were performed using the Tas20 H3N2 virus stocks (BIOQUAL, Inc.). Sera were treated with receptor-destroying enzyme (RDE) by diluting one part serum with five parts enzyme and incubated overnight in a 37°C water bath. Enzyme was inactivated by a 45-60-m inute incubation period at 56°C followed by addition of six parts PBS for a final dilution of 1/10. HAI assays were performed in V-bottom 96-well plates using four hemagglutinating units (HAU) of virus and 0.5% turkey RBC. The reference serum for each strain was included as a positive control on every assay plate.
  • RDE receptor-destroying enzyme
  • Each plate also included a back-titration to confirm the antigen dose (4 HAU/25pl) as well as a negative control sample (PBS or naive control serum).
  • the HAI titer was determined as the highest dilution of serum resulting in complete inhibition of hemagglutination. Results were only valid for plates with the appropriate back-titration result (verifying 4 HAU/25 pl added) and a reference serum titer within 2-fold of the expected titer.
  • HSMM Cell Culture and Transfection The day before transfection, 10,000 cells/well (Lonza HSMM, CC-2580) were plated in a 96-well plate (100 pL cell suspension/well) in SKGM complete media and incubated overnight at 37°C with 5% CO2. The following day, the cell media was replaced with 100 pl of fresh SKGM media. LNPs encapsulating mRNA encoding for the HA (H3) antigen were diluted to 5 ng/pl working stock in LNP storage buffer. LNPs were transfected at a dose of 10 ng/well and the plate was incubated at 37°C for 20-24 hours. Following incubation, IF imaging was performed to measure protein expression.
  • Example 8 HSMMs Can be Used to Assess the Potency of Modified and Unmodified mRNAs
  • smFISH spot intensities of unformulated mRNA in fixed and permeabilized cells were used for defining single LNP-released single-mRNAs (cytosolic faint spots) and intact LNPs (bright spots).
  • Endosomal escape ratio (ER) is defined herein as the ratio of faint spots to bright spots, which is used to compare the relative endosomal escape efficiencies of different LNP-mRNAs.

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Abstract

La présente invention concerne un système in vitro comprenant une pluralité de cellules musculaires humaines isolées et une pluralité d'ARN messagers (ARNm) encapsulés dans des nanoparticules lipidiques (LNP), et des procédés de criblage d'ARNm encapsulés dans des LNP pour l'expression polypeptidique avec ledit système.
PCT/IB2025/053600 2024-04-05 2025-04-04 Dosages de contrôle de qualité de lnp-arnm de cellules musculaires Pending WO2025210592A1 (fr)

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