WO2024028492A1 - Évaluation quantitative d'encapsulation d'arn - Google Patents

Évaluation quantitative d'encapsulation d'arn Download PDF

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WO2024028492A1
WO2024028492A1 PCT/EP2023/071712 EP2023071712W WO2024028492A1 WO 2024028492 A1 WO2024028492 A1 WO 2024028492A1 EP 2023071712 W EP2023071712 W EP 2023071712W WO 2024028492 A1 WO2024028492 A1 WO 2024028492A1
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rna
fluorophore
lnps
mrna
nucleic acid
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PCT/EP2023/071712
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English (en)
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Adrien NOUGAREDE
Charlène VALADON
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Sanofi
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars

Definitions

  • the present invention relates to the field of RNA analysis and more particularly to the determination of RNA encapsulation efficiency.
  • MRT messenger RNA therapy
  • mRNA messenger RNA
  • LNPs lipid nanoparticles
  • RNA encapsulation efficiency is generally determined using the RiboGreen Assay, which involves a step of LNP lysis with a detergent, typically T riton X-100, to liberate encapsulated nucleic acids such that they can be detected by the RiboGreen® fluorophore.
  • a detergent typically T riton X-100
  • the use of detergent leads to foaming. This is particularly undesirable in high throughput assay conditions where sample volumes are low.
  • the present invention provides improved methods for determining RNA encapsulation efficiency in lipid particles.
  • the methods provide a simple, quick, and cost- effective approach for determining encapsulation efficiency.
  • encapsulation efficiency is an important quality control parameter when generating RNA-based therapeutics. These methods are particularly suitable for use in quality control during or following RNA encapsulation in LNPs and batch release.
  • they may be used in high-throughput and/or automated screening techniques.
  • a method of determining the efficiency of RNA encapsulation in lipid nanoparticles comprising: a) contacting a sample comprising RNA encapsulated in LNPs with a first fluorophore and a second fluorophore, thereby forming fluorophore-RNA complexes, and b) detecting the fluorescence signals of the complexed first and second fluorophores, wherein the first fluorophore permeates the LNPs and wherein the second fluorophore does not permeate the LNPs.
  • the first fluorophore is Quant-iTTM HS or SYBR® Green II.
  • the second fluorophore is RiboGreen® or SYBR® Gold.
  • the RNA is from 10 to 50000 nucleotides in length.
  • the RNA is from 300 to 10000 nucleotides in length.
  • the RNA is from 500 to 5000 nucleotides in length.
  • the RNA is double-stranded RNA or single-stranded RNA.
  • the RNA comprises mRNA, miRNA, shRNA, rRNA, tRNA, snRNA, snoRNA, asRNA, siRNA, aiRNA, gRNA, and/or piRNA.
  • the first fluorophore and the second fluorophore are added to the sample simultaneously.
  • the first fluorophore and the second fluorophore are added to the sample sequentially.
  • RNA is present at a final concentration of at least 0.25 pg/mL, optionally at a final concentration of at least 1 pg/mL. In certain embodiments, RNA is present at a final concentration of comprised between 0.25 and 10 pg/mL.
  • the ratio of the first fluorophore to the second fluorophore is 2:1.
  • the first fluorophore is present at a final concentration of 0.5X.
  • the second fluorophore is present at a final concentration of 0.25X.
  • the method further comprises determining the ratio of the second fluorescent signal to the first fluorescent signal (i.e., ratio of the fluorescent signal of second fluorophore to the fluorescent signal of first fluorophore).
  • the method comprises generating a standard curve for the first and second fluorescent signals.
  • the method comprises determining the absolute amount of encapsulated RNA by matching each fluorescent signal detected in step b) with the corresponding standard curve.
  • a method of manufacturing LNPs encapsulating RNA comprising: a) encapsulating RNA in LNPs, and b) determining efficiency of RNA encapsulation in LNPs according to the method provided herein.
  • the RNA is mRNA.
  • the method further comprises a step of synthesizing mRNA in vitro prior to step a).
  • the LNPs comprise one or more ionizable lipids, one or more helper lipids, and one or more PEG-modified lipids.
  • the in vitro synthesized mRNA is purified prior to encapsulation in LNPs.
  • the encapsulation efficiency is at least 80%.
  • the method is conducted before releasing a batch of LNPs encapsulating RNA.
  • kits for determining efficiency of RNA encapsulation in LNPs comprising:
  • Quant-iTTM HS in quantifying LNP-encapsulated RNA is provided.
  • Fig. 1 Determination of mRNA encapsulation efficiency by the RiboGreen Assay or the method of the invention (the ‘dual RNA encapsulation’ assay). Following encapsulation in LNPs, mRNA is generally present in a sample in two different states: as nonencapsulated or ‘free’ mRNA and as mRNA that has been encapsulated in LNPs (mRNA-LNPs). ‘Total mRNA’ refers to the combination of both free and encapsulated mRNAs.
  • the RiboGreen Assay uses the RiboGreen® fluorophore (black circles), which labels RNA but does not permeate LNPs.
  • non-encapsulated mRNA can be measured with RiboGreen® alone (i.e., in TE buffer) while total mRNA can be measured when RiboGreen® is present in combination with a detergent, such as Triton, which lyses the LNPs.
  • the principle of the present invention is based on the use of two fluorophores labeling RNA (right panel), one of which is nonpermeant (here, RiboGreen®, black circles) while the other is permeant (here, Quant-iTTM HS, white circles).
  • both fluorophores can be used in the same well without overlap of spectral detection or detrimental competition.
  • Fig. 2 Determination of LNP permeability to Quant-iTTM HS. Quant-iTTM HS fluorescence emission was measured for unencapsulated (free) mRNA or encapsulated mRNA at different total mRNA concentrations.
  • Fig. 3 Establishment of linear regression curves for RiboGreen® and Quant-iTTM HS fluorophores when in presence of each other. Eight different concentrations of unencapsulated mRNA were labeled with both RiboGreen® and Quant-iTTM HS. Simple linear regression curves, and the corresponding equations (Y) and R 2 , were determined from these eight points for each fluorophore.
  • Fig. 4 Determination of RiboGreen® and Quant-iTTM HS emissions ratio for the same concentration of labeled mRNA.
  • the RiboGreen® (R) fluorophore does not permeate LNPs; thus, it labelled only non-encapsulated mRNA.
  • the Quant-iTTM HS (Q) fluorophore permeates LNPs; thus, it labelled both encapsulated and non-encapsulated mRNA.
  • the Coefficient of Fluorescence (Ct) between RiboGreen® and Quant-iTTM HS was determined for different concentrations (x) of mRNA labeled with a mixture of RiboGreen® and Quant-iTTM HS.
  • Fig. 5 Determination of encapsulation efficiency without standard RNA curve.
  • the percentage of free mRNA, and thus encapsulation efficiency, can be calculated from the ratio of the RiboGreen® and Quant-iTTM HS emissions in the same well as illustrated.
  • the Ct between RiboGreen® and Quant-iTTM HS for the same concentration of labeled mRNA was experimentally determined to be constant (0.01 ).
  • Fig. 6 Evaluation of the SYTO® 17 fluorophore.
  • A-B Standard mRNA concentration range for RiboGreen® fluorophore alone (A) or with SYTO® 17 in the same well (B). Five different concentrations of free mRNA were labeled with RiboGreen® (0.5X) in TE buffer with or without SYTO® 17 (1 pM). The simple linear regression curves, and the corresponding equations (Y) and R 2 , were determined from the 5 points of the standard mRNA dilution.
  • C Determination of LNP permeability to SYTO® 17. SYTO® 17 fluorescence emission was measured for free mRNA or encapsulated mRNA at different total mRNA concentrations.
  • Fig. 7 Evaluation of the SYBR® Green II and SYBR® Gold fluorophores.
  • SYBR® Green II and SYBR® Gold fluorophore permeability to LNPs was evaluated to determine if these fluorophores could be suitable for use in the dual RNA encapsulation assay.
  • A When using SYBR® Green II, the emission curves obtained for free mRNA and encapsulated mRNA were identical, indicating that this fluorophore permeates LNPs.
  • B When using SYBR® Gold, an emission curve was obtained for free mRNA, with almost no fluorescence emissions detected for encapsulated mRNA, indicating that this fluorophore does not permeate LNPs.
  • the simple linear regression curves determined when labeling mRNA with SYBR® Green II or SYBR® Gold indicate that these fluorophores may be utilized in the dual RNA encapsulation assay in combination with compatible fluorophores.
  • Fig. 8 Automation of Dual RNA encapsulation assay: validation of the method with two different spectrophotometers. Encapsulation efficiency was determined using an automated system and with two different spectrophotometers: Cytation 7 and Spectramax i3. The results were shown to be accurate independently of the spectrophotometer used and also illustrate that method according to the invention is automatable.
  • the present disclosure is directed, inter alia, to methods of determining RNA encapsulation efficiency.
  • a or “an” entity refers to one or more of that entity; for example, “a nucleotide sequence” is understood to represent one or more nucleotide sequences.
  • the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.
  • the term "about” can modify a numerical value above and below the stated value by a variance of, e.g., 10 percent, up or down (higher or lower). In some embodiments, the term indicates deviation from the indicated numerical value by ⁇ 10%, ⁇ 5%, ⁇ 4%, ⁇ 3%, ⁇ 2%, ⁇ 1%, ⁇ 0.9%, ⁇ 0.8%, ⁇ 0.7%, ⁇ 0.6%, ⁇ 0.5%, ⁇ 0.4%, ⁇ 0.3%, ⁇ 0.2%, ⁇ 0.1%, ⁇ 0.05%, or ⁇ 0.01 %. In some embodiments, “about” indicates deviation from the indicated numerical value by ⁇ 10%. In some embodiments, “about” indicates deviation from the indicated numerical value by ⁇ 5%.
  • “about” indicates deviation from the indicated numerical value by ⁇ 4%. In some embodiments, “about” indicates deviation from the indicated numerical value by ⁇ 3%. In some embodiments, “about” indicates deviation from the indicated numerical value by ⁇ 2%. In some embodiments, “about” indicates deviation from the indicated numerical value by ⁇ 1 %. In some embodiments, “about” indicates deviation from the indicated numerical value by ⁇ 0.9%. In some embodiments, “about” indicates deviation from the indicated numerical value by ⁇ 0.8%. In some embodiments, “about” indicates deviation from the indicated numerical value by ⁇ 0.7%. In some embodiments, “about” indicates deviation from the indicated numerical value by ⁇ 0.6%. In some embodiments, “about” indicates deviation from the indicated numerical value by ⁇ 0.5%.
  • “about” indicates deviation from the indicated numerical value by ⁇ 0.4%. In some embodiments, “about” indicates deviation from the indicated numerical value by ⁇ 0.3%. In some embodiments, “about” indicates deviation from the indicated numerical value by ⁇ 0.1%. In some embodiments, “about” indicates deviation from the indicated numerical value by ⁇ 0.05%. In some embodiments, “about” indicates deviation from the indicated numerical value by ⁇ 0.01%.
  • nucleic acid or “nucleic acid molecule” refers to a polynucleotide chain comprising individual nucleic acid residues.
  • a “nucleic acid” encompasses single and/or double-stranded DNA and/or cDNA, as well as single and/or double-stranded RNA.
  • a “nucleic acid,” “DNA,” “RNA,” and/or similar terms include nucleic acid analogs, i.e., analogs having other than a phosphodiester backbone.
  • Nucleic acid sequences that encode proteins and/or RNA may include introns.
  • Nucleic acid may be of any origin, e.g., viral, bacterial, archae-bacterial, fungal, ribosomal, eukaryotic or prokaryotic.
  • Nucleic acid may be purified from natural sources (e.g., from any biological sample and any organism, tissue, cell, or sub-cellular compartment), produced using recombinant expression systems and optionally purified, chemically synthesized, etc.
  • nucleic acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc.
  • a nucleic acid comprises natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3- methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2- aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-prop
  • the nucleic acid is composed of “unmodified nucleic acids,” meaning nucleic acids (e.g., polynucleotides and residues, including nucleotides and/or nucleosides) that have not been chemically modified.
  • the nucleic acid comprises at least one chemical modification.
  • the nucleic acid is RNA. In some embodiments, the nucleic acid is mRNA.
  • mRNA messenger RNA
  • mRNA refers to a polynucleotide that encodes at least one peptide, polypeptide or protein.
  • mRNA may contain one or more coding and non-coding regions.
  • a coding region is alternatively referred to as an open reading frame (ORF).
  • Non-coding regions in mRNA include the 5’ cap, 5’ untranslated region (UTR), 3’ UTR, and a polyA tail.
  • mRNA as used herein encompasses both modified and unmodified RNA.
  • the mRNA disclosed herein may contain one or more modifications that typically enhance RNA stability. Exemplary modifications can include backbone modifications, sugar modifications, or base modifications.
  • the disclosed mRNA may be synthesized from naturally occurring nucleotides and/or nucleotide analogues (modified nucleotides) including, but not limited to, purines (adenine (A) and guanine (G)) or pyrimidines (thymine (T), cytosine (C), and uracil (U)).
  • the disclosed mRNA may be synthesized from modified nucleotide analogues or derivatives of purines and pyrimidines, such as, e.g., 1 -methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl- adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5- methyl-cytosine, 2,6-diaminopurine, 1 -methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1 -methyl-inosine, pseudouracil (5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydroxy
  • the mRNA may comprise at least one chemical modification including, but not limited to, pseudouridine, N1 -methylpseudouridine, 2-thiouridine, 4’-thiouridine, 5-methylcytosine, 2-th io- 1 -methyl-1 -deaza-pseudouridine, 2-thio- 1 -methyl-pseudouridine, 2-th io- 5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4- methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1 -methyl-pseudouridine, 4-thio- pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5- methoxyuridine, and 2’-O-methyl uridine.
  • pseudouridine N1 -methylpseud
  • the chemical modification is selected from pseudouridine, N1 -methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and any combination thereof. In some embodiments, the chemical modification comprises N1 - methylpseudouridine.
  • At least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in the mRNA are chemically modified.
  • at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in the ORF are chemically modified.
  • mRNA can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc.
  • an mRNA is or comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2- aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5- methylcytidine, C-5 propynyl-cytidine, C-5propynyl-uridine, 2-aminoadenosine, C5- bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5- methylcytidine,
  • natural nucleosides e.g., adenosine, guanosine
  • RNA in addition to messenger RNA, several non-coding types of RNA exist which may be involved in regulation of transcription and/or translation, and immunostimulation.
  • RNA further encompasses any type of single stranded (ssRNA) or double stranded RNA (dsRNA) molecule known in the art, such as viral RNA, retroviral RNA and replicon RNA, messenger RNA (mRNA), microRNA (miRNA), small hairpin RNA (shRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), antisense RNA (asRNA), small interfering RNA (siRNA), asymmetrical interfering RNA (aiRNA), CRISPR/Cas9 guide RNA (gRNA), Piwi-interacting RNA (piRNA), dicer-substrate RNA, ribozymes, aptamers, riboswit
  • the RNA is double-stranded RNA. In another embodiment, the RNA is single-stranded RNA. In cases where RNA is single stranded, it may further comprise one or more secondary structures, such as hairpins.
  • RNA is circular RNA (circRNA). In one embodiment, RNA is linear RNA. In one embodiment, the RNA may be any type of RNA provided herein. In one embodiment, the RNA is selected from mRNA, miRNA, shRNA, rRNA, tRNA, snRNA, snoRNA, asRNA, siRNA, aiRNA, gRNA, piRNA and any combination thereof. In one embodiment, the RNA is mRNA. In one embodiment, the RNA is miRNA. In one embodiment, the RNA is siRNA. In one embodiment, the RNA is a combination of mRNA and a second type of RNA, such as siRNA or gRNA. In one embodiment, the mRNA is synthesized in vitro.
  • lipid nanoparticle is a composition comprising one or more lipids.
  • Lipids present in an LNP may include one or more cationic/ionizable, PEGylated, helper, or other lipids, such as phospholipids.
  • LNPs are typically sized on the order of micrometers or smaller and may include a lipid bilayer.
  • Lipid nanoparticles encompass lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes.
  • an LNP may be a liposome having a lipid bilayer with a diameter of 500 nm or less.
  • LNPs are in the shape of a hollow sphere, encapsulating an aqueous compartment.
  • the “lipid component” is that component of an LNP that includes one or more lipids.
  • an LNP can be formed by mixing one or more lipids or by mixing one or more lipids and polymer(s).
  • An LNP may notably contain ionizable/cationic lipid(s) and optionally non-cationic lipid(s), optionally cholesterol-based lipid(s), and/or optionally PEGylated lipid(s).
  • a “noncationic lipid” or “helper lipid” refers to any neutral, zwitterionic, or anionic lipid.
  • a “PEG lipid” or “PEGylated lipid” refers to a lipid comprising a polyethylene glycol component.
  • encapsulation and its grammatical equivalents refer to the process of confining nucleic acid within a nanoparticle. Encapsulation may notably be complete, substantial, or partial. Nucleic acid may be located in an aqueous phase within the liposome or integrated into a lipid layer. As used herein, an “empty” LNP may refer to a nanoparticle that is substantially free of a nucleic acid. As used herein, an “empty” LNP may refer to a nanoparticle that consists substantially of only lipid components. Nucleic acid that is not encapsulated in an LNP is referred to herein as “unencapsulated,” “non-encapsulated, or “free” nucleic acid.
  • encapsulation efficiency and “efficiency of encapsulation” refer to the amount of a nucleic acid that is integrated into the internal structure of an LNP (i.e., that is not accessible to hydrophilic solvents or molecules when the LNP is intact), relative to the initial total amount of nucleic acid used in the preparation of an LNP.
  • the encapsulation efficiency may be given as 95%.
  • Total initial nucleic acid may notably be determined by adding the amount of encapsulated nucleic acid to the amount of nonencapsulated nucleic acid in a given sample.
  • sample comprising nucleic acid encapsulated in LNPs denotes a sample that comprises nucleic acid, e.g., RNA or mRNA, encapsulated in LNPs.
  • the sample comprising nucleic acid encapsulated in LNPs comprises a mixture of nucleic acid encapsulated in LNPs and of unencapsulated nucleic acid.
  • the sample comprising nucleic acid (e.g., RNA or mRNA) encapsulated in LNPs is for example a batch of RNA-LNPs or mRNA-LNPs as obtained by a method of manufacturing LNPs encapsulating RNA or mRNA.
  • RNA-LNP refers to RNA encapsulated in LNP.
  • mRNA-LNP refers to mRNA encapsulated in LNP.
  • labeled refers to attachment of a detectable signal, agent or moiety, such as a fluorophore, to a molecule, such as a nucleic acid molecule.
  • a “fluorophore” or “fluorescent dye” as used herein refers to a chemical group that absorbs light at a particular wavelength (excitation frequency) and subsequently emits light of a longer wavelength (emission frequency), i.e., it fluoresces. Fluorophores may contain substituents that alter the solubility, spectral properties, or physical properties of the fluorophore.
  • a fluorophore may be conditionally fluorescent, i.e., the level of fluorescence increases when the fluorophore binds to its target as compared to the level of fluorescence when the fluorophore is in its unbound form.
  • fluorophores include, but are not limited to, a coumarin, a cyanine dye, a phenanthridinium dye, a bisbenzimide dye, a bisbenzimidazole dye, an acridine dye, a chromomycinone dye, a benzofuran dye, a quinoline dye, a quinazolinone dye, an indole dye, a pyrene dye, a merocyanine dye, a benzocyanine dye, a penzopyrilium dye, a benzazole dye, a borapolyazaindacene dye, a xanthene dye including fluoroscein, rhodamine, or rhodol, as well as other fluorophores described in The Molecular Probes® Handbook: A Guide to Fluorescent Probes and Labeling Technologies (11 th edition, 2010), and US 2005/0208534.
  • detectably distinct refers to a signal that is distinguishable or separable by a physical property either by observation or by instrumentation.
  • a fluorophore is readily distinguishable from another fluorophore in a sample, as well as from additional materials that are optionally present, by spectral characteristics, i.e., excitation and emission spectra.
  • sensitivity range or “sensitivity scale” refers to the range of RNA concentrations for which a given labeling fluorophore gives a linear curve of emission.
  • Permeability refers to the material properties that enable one or more substances to pass through a material.
  • Selectively permeable refers to the material properties that allow specific substances (e.g., fluorophores) to pass through the material while preventing other substances from passing through the material.
  • permeate refers to the ability of a substance (i.e., a fluorophore) to penetrate or pass through a lipid structure, such as the lipid component of an LNP.
  • the ability of a fluorophore to permeate (or not) LNPs can be easily determined by comparing the level of fluorescence detected between two samples: a first sample comprising free mRNA and LNP encapsulated mRNA (mRNA-LNP) with known concentrations of free mRNA / mRNA-LNP (e.g. as can be determined by the RiboGreen assay), and a second sample which has a concentration of free mRNA identical to either the concentration of free mRNA in the first sample, or to the total concentration of mRNA (free mRNA + mRNA-LNP) in the first sample.
  • mRNA-LNP free mRNA and LNP encapsulated mRNA
  • second sample which has a concentration of free mRNA identical to either the concentration of free mRNA in the first sample, or to the total concentration of mRNA (free mRNA + mRNA-LNP) in the first sample.
  • fluorescence levels are the same for the first sample and for a second sample having a concentration of free mRNA identical to the total concentration of mRNA in the first sample, then the fluorophore is permeating. If fluorescence levels are the same for the first sample and for a second sample having a concentration of free mRNA identical to the concentration of free mRNA in the first sample, then the fluorophore is non-permeating.
  • contacting refers to the mixing of two or more components such that those components are capable of interacting (e.g., contacting an RNA-LNP with fluorophores).
  • the two or more components may be incubated for any time sufficient to produce a desired effect (e.g., such that they form a complex).
  • control refers to a standard against which results may be compared. Typically, controls are used to augment integrity in experiments by isolating variables in order to make a conclusion about such variables.
  • a control is a reaction or assay that is performed simultaneously with a test reaction or assay to provide a comparator. In one experiment, the "test” (i.e., the variable being tested) is applied. In the second experiment, the "control," the variable being tested is not applied.
  • a control is a historical control (i.e., of a test or assay performed previously, or an amount or result that is previously known).
  • a control is or comprises a printed or otherwise saved record. A control may be a positive control or a negative control.
  • kits refers to any delivery system for delivering materials. Such delivery systems may include systems that allow for the storage, transport, or delivery of various diagnostic or therapeutic reagents (e.g., oligonucleotides, antibodies, enzymes, etc. in the appropriate containers) and/or supporting materials (e.g., buffers, written instructions for performing the assay etc.) from one location to another.
  • diagnostic or therapeutic reagents e.g., oligonucleotides, antibodies, enzymes, etc. in the appropriate containers
  • supporting materials e.g., buffers, written instructions for performing the assay etc.
  • kits include one or more enclosures (e.g., boxes) containing the relevant reaction reagents and/or supporting materials.
  • fragment kit refers to delivery systems comprising two or more separate containers that each contains a sub-portion of the total kit components. The containers may be delivered to the intended recipient together or separately.
  • a first container may contain an enzyme for use in an assay, while a second container contains oligonucleotides.
  • fragment kit is intended to encompass kits containing Analyte Specific Reagents (ASR's) regulated under section 520(e) of the Federal Food, Drug, and Cosmetic Act, but are not limited thereto. Indeed, any delivery system comprising two or more separate containers that each contains a sub-portion of the total kit components are included in the term “fragmented kit.”
  • a “combined kit” refers to a delivery system containing all of the components in a single container (e.g., in a single box housing each of the desired components).
  • kit includes both fragmented and combined kits.
  • the present invention is based on the use of two fluorophores having different permeability profiles to LNPs and that bind to nucleic acid. Specifically, a first fluorophore permeates the LNP encapsulating the nucleic acid (e.g., an mRNA- LNP) while a second fluorophore does not permeate the LNP. The first fluorophore forms complexes with encapsulated and unencapsulated nucleic acid that may be present in the sample, while the second fluorophore forms complexes with unencapsulated nucleic acid that may be present in the sample.
  • a first fluorophore permeates the LNP encapsulating the nucleic acid (e.g., an mRNA- LNP) while a second fluorophore does not permeate the LNP.
  • the first fluorophore forms complexes with encapsulated and unencapsulated nucleic acid that may be present in the sample, while the second fluorophor
  • the method of the invention reduces the number of samples needed for determining encapsulation efficiency by at least two-fold, as both fluorescent measurements can be performed on a single sample.
  • the method can be used in high-throughput screening, as foaming is no longer an issue in the absence of detergent.
  • the present invention provides a simple, reliable, and efficient quantitative or semi-quantitative approach for assessing RNA encapsulation efficiency.
  • the present invention is particularly useful for quality control during manufacturing and for characterization of encapsulated nucleic acid, such as mRNA, as a pharmaceutical ingredient in final therapeutic products.
  • the present invention provides a method for determining the efficiency of nucleic acid encapsulation in LNPs, said method comprising the steps of: a) contacting a sample comprising nucleic acid encapsulated in LNPs with a first fluorophore and a second fluorophore, thereby forming fluorophore-nucleic acid complexes, and b) detecting the fluorescence signals of the complexed first and second fluorophore, wherein the first fluorophore permeates the LNPs and wherein the second fluorophore does not permeate the LNPs.
  • encapsulation efficiency is desirably high (e.g., close to 100%), e.g., for encapsulated nucleic acid that is to be used in pharmaceutical products
  • an encapsulation efficiency ranging from 0 to 100% may be determined.
  • the encapsulation efficiency that is determined is from 0 to 100%.
  • the encapsulation efficiency is at least about 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.
  • the encapsulation efficiency is at least 80%.
  • the encapsulation efficiency is at least 90%. In some embodiments, the encapsulation efficiency is at least 91%. In some embodiments, the encapsulation efficiency is at least 92%. In some embodiments, the encapsulation efficiency is at least 93%. In some embodiments, the encapsulation efficiency is at least 94%. In some embodiments, the encapsulation efficiency is at least 95%. In some embodiments, the encapsulation efficiency is at least 96%. In some embodiments, the encapsulation efficiency is at least 97%. In some embodiments, the encapsulation efficiency is at least 98%. In some embodiments, the encapsulation efficiency is at least 99%. In some embodiments, the encapsulation efficiency is from 80 to 100%.
  • nucleic acid is present at a final concentration of at least 0.25 pg/mL. In one embodiment, nucleic acid is present at a final concentration comprised between 0.25 and 10 pg/mL. In one embodiment, nucleic acid is present at a final concentration comprised between 0.25 and 5 pg/mL. In one embodiment, nucleic acid is present at a final concentration comprised between 0.25 and 1 .25 pg/mL.
  • nucleic acid is present at a final concentration comprised between 0.25 and 1 pg/mL.
  • a sample comprising nucleic acid encapsulated in LNPs may be subjected to one or more dilutions to provide a nucleic acid concentration that falls within the sensitivity range of the fluorophores.
  • the nucleic acid encapsulated in the LNPs may be any type of nucleic acid as provided herein.
  • Encapsulated nucleic acid molecules e.g., RNA
  • a nucleic acid molecule is at least about 10 nucleotides (nt) in length.
  • a nucleic acid molecule is at least about 20 nt in length.
  • a nucleic acid molecule is at least about 50 nt in length.
  • a nucleic acid molecule is at least about 100 nt in length.
  • a nucleic acid molecule is at least about 500 nt in length.
  • a nucleic acid molecule is from about 10 to 50000 nt in length. In one embodiment, a nucleic acid molecule is from about 20 to 25000 nt in length. In one embodiment, a nucleic acid molecule is from about 300 to 10000 nt in length. In one embodiment, a nucleic acid molecule is from about 300 to 10000 nt in length. In one embodiment, a nucleic acid molecule is from about 400 to 8000 nt in length. In one embodiment, a nucleic acid molecule is from about 500 to 5000 nt in length. In some embodiments, the LNP comprises 1 -20, optionally 5-10 or 6-8, nucleic acid molecules.
  • the first fluorophore provided in the context of the method permeates LNPs.
  • the first fluorophore is the Quant-iTTM High Sensitivity reagent, also referred to herein as Quant-iTTM HS.
  • the Quant-iTTM HS reagent is composed of the RiboRed fluorophore and can therefore alternatively be referred to herein as “RiboRed”.
  • the excitation spectra of Quant-iTTM HS is comprised within the range of 640 nm to 648 nm. In one embodiment, the excitation spectra of Quant-iTTM HS is 644 nm.
  • excitation is performed with a bandwidth of 9 nm (e.g., 644 nm +/- 4.5 nm).
  • the emission spectra of Quant-iTTM HS is comprised within the range of 666 nm to 680 nm.
  • the emission spectra of Quant-iTTM HS is 673 nm.
  • emission spectra is collected with a bandwidth of 15 nm (e.g. 673 nm +/- 7.5 nm).
  • the excitation and emission spectra of Quant-iTTM HS are 644 and 673 nm, respectively.
  • the first fluorophore is the SYBR® Green II fluorophore.
  • the excitation spectra of SYBR® Green II is comprised within the range of 491 nm to 499 nm. In one embodiment, the excitation spectra of SYBR® Green II is 495 nm. In one embodiment, excitation is performed with a bandwidth of 9 nm (e.g., 495 nm +/- 4.5 nm). In one embodiment, the emission spectra of SYBR® Green II is comprised within the range of 513 nm to 527 nm. In one embodiment, the emission spectra of SYBR® Green II is 520 nm.
  • emission spectra is collected with a bandwidth of 15 nm (e.g., 520 nm +/- 7.5 nm).
  • the excitation and emission spectra of SYBR® Green II are 495 and 520 nm, respectively.
  • the second fluorophore provided in the context of the method does not permeate LNPs.
  • the second fluorophore should be detectably distinct from the first fluorophore, to ensure that the fluorescence of each fluorophore can be measured from a single sample.
  • the first fluorophore is a cyanine dye.
  • the second fluorophore is a cyanine dye.
  • the second fluorophore is RiboGreen® (see e.g., Jones et al., Analytical Biochemistry. (1998) 265:368-374). RiboGreen® is detectably distinct from Quant-iTTM HS.
  • RiboGreen® excitation may be performed at 485 ⁇ 10 nm. In one embodiment, RiboGreen® excitation may be performed at 486 ⁇ 5 nm. In one embodiment, the excitation spectra of RiboGreen® is comprised within the range of 475 nm to 495 nm. In one embodiment, the excitation spectra of RiboGreen® is comprised within the range of 470 nm to 491 nm. In one embodiment, the excitation spectra of RiboGreen® is 485 nm. In one embodiment, excitation is performed with a bandwidth of 9 nm (e.g., 485 nm +/- 4.5 nm).
  • RiboGreen® fluorescence emission may be collected at 530 ⁇ 15 nm.
  • the emission spectra of RiboGreen® is comprised within the range of 515 nm to 545 nm.
  • the emission spectra of RiboGreen® is 525 nm.
  • emission spectra is collected with a bandwidth of 15 nm (e.g. 525 nm +/- 7.5 nm).
  • the excitation and emission spectra of RiboGreen® are 485 nm and 525 nm, respectively.
  • the second fluorophore is SYBR® Gold.
  • SYBR® Gold is also referred to as [2-(4- ⁇ [diethyl(methyl)ammonio]methyl ⁇ phenyl)-6-methoxy-1 - methyl-4- ⁇ [(2Z)-3-methyl-1 ,3-benzoxazol2-ylidene]methyl ⁇ quinolin-1 -ium].
  • the excitation spectra of SYBR® Gold is comprised within the range of 491 to 499 nm.
  • the excitation spectra of SYBR® Gold is 495 nm.
  • excitation is performed with a bandwidth of 9 nm (e.g., 495 nm +/- 4.5 nm).
  • the emission spectra of SYBR® Gold is 537 nm. In one embodiment, emission spectra is collected with a bandwidth of 15 nm (e.g. 537 nm +/- 7.5 nm). In one embodiment, the emission spectra of SYBR® Gold is comprised within the range of 530 to 544 nm. In one embodiment, the excitation and emission spectra of SYBR® Gold are 495 and 537 nm, respectively.
  • fluorophores should not generate non-specific fluorescence, e.g., resulting from interactions of the fluorophore with the lipid component of the LNPs.
  • a control sample that does not comprise nucleic acid may be used to determine baseline fluorescence. This baseline may be subtracted from fluorescence emissions detected in corresponding sample(s) comprising LNPs encapsulating nucleic acid.
  • one or both fluorophores are conditionally fluorescent upon binding with nucleic acid.
  • the fluorescent signals of the complexed first and second fluorophores should be detectably distinct.
  • the fluorophores used in the method do not have overlapping excitation and emission spectra. In cases where spectra may overlap, an appropriate cutoff may be used to distinguish between the signals.
  • the first and second fluorophores should not compete for binding to the nucleic acid.
  • the sensitivity range of said first and second fluorophores should encompass the total concentration of nucleic acid (i.e., both unencapsulated and encapsulated) present in the sample.
  • Fluorophore concentrations may be expressed in units (e.g., pg/mL) or as a dilution factor with regards to the initial concentration provided by a manufacturer. For example, the dilution of a 200X concentrated solution to a 1X concentration may be expressed as a dilution of 1 :200. Alternatively, the fluorophore concentrations may be expressed as the final concentration used (e.g., 0.5X, 1X, etc.).
  • the first fluorophore is present at a final concentration of 0.05X to 10X, with regard to the concentration provided by the manufacturer. In one embodiment, the first fluorophore is present at a final concentration of 0.1X to 5X. In one embodiment, the first fluorophore is present at a final concentration of 0.2X to 1X. In one embodiment, the first fluorophore is present at a final concentration of 0.5X. In one embodiment, the second fluorophore is present at a final concentration of 0.05 to 10X. In one embodiment, the second fluorophore is present at a final concentration of 0.075X to 5X. In one embodiment, the second fluorophore is present at a final concentration of 0.1X to 1X.
  • the second fluorophore is present at a final concentration of 0.1 X to 0.5X. In one embodiment, the second fluorophore is present at a final concentration of 0.25X. In one embodiment, the ratio of the first fluorophore to the second fluorophore is comprised within the range of 1 :1 to 20:1 . In one embodiment, the ratio of the first fluorophore to the second fluorophore comprised within the range of 1.5:1 to 10:1. In one embodiment, the ratio of the first fluorophore to the second fluorophore comprised within the range of 2:1 to 4:1. In one embodiment, the ratio of the first fluorophore to the second fluorophore is 4:1 . In one embodiment, the ratio of the first fluorophore to the second fluorophore is 2:1 .
  • the first and second fluorophores are Quant-iTTM HS and RiboGreen®, respectively.
  • the ratio of Quant-iTTM HS to RiboGreen® is 2:1.
  • Quant-iTTM HS is provided at a final concentration of 0.5X and RiboGreen® is provided at a final concentration of 0.25X.
  • fluorophores Prior to the measurement of fluorescence emissions, samples are contacted with the fluorophores for a quantity of time sufficient to allow fluorophore binding to nucleic acid, such that they form a complex.
  • fluorophores are incubated with a sample for more than 5 min. In one embodiment, fluorophores are incubated with a sample for more than 10 min. In one embodiment, fluorophores are incubated with a sample for 15-30 min. In one embodiment, fluorophores are incubated with a sample for 20 min. In one embodiment, fluorophores are incubated with a sample for 25 min. In one embodiment, incubation occurs in the dark. In one embodiment, incubation occurs at room temperature.
  • fluorophores are incubated with a sample for 15-30 min at room temperature in the dark.
  • the above-mentioned fluorophores bind to RNA.
  • Alternative fluorophores may notably be used when the nucleic acid is not RNA.
  • the PicoGreen® fluorophore may be envisaged when the nucleic acid is dsDNA.
  • the first and second fluorophores are added to the sample simultaneously, i.e., in a pre-mixed solution.
  • the first and second fluorophores are added to the sample separately.
  • both fluorophores are added to the sample prior to measurement of fluorescence.
  • addition of the first fluorophore and measurement of the first fluorescent signal of said first fluorophore is followed by addition of the second fluorophore and measurement of the second fluorescent signal, or vice versa.
  • Controls may be used to quantitate the amount of encapsulated nucleic acid.
  • the control comprises a sample with a predetermined amount of nucleic acid.
  • the control comprises a predetermined amount of free nucleic acid.
  • the control comprises a predetermined amount of encapsulated nucleic acid.
  • the control comprises a predetermined amount of total nucleic acid.
  • the control comprises a predetermined amount of both free and encapsulated nucleic acid.
  • the control may be a predetermined amount of free RNA.
  • the control may be an RNA-LNP comprising a predetermined amount of encapsulated RNA.
  • Efficiency of nucleic acid (e.g., RNA or mRNA) encapsulation in lipid nanoparticles is determined based on the detected fluorescence signals of the complexed first and second fluorophores.
  • assays are made quantitative by establishing a calibration curve (also referred to as a standard curve).
  • encapsulation efficiency can be determined qualitatively or semi-quantitatively by comparison of the fluorescent signals detected in unknown samples with known standards or quantitatively by comparison with standard curves prepared using a number of samples of known nucleic acid concentration.
  • quantitation may be performed by making a set of standards or calibrators having a known quantity of total nucleic acid and/or a known quantity of free nucleic acid. These standards or calibrators can be serially diluted, and the resulting signal value from each tested concentration of standard or calibrator is used to generate a standard curve; plotting the concentration of standards versus the resulting signal values.
  • the method further comprises generating a standard curve for the first and second fluorescent signals. In one embodiment, the method further comprises determining the absolute amount of encapsulated RNA by plotting each fluorescent signal that is detected in step b) with the corresponding standard curve.
  • the amount of non-encapsulated nucleic acid (in percent) can then be determined by dividing the amount of nucleic acid detected with the second fluorophore (i.e., the fluorophore that does not permeate LNPs) by that detected with the first fluorophore (i.e., the fluorophore that permeates LNPs).
  • the corresponding equation is illustrated below:
  • Encapsulation efficiency i.e., the relative amount (in percent) of encapsulated nucleic acid
  • Encapsulation efficiency can then be determined by subtracting the quantity of free (unencapsulated) nucleic acid from 100, as illustrated in the equation below:
  • the ratio of fluorescence between the two fluorophores may be used to directly determine encapsulation efficiency (i.e., without the need to generate standard curves).
  • a ratio of fluorescence is first established between the two fluorophores over a range of nucleic acid concentrations. By dividing the fluorescent signal of the second fluorophore by the fluorescent signal of the first fluorophore at each nucleic acid concentration, a coefficient of fluorescence (Ct) can be obtained. In some embodiments, the Ct is 0.01.
  • the fluorescent signal provided by the second fluorophore i.e., the fluorophore that does not permeate LNPs, and that thus binds unencapsulated nucleic acid
  • the fluorescent signal provided by the first fluorophore i.e., the fluorophore that permeates LNPs, and that thus binds total nucleic acid. This allows for a relative amount (in percent) of free nucleic acid to be determined.
  • An exemplary equation is illustrated below:
  • Encapsulation efficiency (in percent) can then be determined by subtracting the quantity of free nucleic acid from 100, as described above.
  • a sample which does not comprise any nucleic acid may be measured, and the corresponding fluorescent signal subtracted from the signal(s) measured in a corresponding sample comprising nucleic acid.
  • the blank may comprise empty LNPs.
  • the ratio of fluorescence is determined at two RNA-LNP dilutions which are then averaged to provide the amount of free nucleic acid (in percent).
  • the estimated final concentration of RNA in a dilution i.e., total RNA is comprised between 0.25 and 10 pg/mL.
  • the estimated final concentration of RNA in a dilution is comprised between 0.25 and 5 pg/mL. In some embodiments, the estimated final concentration of RNA is about 1 and about 1.25 pg/mL for the two RNA-LNP dilutions, respectively.
  • the method provided herein is used to characterize encapsulation of a batch of RNA-LNPs.
  • the method provided herein is performed before releasing a batch of LNPs encapsulating nucleic acid.
  • the encapsulation efficiency determined according to the method provided herein is at least 80% for batch release.
  • a method for batch release comprising: a) determining the efficiency of RNA encapsulation in LNPs according to the method provided herein and b) releasing a batch of RNA-LNPs when the encapsulation efficiency is at least 80%.
  • the present invention further relates to a method of manufacturing LNPs encapsulating nucleic acid, comprising a step a) of encapsulating nucleic acid in LNPs, and a step b) of determining efficiency of nucleic acid encapsulation in LNPs according to the method provided herein.
  • the nucleic acid is RNA, such as mRNA, as described herein.
  • a method of manufacturing LNPs encapsulating RNA comprising a step a) of encapsulating RNA in LNPs, and a step b) of determining efficiency of RNA encapsulation in LNPs is according to the method provided herein is further disclosed.
  • the LNPs comprise one or more ionizable lipids, one or more helper lipids, and one or more PEG-modified lipids.
  • the nucleic acid molecule and/or LNP corresponds to that disclosed in US 2022/0142923, incorporated herein by reference in its entirety.
  • the LNPs may comprise four categories of lipids: (i) an ionizable lipid; (ii) a PEGylated lipid; (iii) a cholesterol- based lipid, and (iv) a helper lipid.
  • An ionizable lipid facilitates mRNA encapsulation and may be a cationic lipid.
  • a cationic lipid affords a positively charged environment at low pH to facilitate efficient encapsulation of the negatively charged mRNA drug substance.
  • the cationic lipid is OF-02.
  • OF-02 is a non-degradable structural analog of OF-Deg-Lin.
  • OF-Deg-Lin contains degradable ester linkages to attach the diketopiperazine core and the doubly-unsaturated tails
  • OF-02 contains non-degradable 1 ,2-amino-alcohol linkages to attach the same diketopiperazine core and the doubly-unsaturated tails (Fenton et al, Adv Mater. (2016) 28:2939; U.S. Pat. 10,201 ,618).
  • the cationic lipid is cKK-E10 (Dong et al., PNAS (2014) 11 1 (11 ):3955-60; U.S. Pat. 9,512,073).
  • the cationic lipid is GL-HEPES-E3-E10-DS-3-E18-1 (2-(4-(2-((3- (Bis((Z)-2-hydroxyoctadec-9-en-1 -yl)amino)propyl)disulfaneyl)ethyl)piperazin-1 -yl)ethyl 4-(bis(2- hydroxydecyl)amino)butanoate), GL-HEPES-E3-E12-DS-4-E10 (2-(4-(2-((3-(bis(2- hydroxydecyl)amino)butyl)disulfaneyl)ethyl)piperazin-1 -yl)ethyl 4-(bis(2- hydroxydodecyl)amino)butanoate), or GL-HEPES-E3-E12-DS-3-E14 (2-(4-(2-((3-(Bis((Z)-2-hydroxy
  • cationic lipids that can be used include those described in Dong, supra; and U.S. Pat. 10,201 ,618.
  • a PEGylated lipid component provides 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 (1990) 268(l):235-7).
  • These components may be selected to rapidly exchange out of the pharmaceutical composition in vivo (see, e.g., U.S. Pat. 5,885,613).
  • Contemplated PEGylated lipids include, but are not limited to, a polyethylene glycol
  • C6-C20 e.g., Cs, C , C12, C14, C15, or C
  • derivatized ceramide e.g., N-octanoyl- sphingosine-1 -[succinyl(methoxypoly ethylene glycol)] (C8 PEG ceramide)
  • the PEGylated lipid is 1 ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG); 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DSPE- PEG); 1 ,2-dilauroyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DLPE-PEG); or 1 .2- distearoyl-rac-glycero-polyethelene glycol (DSG-PEG).
  • DMG-PEG 1 ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol
  • DSPE- PEG 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol
  • DLPE-PEG ,2-dilauroyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol
  • the PEG has a high molecular weight, e.g., 2000-2400 g/mol.
  • the PEG is PEG2000 (or PEG-2K).
  • the PEGylated lipid herein is DMG-PEG2000, DSPE-PEG2000, DLPE-PEG2000, DSG-PEG2000, or C8 PEG2000.
  • a cholesterol component provides stability to the lipid bilayer structure within the nanoparticle.
  • the LNPs comprise one or more cholesterol-based lipids.
  • Suitable cholesterol-based lipids include, for example: DC-Choi (N,N-dimethyl-N- ethylcarboxamidocholesterol), 1 ,4-bis(3-N-oleylamino-propyl)piperazine (Gao et aL, Biochem Biophys Res Comm. (1991 ) 179:280; Wolf et aL, BioTechniques (1997) 23:139; U.S. Pat.
  • a cholesterol-based lipid used in the LNPs is cholesterol.
  • a helper lipid enhances the structural stability of the LNP and helps the LNP in endosome escape. It improves uptake and release of the mRNA drug payload.
  • a helper lipid is a neutral lipid, i.e., a lipid that does not carry a net charge in the conditions under which the composition is formulated and/or administered.
  • a helper lipid is an “anionic lipid", i.e., a lipid that carries a net negative charge at a selected pH, such as physiological pH.
  • 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), 1 ,2-dilauroyl-sn-glycero-3- phosphocholine (DLPC), 1 ,2-distearoylphosphatidylethanolamine (DSPE), and 1 ,2-dilauroyl-sn- glycero-3- phosphoethanolamine (DLPE).
  • DOPE 1,2-dioleoyl
  • helper lipids are dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmito yloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1 -carboxylate (DOPE- mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), phosphatidylserine, sphingolipids, cerebrosides, gangliosides, 16-O-monomethyl PE, 16-0- dimethyl PE, 18-1 -trans PE, 1 -stearoyl-2-oleoyl-phosphat
  • the helper lipid is DOPE.
  • the LNPs comprise (i) a cationic lipid selected from OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1 , GL- HEPES-E3-E12-DS-4-E10, and GL-HEPES-E3-E12-DS-3-E14; (ii) DMG-PEG2000; (iii) cholesterol; and (iv) DOPE.
  • the molar ratio of the cationic lipid in the LNPs relative to the total lipids i.e., A
  • the molar ratio of the PEGylated lipid component relative to the total lipids is 0.25-2.75% (e.g., 1 - 2% such as 1.5%).
  • the molar ratio of the cholesterol-based lipid relative to the total lipids is 20-35% (e.g., 27-30% such as 28.5%).
  • the molar ratio of the helper lipid relative to the total lipids i.e., D
  • the (PEGylated lipid + cholesterol) components have the same molar amount as the helper lipid.
  • the LNPs contain a molar ratio of the cationic lipid to the helper lipid that is more than 1 .
  • the LNPs contain a cationic lipid, a PEGylated lipid, a cholesterol-based lipid, and a helper lipid at a molar ratio of 40: 1.5: 28.5: 30.
  • the LNPs contain (i) OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1 , GL- HEPES-E3-E12-DS-4-E10, or GL-HEPES-E3-E12-DS-3-E14; (ii) DMG-PEG2000; (iii) cholesterol; and (iv) DOPE at 40: 1 .5: 28.5: 30.
  • 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.
  • nucleic acid is encapsulated in an LNP comprising: a cationic lipid at a molar ratio between 35% and 45%, a polyethylene glycol (PEG) conjugated (PEGylated) lipid at a molar ratio between 0.25% and 2.75%, a cholesterol-based lipid at a molar ratio between 20% and 35%, and a helper lipid at a molar ratio of between 25% and 35%, wherein all the molar ratios are relative to the total lipid content of the LNP.
  • PEG polyethylene glycol
  • PEGylated polyethylene glycol
  • cholesterol-based lipid at a molar ratio between 20% and 35%
  • helper lipid at a molar ratio of between 25% and 35%
  • the cationic lipid is OF-02, CKK-E10, GL-HEPES-E3-E10-DS-3-E18-1 , GL-HEPES-E3-E12-DS-4-E10, or GL- HEPES-E3-E12-DS-3-E14.
  • 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 cationic lipid is OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3- E18-1 , GL-HEPES-E3-E12-DS-4-E10, or GL-HEPES-E3-E12-DS-3-E14
  • the PEGylated lipid is dimyristoyl-PEG2000 (DMG-PEG2000)
  • the cholesterol-based lipid is cholesterol
  • the helper lipid is l,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE).
  • the LNP comprises OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1 , GL- HEPES-E3-E12-DS-4-E10, or GL-HEPES-E3-E12-DS-3-E14 at a molar ratio of 40%, DMG- PEG2000 at a molar ratio of 1.5%, cholesterol at a molar ratio of 28.5%, and DOPE at a molar ratio of 30%.
  • the LNP may have a mean diameter of from about 30 nm to about 200 nm, from about 80 nm to about 150 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm, or 200 nm.
  • the LNPs are substantially non-toxic.
  • the one or more nucleic acid molecules, when present in one or more LNPs, are typically resistant to degradation with a nuclease in aqueous solution.
  • RNA-LNPs have an N/P-ratio of 1 -20, 1 -15, 1 -10, 2-8, 2-6, or 2-4.
  • N/P ratio refers to a molar ratio of positively charged molecular units in the cationic lipids in an LNP relative to negatively charged molecular units in the RNA encapsulated within that LNP.
  • N/P ratio is typically calculated as the ratio of moles of amine groups in cationic lipids in a LNP relative to moles of phosphate groups in RNA encapsulated within that LNP.
  • the N/P ratio is from about 1 to about 20, from about 1 to about 18, from about 1 to about 16, from about 1 to about 14, from about 1 to about 12, from about 1 to about 10, from about 1 to about 8, or from about 1 to about 6.
  • the N/P ratio is from about 2 to about 20, from about 2 to about 16, from about 2 to about 12, about 2 to about 8, or from about 2 to about 4.
  • the N/P ratio is from about 4 to about 20, from about 4 to about 16, or from about 4 to 8.
  • the N/P-ratio is above 1 , about 1 , about 2, about 3, about 4, about 5, about 6, about 7, or about 8.
  • the RNA-LNPs have an N/P ratio of 8.
  • the RNA-LNPs have an N/P ratio of 4.
  • the RNA-LNPs have an N/P ratio of 2.
  • the LNP comprises one or more mRNA molecules encoding an antigen (e.g., a viral antigen such as an influenza viral antigen, or a bacterial antigen).
  • an antigen e.g., a viral antigen such as an influenza viral antigen, or a bacterial antigen.
  • LNPs can be prepared by various techniques presently known in the art.
  • 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.
  • Various methods are described in US 2011/0244026, US 2016/0038432, US 2018/0153822, US 2018/0125989, and US 2021/0046192 and can be used to practice the present disclosure.
  • One exemplary process entails encapsulating mRNA by mixing it with a mixture of lipids, without first pre-forming the lipids into lipid nanoparticles, as described in US 2016/0038432.
  • Another exemplary process entails encapsulating mRNA by mixing preformed LNPs with mRNA, as described in US 2018/0153822.
  • nucleic acid is 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, 150 mM NaCI, pH 4.5.
  • preformed LNPs are mixed with nucleic acid under conditions that allow formation of nucleic acid-LNPs in step a).
  • the method comprises a step of synthesizing mRNA in vitro prior to step a).
  • the in vitro synthesized mRNA is purified prior to encapsulation in LNPs.
  • encapsulation efficiency is at least 80%.
  • the methods provided herein may further comprise a step of removing nucleic acid molecules which are not properly encapsulated in an LNP, but which are instead bound to the outer surface of the LNP.
  • RNA molecules that are bound to the outer surface of LNPs may be removed by contacting RNA-LNPs with high salt.
  • the methods provided herein may further comprise a step of dissociating RNA that is bound to the outer surface of LNPs.
  • the step of dissociating RNA may comprise contacting the RNA-LNPs with high salt prior to determining encapsulation efficiency.
  • “High salt” as used herein may refer to salt that is provided at a final concentration ranging from 500 mM to 5 M.
  • salt is provided at a final concentration of 1 M to 5 M. In some embodiments, salt is provided at a final concentration of 0.75 M to 3 M.
  • the salt may be NaCI. In some embodiments, NaCI is used at a final concentration ranging from about 500 mM to 5 M, optionally from 1 to 2 M.
  • the step of dissociating the RNA that is bound to the outer surface of LNPs may additionally require heating.
  • a suitable temperature for the dissociation step may be from about 60°C to 95°C. In some embodiments, the dissociation step is performed at a temperature from about 70°C to about 90°C. In some embodiments, the dissociation step is performed at a temperature from about 80°C to 90°C. In some embodiments, the dissociation step is performed at a temperature of about 85°C.
  • the present invention further provides kits comprising various reagents and materials useful for carrying out inventive methods according to the present invention.
  • the quantitative procedures described herein may be performed by diagnostic laboratories, experimental laboratories, or commercial laboratories.
  • the invention provides kits which can be used in these different settings.
  • kits for quantifying RNA encapsulation efficiency in a sample according to the methods provided herein may be assembled together in a kit.
  • Each kit preferably comprises the reagents which render the procedure specific.
  • a kit comprises two fluorophores, wherein a first fluorophore permeates LNPs and a second fluorophore does not permeate LNPs, such as those described herein.
  • a kit optionally comprises additional reagents, such as a buffer, and instructions for using the kit according to a method of the invention.
  • the present disclosure further provides a kit for determining efficiency of RNA encapsulation in LNPs, comprising: a first fluorophore that permeates LNPs; a second fluorophore that does not permeate LNPs; and optionally, instructions for use according to the method provided herein.
  • Kits or other articles of manufacture according to the invention may include one or more containers to hold various reagents.
  • Suitable containers include, for example, bottles, vials, syringes (e.g., pre-filled syringes), ampules.
  • the container may be formed from a variety of materials such as glass or plastic.
  • kits of the present invention may include suitable control levels or control samples for determining control levels as described herein.
  • the kit may comprise RNA at a known concentration and/or LNPs having a known level of RNA encapsulation.
  • kits of the invention may include instructions for using the kit according to the method provided herein.
  • kits of the invention may further comprise instructions for RNA encapsulation in LNPs.
  • the present invention further relates to the use of Quant-iTTM HS in quantifying LNP- encapsulated RNA.
  • the present invention further relates to the use of Quant-iTTM HS and RiboGreen® in quantifying LNP-encapsulated RNA.
  • the RNA is present at a final concentration of at least 0.25 pg/mL. In one embodiment, the RNA is present at a final concentration comprised between 0.25 pg/mL and 10 pg/mL.
  • the methods provided may be used in the quality control of LNP-encapsulated RNA and batch release of RNA-LNP compositions. Indeed, the present invention is particularly useful for quality control during manufacture of LNP-encapsulated mRNA and for characterization of LNP- encapsulated mRNA as an active pharmaceutical ingredient (API) in final therapeutic products.
  • API active pharmaceutical ingredient
  • the present invention comprises the following embodiments.
  • Embodiment 1 A method of determining the efficiency of RNA encapsulation in lipid nanoparticles (LNPs), comprising: a) contacting a sample comprising RNA encapsulated in LNPs with a first fluorophore and a second fluorophore, thereby forming fluorophore-RNA complexes, and b) detecting the fluorescence signals of the complexed first and second fluorophores, wherein the first fluorophore permeates the LNPs and wherein the second fluorophore does not permeate the LNPs.
  • LNPs lipid nanoparticles
  • Embodiment 2 The method of embodiment 1 , wherein the first fluorophore is Quant- iTTM HS or SYBR® Green II.
  • Embodiment 3 The method of embodiment 1 or 2, wherein the second fluorophore is RiboGreen® or SYBR® Gold.
  • Embodiment 4 The method of any one of the preceding embodiments, wherein the RNA is from 10 to 50000 nucleotides in length.
  • Embodiment 5 The method of any one of the preceding embodiments, wherein the RNA is from 300 to 10000 nucleotides in length.
  • Embodiment 6 The method of any one of the preceding embodiments, wherein the RNA is from 500 to 5000 nucleotides in length.
  • Embodiment 7 The method of any one of the preceding embodiments, wherein the RNA is double-stranded RNA or single-stranded RNA.
  • Embodiment 8 The method of any one of the preceding embodiments, wherein the RNA comprises mRNA, miRNA, shRNA, rRNA, tRNA, snRNA, snoRNA, asRNA, siRNA, aiRNA, gRNA, and/or piRNA.
  • Embodiment 9 The method of any one of the preceding embodiments, wherein the first fluorophore and the second fluorophore are added to the sample simultaneously.
  • Embodiment 10 The method of any one of embodiments 1 -8, wherein the first fluorophore and the second fluorophore are added to the sample sequentially.
  • Embodiment 1 1 The method of any one of the preceding embodiments, wherein RNA is present at a final concentration of at least 0.25 pg/mL, optionally at a final concentration comprised between 0.25 and 10 pg/mL.
  • Embodiment 12 The method of any one of the preceding embodiments, wherein the ratio of the first fluorophore to the second fluorophore is 2:1 .
  • Embodiment 13 The method of any one of the preceding embodiments, wherein the first fluorophore is present at a final concentration of 0.5X.
  • Embodiment 14 The method of any one of the preceding embodiments, wherein the second fluorophore is present at a final concentration of 0.25X.
  • Embodiment 15 The method of any one of the preceding embodiments, wherein the method further comprises generating a standard curve for the first and second fluorescent signals.
  • Embodiment 16 The method of embodiment 15, wherein the method comprises determining the absolute amount of encapsulated RNA by matching each fluorescent signal detected in step b) with the corresponding standard curve.
  • Embodiment 17 The method of any one of embodiments 1 -14, wherein the method further comprises determining the ratio of the second fluorescent signal to the first fluorescent signal.
  • Embodiment 18 A method of manufacturing LNPs encapsulating RNA, comprising: a) encapsulating RNA in LNPs, and b) determining efficiency of RNA encapsulation in LNPs according to the method of any one of embodiments 1 -17.
  • Embodiment 19 The method of embodiment 18, wherein the RNA is mRNA.
  • Embodiment 20 The method of embodiment 19, wherein the method further comprises a step of synthesizing mRNA in vitro prior to step a).
  • Embodiment 21 The method of embodiment 20, wherein the in vitro synthesized mRNA is purified prior to encapsulation in LNPs.
  • Embodiment 22 The method of any one of embodiments 18-21 , wherein lipids are mixed with RNA under conditions that allow formation of LNPs encapsulating RNA.
  • Embodiment 23 The method of any one of embodiments 18-22, wherein the encapsulation efficiency is at least 80%.
  • Embodiment 24 The method of any one of embodiments 18-23, wherein the method is conducted before releasing a batch of LNPs encapsulating RNA.
  • Embodiment 25 The method of any one of the preceding embodiments, wherein the LNPs comprise one or more ionizable lipids, one or more helper lipids, and one or more PEG- modified lipids.
  • Embodiment 26 A kit for determining efficiency of RNA encapsulation in LNPs, comprising:
  • Embodiment 27 Use of Quant-iTTM HS in quantifying LNP-encapsulated RNA.
  • Example 1 Materials & Methods
  • mRNAs were produced as previously described (see Kalnin et al (2021 ), NPJ Vaccines 6(1 ):61 and US 2022/0142923). Briefly, mRNAs incorporating coding sequences were synthesized by in vitro transcription employing RNA polymerase with a plasmid DNA template encoding a desired gene using unmodified or modified nucleotides.
  • An exemplary mRNA (mRNA 1 ) is about 2000 nucleotides in length.
  • the resulting purified precursor mRNA was reacted further via enzymatic addition of a 5' cap structure (Cap 1 ) and a 3' poly(A) tail of approximately 200 nucleotides in length as determined by gel electrophoresis and purified. All mRNA preparations were analyzed for purity, integrity, and percentage of Cap 1 before storage at -80°C.
  • lipids cationic/ionizable lipid, helper lipid, cholesterol and polyethylene glycol-lipid
  • aqueous buffered solution of target mRNA at an acidic pH under controlled conditions to yield a suspension of uniform LNPs.
  • the resulting nanoparticle suspensions were diluted to final concentration, filtered, and stored frozen at -80°C until use.
  • the mRNA-LNP formulations were characterized for size by dynamic light scattering and encapsulation efficiency using the RiboGreen Assay.
  • LNPs were composed of cationic lipid (40%), 1 ,2-dioleoyl-SN-glycero-3- phosphoethanolamine (DOPE; 30%), cholesterol (28.5%), and dimyristoyl-PEG2000 (DMG- PEG; 1.5%).
  • DOPE 1 ,2-dioleoyl-SN-glycero-3- phosphoethanolamine
  • DMG- PEG dimyristoyl-PEG2000
  • Fluorescence emissions also referred to herein as fluorescence
  • fluorescence Fluorescence emissions
  • excitation bandwidth was 9 nm and emission bandwidth was 15 nm.
  • mRNA-LNPs free mRNA and mRNA encapsulated in LNPs
  • TE buffer 10 mM Tris-HCI, 1 mM EDTA, pH 7.5
  • mRNA-LNPs were diluted in TET buffer (TE buffer comprising 0.5% Triton X-100) to a final concentration of 2 ng/pL mRNA.
  • TET buffer TE buffer comprising 0.5% Triton X-100
  • a standard scale of 8 independent dilutions of free mRNA (from 0 to 1 ng/pL) in TE or TET buffers was performed, with 4 independent dilutions of mRNA-LNPs at 20 ng/pL (from 0.5 to 20 ng/pL) in TE and 4 independent dilutions of mRNA-LNPs at 2 ng/pL (from 0.05 to 0.4 ng/pL) in TET.
  • RiboGreen® reagent was diluted to 1 X in TE buffer extemporaneously and 100 pL of the 1X solution was added in each well, such that RiboGreen® was present at a final concentration of 0.5X. After incubation for 10 min in the dark at room temperature, RiboGreen® fluorescence was measured as described in Example 1 . RNA quantification was performed based on a standard simple linear regression curve calculated from RiboGreen® fluorescence emissions of mRNA- LNPs in TE or TET buffers.
  • the non-encapsulated mRNA concentration was determined by RiboGreen® fluorescence in mRNA-LNPs in TE buffer, and the total mRNA concentration by RiboGreen® emission in LNPs in TET buffer. Encapsulation efficiency was then calculated using the following equations:
  • mRNA-LNPs and corresponding free mRNA was diluted in TE at different total concentrations in 100 pL as final volume in a black 96- well microplate with a flat clear bottom.
  • 100 pL of Quant-iTTM HS (2X) or 100pL of SYTO® 17 (2pM) were added in each well and samples were incubated for 20 min at room temperature in the dark. Fluorescence emissions were measured as described above.
  • the fluorescence standard curves were obtained and compared between free mRNA and mRNA-LNP ranges.
  • SYTO® 17 was provided at a final concentration of 1 pM
  • Quant-iTTM HS was provided at a final concentration of 1X.
  • Table 1 Characteristics of fluorophores used in the assay. Quant-iTTM HS was coupled with RiboGreen®.
  • the assay was performed in duplicate for each point from an initial concentration of 2.5 ng/pL for free mRNA and 100 ng/pL for mRNA-LNPs in TE buffer. 8 independent dilutions of free mRNA (from 0 to 2.5 ng/pL) and 8 independent dilutions of mRNA-LNPs (from 0.5 to 20 ng/pL) were performed in a black 96-well microplate with a flat clear bottom using 100 pL as final volume.
  • RiboGreen® and Quant-iTTM HS reagents were diluted extemporaneously to 0.5X and 1X, respectively, in TE buffer and 10OpL of the fluorophore mixture was added in each well, such that RiboGreen® and Quant-iTTM HS were present at a final concentration of 0.25X and 0.5X, respectively. Fluorescence was measured as described in Example 1. Standard simple linear regression curves were established for each fluorophore (Fig. 3). [0184] RNA quantification was performed based on the two standard curves calculated from RiboGreen® and Quant-iTTM HS fluorescence using free mRNA in TE (see Fig. 3).
  • Free mRNA concentration was then determined by measuring RiboGreen® fluorescence in a sample containing mRNA-LNPs, and the total mRNA concentration by measuring Quant-iTTM HS fluorescence in a sample containing mRNA-LNPs.
  • Table 2 Summary of fluorophore concentrations and free and encapsulated mRNA ranges, with the number of data points of dilution analyzed. Free mRNA dilutions of known concentration were used to establish a standard curve needed to quantify free and total mRNA concentrations in the samples. Cone.: concentration, TE: Tris-EDTA, TET: Tris-EDTA-Triton.
  • the assay was performed in triplicate for each point using mRNA-LNP samples at an initial concentration of 20 ng/pL in TE buffer. Two independent dilutions of mRNA-LNP samples (to 2 and 2.5 ng/pL) were performed in TE buffer in a black 96-well microplate with flat clear bottom with 100pL as final volume. A blank without mRNA-LNP was also included to remove background fluorescence. RiboGreen® and Quant-iTTM HS reagents were diluted to 0.5X and 1X respectively in TE buffer extemporaneously and 100pL of the fluorophore mixture was added in each well. RiboGreen® and Quant-iTTM HS emissions were measured as described above.
  • Free mRNA (%) — - > - > — Quant iT HS Emission in mRNA-LNPs — corresponding blank)
  • Encapsulation efficiency (%) 100 — Free mRNA (%)
  • RNA encapsulation efficiency also referred to herein as a ‘dual RNA encapsulation assay’.
  • Fig. 1 An illustration of the principle of the method, as compared to the RiboGreen assay, is provided in Fig. 1 .
  • compatible fluorophores were selected for use in the assay.
  • the two selected fluorophores should not show a spectral overlap (i.e., in excitation and emission wavelengths).
  • the two selected fluorophores should also detect similar mRNA concentration ranges (i.e., have a similar sensitivity scale). This is notably the case for RiboGreen® and Quant-iTTM HS (see Table 1 above).
  • RiboGreen® does not permeate LNPs, given that in the RiboGreen assay a detergent must be added to lyse LNPs in view of measuring fluorescence of total RNA emissions. Thus, the second fluorophore should be able to permeate LNPs.
  • Quant-iTTM HS The emissions of Quant-iTTM HS were measured in the presence of free mRNA or encapsulated mRNA (i.e., mRNA-LNPs) to determine its ability to label total mRNA in LNPs. Quant-iTTM HS emissions were the same between free and encapsulated mRNA demonstrating the permeability properties of this fluorophore as concerns LNPs (see Fig. 2). These results further illustrate that Quant-iTTM HS does not interact with lipids comprising the LNPs.
  • the two fluorophores should also not show any binding to lipids or competition with one another.
  • the quantification of non-encapsulated or total mRNA concentrations is based on a standard free mRNA curve, which must fit with a standard linear regression.
  • RiboGreen® and Quant-iTTM HS were thus tested in the same well for their ability to concomitantly label free mRNA at different concentrations (see Fig. 3). Results showed that emissions from both fluorophores resulted in linear regressions that could be used as standard curves. Moreover, neither RiboGreen® nor Quant-iTTM HS lost their sensitivity range in the presence of the other. Thus, RiboGreen® and Quant-iTTM HS were the two fluorophores selected to further evaluate the dual labeling assay.
  • Example 3 Evaluation of the dual RNA encapsulation assay
  • Table 3 Encapsulation efficiency as determined with the RiboGreen assay or the assay of the present invention.
  • the RiboGreen assay and the dual RNA encapsulation assay were performed as described above using unmodified free mRNA and mRNA encapsulated in LNPs comprising the OF-02 cationic lipid.
  • mRNA concentration was determined based on the standard linear regression lines established for each assay. Standard deviation (%) was calculated for both mRNA concentrations from 4 independent dilution points of mRNA-LNPs in triplicate.
  • Example 4 Determination of the range of encapsulation efficiency that can be measured with the dual RNA encapsulation assay
  • the ability of the dual RNA encapsulation assay to detect different levels of encapsulation efficiency was evaluated. As illustrated in Table 4, below, the assay of the invention was able to accurately quantify both free and total mRNA concentrations in samples comprising LNPs, without the addition of detergent, in contrast to what is required in the classic RiboGreen assay. The determination of encapsulation efficiency was accurate at various encapsulation rates (here, as low as 50%).
  • Table 4 Different encapsulation efficiencies of as determined by the dual RNA encapsulation assay.
  • the dual RNA encapsulation assay was performed as described above using unmodified free mRNA encapsulated in LNPs comprising the OF-02 cationic lipid.
  • Free mRNA was added to the sample to artificially modulate encapsulation efficiency to 70% and 50%.
  • the dual RNA encapsulation assay uses a standard linear regression line calculated from the labeling of free mRNA by both the RiboGreen® and Quant-iTTM HS fluorophores.
  • the non-encapsulated (free) and total mRNA concentrations are then determined respectively by RiboGreen® and Quant-iTTM HS fluorescence. Finally, encapsulation efficiency was determined by the ratio between the concentration of free mRNA and total mRNA. 4 independent dilution points of mRNA-LNPs were measured for each concentration in triplicate.
  • Example 5 Establishment of a high-throughput dual RNA encapsulation assay
  • Table 5 Coefficient of fluorescence between the RiboGreen® and the Quant-iTTM HS fluorophores for the same amount of labeled mRNA.
  • LNPs encapsulating unmodified mRNA were diluted at different rates and the mRNA labeled with both the RiboGreen® and Quant-iTTM HS fluorophores.
  • Table 6 Standard and Dual RNA Encapsulation assays’ biological materia s.
  • the table summarizes fluorophore concentrations and the free and encapsulated mRNA (LNP) ranges used to quantify free and total mRNA concentrations in the mRNA-LNP samples.
  • High-throughput (HT) screening based on the dual RNA encapsulation protocol provided in Example 4, but without absolute quantification of mRNA concentration, was performed.
  • Table 7 Encapsulation efficiency of mRNA-LNPs using a simplified assay.
  • the mRNA- LNP source sample is known to have an encapsulation efficiency of 95%. Free mRNA was added to the sample to artificially modulate encapsulation efficiency to 50%.
  • Example 6 Evaluation of encapsulation efficiency with various mRNA-LNPs
  • Table 8 Encapsulation efficiency as determined by the RiboGreen assay and the dual RNA encapsulation assay of the invention.
  • the encapsulation efficiency of mRNA in 5 different mRNA-LNPs (comprising 3 different cationic lipids and 4 different mRNAs with or without chemically modified nucleosides) were determined in parallel.
  • the standard deviation of encapsulation efficiency was calculated from two different LNP dilutions as described in Example 5 (see Table 6).
  • SYTO® 17 was evaluated to determine if it may be used as an alternative fluorophore in the method of the invention. While a linear standard curve is obtained with RiboGreen® alone, this is no longer the case when RiboGreen® is combined with SYTO® 17 (Fig 6., compare panels A and B). This indicates that RiboGreen® fluorescence is modified by the presence of SYTO® 17. Furthermore, as shown in Fig. 6C, the standard curve established for SYTO® 17 differed for free mRNA and mRNA-LNPs. In particular, fluorescence is higher for mRNA-LNPs than with free mRNA. Without being bound by theory, this suggests that SYTO® 17 may interact with a lipid component of the LNPs.
  • SYBR® Gold and SYBR® Green II were also evaluated to determine if they may be used as alternative fluorophores. As illustrated in Fig. 7, the standard curve obtained for free mRNA and encapsulated mRNA were identical when using SYBR® Green II, indicating that this fluorophore permeates LNPs. In contrast, the standard curve obtained for SYBR® Gold varied according to the sample tested (i.e., free mRNA only or mRNA-LNPs). In particular, almost no fluorescence emissions were detected when mRNA-LNPs were contacted with SYBR® Gold. This suggests that this fluorophore does not permeate LNPs.
  • Example 8 Automation of dual RNA encapsulation assay and validation of the method with two different spectrophotometers
  • the dual RNA encapsulation assay was implemented on a Starlet Hamilton robotic platform in view of automating the method.
  • a source sample comprising unmodified mRNA encapsulated in LNPs comprising the OF-02 cationic lipid known to have a total mRNA concentration of 1000 pg/mL and an encapsulation efficiency of 95% (as determined by the RiboGreen assay described in Example 1 above) was used.
  • Unmodified free mRNA was added to the source sample to artificially modulate encapsulation efficiency to 70% and 50%.
  • Various concentrations of these mRNA-LNPs (ranging from 20 to 200 ng/pL) were loaded in a PCR 96- well plate using 40 pL as final volume.
  • the method of the invention represents an improved method of determining encapsulation efficiency.
  • the number of samples required is reduced by at least 2-fold, as dual measurements are able to be determined from a single well.
  • the method can be used in both quantitative and high-throughput screening approaches and shows a high level of accuracy in determining encapsulation efficiency of nucleic acid in LNPs.
  • the ratiometric approach provided in Example 5 for measurement of LNP-RNA encapsulation efficiency
  • many different RNAs can be evaluated without the need to provide a standard curve for each sample for absolute quantification of free and encapsulated mRNA.
  • the protocol is fully automated with up to 95 samples processed in less than two hours and requires only limited amounts of raw materials (e.g. mRNA-LNP samples, fluorophores).

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Abstract

La présente invention concerne des procédés de détermination de l'efficacité d'encapsulation d'ARN dans des nanoparticules lipidiques (LNP). Dans certains modes de réalisation, les procédés selon la présente invention comprennent une étape a) de mise en contact d'un échantillon comprenant de l'ARN encapsulé dans des LNP avec un premier fluorophore et un second fluorophore, formant ainsi des complexes fluorophore-ARN, et une étape b) de détection des signaux de fluorescence des premier et second fluorophore complexés, le premier fluorophore pénéatrant les LNP et le second fluorophore ne pénétrant pas les LNP.
PCT/EP2023/071712 2022-08-04 2023-08-04 Évaluation quantitative d'encapsulation d'arn WO2024028492A1 (fr)

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