Quantitative assessment of RNA encapsulation

Abstract

The present invention relates to a method for determining the efficiency of RNA encapsulation in lipid nanoparticles (LNPs). In some embodiments, a method according to the invention comprises the steps of a) contacting a sample comprising RNA encapsulated in LNP with a first fluorophore and a second fluorophore, thereby forming a fluorophore-RNA complex, and b) detecting a fluorescence signal of the complexed first and second fluorophore, wherein the first fluorophore permeates the LNPs and wherein the second fluorophore does not permeate the LNPs.

Description

Technical Field

[0001] The present invention relates to the field of RNA analysis and more particularly to the determination of RNA encapsulation efficiency. Background Art

[0002] Messenger RNA therapy (MRT) is becoming an increasingly important method for treating a variety of diseases. MRT involves administering messenger RNA (mRNA) to patients in need of the therapy to produce proteins encoded by the mRNA in the patient. In order to ensure that RNA is effectively delivered to host cells in vivo, RNA is usually encapsulated in a carrier such as lipid nanoparticles (LNPs). Therefore, accurate characterization of RNA encapsulation efficiency is particularly important for determining the quality of mRNA for therapeutic applications.

[0003] RNA encapsulation efficiency is usually determined using the RiboGreen assay, which involves a LNP lysis step with a detergent (typically Triton X-100) to release the encapsulated nucleic acids so that they can be accessed by The fluorophore is detected. However, the use of detergents leads to foaming. This is particularly undesirable under high-throughput assay conditions where sample volumes are low. Furthermore, to determine the encapsulation efficiency, it is necessary to perform two separate measurements of the untreated sample and the sample treated with detergent to determine the free and total mRNA content, respectively, which allows calculation of the proportion of the encapsulated mRNA payload.

[0004] Therefore, there remains a need for improved methods for determining RNA encapsulation efficiency. Such improved methods should be particularly rapid, robust, cost-effective, and easy to perform. Summary of the invention

[0005] The present invention provides an improved method for determining RNA encapsulation efficiency in lipid particles. Advantageously, these methods provide a simple, quick and cost-effective method for determining encapsulation efficiency. As discussed above, when producing RNA-based therapeutic agents, encapsulation efficiency is an important quality control parameter. These methods are particularly useful for RNA encapsulation in LNP and batch release during or after quality control. Advantageously, they can be used in high throughput and / or automated screening techniques.

[0006] Provided herein is a method for determining RNA encapsulation efficiency in lipid nanoparticles (LNPs), the method comprising: a) contacting a sample comprising RNA encapsulated in LNPs with a first fluorophore and a second fluorophore, thereby forming a fluorophore-RNA complex, and b) detecting the fluorescent signal of the composite first and second fluorophores, Wherein the first fluorophore permeates the LNPs, and wherein the second fluorophore does not permeate the LNPs.

[0007] In certain embodiments, the first fluorophore is Quant-iT TM HS or Green II.

[0008] In certain embodiments, the second fluorophore is or Gold.

[0009] In certain embodiments, the RNA is 10 to 50,000 nucleotides in length.

[0010] In certain embodiments, the RNA is 300 to 10,000 nucleotides in length.

[0011] In certain embodiments, the RNA is 500 to 5000 nucleotides in length.

[0012] In certain embodiments, the RNA is double-stranded RNA or single-stranded RNA.

[0013] In certain embodiments, the RNA comprises mRNA, miRNA, shRNA, rRNA, tRNA, snRNA, snoRNA, asRNA, siRNA, aiRNA, gRNA and / or piRNA.

[0014] In certain embodiments, the first fluorophore and the second fluorophore are added to the sample simultaneously.

[0015] In certain embodiments, the first fluorophore and the second fluorophore are added to the sample sequentially.

[0016] In certain embodiments, the RNA is present at a final concentration of at least 0.25 μg / mL, optionally at a final concentration of at least 1 μg / mL. In certain embodiments, the RNA is present at a final concentration comprised between 0.25 and 10 μg / mL.

[0017] In certain embodiments, the ratio of the first fluorophore to the second fluorophore is 2:1.

[0018] In certain embodiments, the first fluorophore is present at 0.5X the final concentration.

[0019] In certain embodiments, the second fluorophore is present at 0.25X the final concentration.

[0020] In certain embodiments, the method further includes determining a ratio of the second fluorescent signal to the first fluorescent signal (ie, a ratio of the fluorescent signal of the second fluorophore to the fluorescent signal of the first fluorophore).

[0021] In certain embodiments, the method includes generating a standard curve for the first and second fluorescent signals.

[0022] In certain embodiments, the method comprises determining the absolute amount of encapsulated RNA by matching each fluorescent signal detected in step b) to a corresponding standard curve.

[0023] In another aspect, a method of making an LNP encapsulating RNA is provided, the method comprising: a) encapsulating RNA in LNPs, and b) Determining the efficiency of RNA encapsulation in LNPs according to the methods provided herein.

[0024] In certain embodiments, the RNA is mRNA.

[0025] In certain embodiments, the method further comprises the step of synthesizing mRNA in vitro prior to step a).

[0026] In certain embodiments, the LNP comprises one or more ionizable lipids, one or more helper lipids, and one or more PEG-modified lipids.

[0027] In certain embodiments, in vitro synthesized mRNA is purified prior to encapsulation in LNPs.

[0028] In certain embodiments, the encapsulation efficiency is at least 80%.

[0029] In certain embodiments, the method is performed prior to releasing a batch of RNA-encapsulated LNPs.

[0030] In another aspect, a kit for determining RNA encapsulation efficiency in LNPs is provided, the kit comprising: - a first fluorophore that permeates the LNP; - a second fluorophore that does not permeate the LNP; and - Instructions for use according to the methods provided herein.

[0031] In another aspect, Quant-iT TM Use of HS for quantification of LNP-encapsulated RNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1: mRNA encapsulation efficiency is determined by RiboGreen assay or the method of the present invention ("dual RNA encapsulation" assay). After encapsulation in LNPs, mRNA is usually present in the sample in two different states: as non-encapsulated or "free" mRNA and mRNA that has been encapsulated in LNPs (mRNA-LNP). "Total mRNA" refers to the combination of both free and encapsulated mRNA. The RiboGreen assay (left panel) uses Fluorophores (black circles) that label RNA but do not permeate LNPs. Therefore, they can be used alone (i.e., in TE buffer) to measure non-encapsulated mRNA, whereas In combination with a detergent that cleaves the LNPs (such as Triton), total mRNA can be measured. The principle of the present invention is based on the use of two fluorophores to label RNA (right panel), one of which is non-permeable (here black circle), and the other is permeabilizing (here Quant-iT TM HS, white circles). Advantageously, both fluorophores can be used in the same well without overlap or deleterious competition in spectral detection.

[0033] Figure 2 : Quant-iT TM Determination of LNP permeability of HS. Quant-iT assay was used to measure the permeability of unencapsulated (free) mRNA or encapsulated mRNA at different total mRNA concentrations. TM HS fluorescence emission.

[0034] Figure 3 :when and Quant-iT TM The linear regression curve was established when HS fluorophores existed with each other. and Quant-iT TM HS and HS labeled eight different concentrations of unencapsulated mRNA. Simple linear regression curve, and the corresponding equation (Y) and R 2 , determined from these eight spots for each fluorophore. (A) In Quant-iT TM In the presence of HS, the Emission as a function of free mRNA concentration. (B) In the presence of TM HS emission as a function of free mRNA concentration.

[0035] Figure 4 : The same concentration of labeled mRNA and Quant-iT TMHS emission ratio determination. (R) The fluorophore does not permeate the LNP; therefore, it only labels non-encapsulated mRNA. Quant-iT TM The HS(Q) fluorophore permeates the LNP; therefore, it labels both encapsulated and non-encapsulated mRNA. and Quant-iT TM The mixture of HS labeled mRNA with different concentrations (x) determined and Quant-iT TM The fluorescence coefficient between HS (C f ). Several factors of mRNA-LNP dilution were used to adjust the xR and xQ labeled mRNAs, allowing calculation of C for different x f .

[0036] Figure 5 : Determination of encapsulation efficiency without standard RNA curve. The percentage of free mRNA, and hence the encapsulation efficiency, can be obtained from the same well as shown and Quant-iT TM For the same concentration of labeled mRNA, and Quant-iT TM C between HS f Determined by experiments as a constant (0.01).

[0037] Figure 6 : Evaluation of 17 fluorophores. (AB) Individual Fluorophore (A) or 17 Standard mRNA concentration range in the same well (B). 17 (1 μM) in TE buffer. (0.5X) Five different concentrations of free mRNA were labeled. Simple linear regression curve, and the corresponding equation (Y) and R 2 , determined from 5 dilutions of standard mRNA. (C) The permeability of LNPs was determined by measuring the permeability of free mRNA or encapsulated mRNA at different total mRNA concentrations. 17 Fluorescence emission.

[0038] Figure 7 : Green II and Evaluation of Gold fluorophores. Green II and The permeability of gold fluorophores into LNPs was investigated to determine whether these fluorophores are suitable for dual RNA encapsulation assays. (A) When using When using fluorophore Green II, the emission curves obtained for free mRNA and encapsulated mRNA are identical, indicating that this fluorophore permeates the LNP. (B) When using 5-mercaptoethanol Gold, emission curves of free mRNA were obtained, and almost no fluorescence emission from encapsulated mRNA was detected, indicating that this fluorophore does not permeate the LNP. Green II or Simple linear regression curves determined when Gold-labeled mRNAs indicate that these fluorophores can be used in combination with compatible fluorophores in dual RNA encapsulation assays.

[0039] Figure 8 : Automation of the dual RNA encapsulation assay: Validation of the method with two different spectrophotometers. The encapsulation efficiency was determined using an automated system and two different spectrophotometers: Cytation 7 and Spectramax i3. The results proved to be accurate, independent of the spectrophotometer used, and also demonstrated that the method according to the invention is automatable. DETAILED DESCRIPTION

[0040] The present disclosure relates particularly to methods of determining RNA encapsulation efficiency.

[0041] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this disclosure should have the meaning commonly understood by those of ordinary skill in the art. Although methods and materials similar to or equivalent to those described herein can also be used in the practice or test of this disclosure, exemplary methods and materials are described below. In the event of a conflict, this specification including the definition shall prevail. Usually, the nomenclature and technology used in conjunction with cell and tissue culture, molecular biology, virology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicine and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization as described herein are those nomenclatures and technologies well known and commonly used in the art. Enzymatic reactions and purification techniques are carried out as usually realized in this area or as described herein according to the manufacturer's instructions. Further, unless the context requires otherwise, singular terms should include plural numbers, and plural terms should include singular numbers. Throughout the present specification and examples, the words "have" and "comprise" or variations such as "has / having", "comprises / comprising" should be understood to mean the inclusion of the stated integer or groups of integers but not the exclusion of any other integer or groups of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0042] It should be noted that the term "a" or "an" entity refers to one or more of the entity; for example, "nucleotide sequence" should be understood to mean one or more nucleotide sequences. Therefore, the terms "a / an", "one or more", and "at least one" can be used interchangeably herein.

[0043] In addition, "and / or" where used herein should be regarded as a specific disclosure of each of the two specified features or components with or without the other. Therefore, the term "and / or" as used in phrases such as "A and / or B" is intended to include "A and B", "A or B", "A" (alone) and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B and / or C" is intended to cover each of the following aspects: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0044] It should be understood that wherever aspects are described herein with the language "comprising," similar aspects described in the form of "consisting of" and / or "consisting essentially of" are also provided.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure relates. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; The Dictionary Of Cell And Molecular Biology, 3rd edition, 1999, Academic Press; and Oxford Dictionary Of Biochemistry And Molecular Biology, Revised edition, 2000, Oxford University Press can provide a general dictionary of many of the terms used in the present disclosure for one of ordinary skill.

[0046] Units, prefixes and symbols are all expressed in a form acceptable to the International System of Units (SI). Numerical ranges include numbers that define the range. The headings provided herein are not limitations on the various aspects of the present disclosure. Therefore, the terms of the definitions immediately below are more fully defined by reference to the specification (in its entirety).

[0047] The term "approximately" or "about" is used herein to mean approximately, roughly, approximately, or about. When the term "approximately" is used in conjunction with a numerical range, it defines the range by extending the boundary above and below the numerical value described. In general, the term "approximately" can limit the numerical value to a change of, for example, 10% above and below (higher or lower) above and below the specified value. In some embodiments, the term represents a deviation of ±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% from the indicated numerical value. In some embodiments, "approximately" represents a deviation of ±10% from the indicated numerical value. In some embodiments, "approximately" represents a deviation of ±5% from the indicated numerical value. In some embodiments, "approximately" represents a deviation of ±4% from the indicated numerical value. In some embodiments, "approximately" represents a deviation of ±3% from the indicated numerical value. In some embodiments, "approximately" represents a deviation of ±2% from the indicated numerical value. In some embodiments, "about" means a deviation of ±1% from the indicated value. In some embodiments, "about" means a deviation of ±0.9% from the indicated value. In some embodiments, "about" means a deviation of ±0.8% from the indicated value. In some embodiments, "about" means a deviation of ±0.7% from the indicated value. In some embodiments, "about" means a deviation of ±0.6% from the indicated value. In some embodiments, "about" means a deviation of ±0.5% from the indicated value. In some embodiments, "about" means a deviation of ±0.4% from the indicated value. In some embodiments, "about" means a deviation of ±0.3% from the indicated value. In some embodiments, "about" means a deviation of ±0.1% from the indicated value. In some embodiments, "about" means a deviation of ±0.05% from the indicated value. In some embodiments, "about" means a deviation of ±0.01% from the indicated value.

[0048] As used herein, the term "nucleic acid" or "nucleic acid molecule" refers to a polynucleotide chain comprising a single nucleic acid residue. "Nucleic acid" includes single-stranded and / or double-stranded DNA and / or cDNA, and single-stranded and / or double-stranded RNA. In addition, "nucleic acid", "DNA", "RNA" and / or similar terms include nucleic acid analogs, i.e., analogs without a phosphodiester backbone. For example, so-called "peptide nucleic acids" known in the art and having peptide bonds rather than phosphodiester bonds in the backbone are considered to be within the scope of the present invention. The nucleic acid sequence encoding protein and / or RNA may include introns. Nucleic acid may be from any source, such as viruses, bacteria, archaea, fungi, ribosomes, eukaryotic or prokaryotic. Nucleic acid can be purified from natural sources (e.g., from any biological sample and any organism, tissue, cell or subcellular compartment), produced using a recombinant expression system, and optionally purified, chemically synthesized, etc. Where appropriate, for example, in the case of chemically synthesized molecules, nucleic acids may include nucleoside analogs, such as analogs with chemically modified bases or sugars, backbone modifications, etc. In some embodiments, the 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, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl -cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); inserted bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite bonds). In some embodiments, the nucleic acid consists of "unmodified nucleic acid", meaning a nucleic acid (e.g., polynucleotides and residues, including nucleotides and / or nucleosides) that has not been chemically modified. In some embodiments, the nucleic acid comprises at least one chemical modification. In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid is mRNA.

[0049] As used herein, the term "messenger RNA (mRNA)" refers to a polynucleotide encoding at least one peptide, polypeptide or protein. An mRNA may contain one or more coding regions and non-coding regions. A coding region may alternatively be referred to as an open reading frame (ORF). Non-coding regions in an mRNA include a 5' cap, a 5' untranslated region (UTR), a 3' UTR and a poly A tail.

[0050] As used herein, mRNA encompasses both modified RNA and unmodified RNA. In certain embodiments, mRNA disclosed herein may contain one or more modifications that typically enhance RNA stability. Exemplary modifications may include backbone modifications, sugar modifications, or base modifications. In certain embodiments, disclosed mRNA may be synthesized by naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides) (including but not limited to purines (adenine (A) and guanine (G)) or pyrimidines (thymine (T), cytosine (C) and uracil (U))). In certain embodiments, the disclosed mRNA can be synthesized from modified nucleotide analogs or derivatives of purines and pyrimidines, such as, for example, 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-carboxy uracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, quercetin, pD-mannosyl-quercetin, phosphoramidate, phosphorothioate, peptide nucleotides, methylphosphonate, 7-deazaguanosine, 5-methylcytosine and inosine.

[0051] In some embodiments, the mRNA may include at least one chemical modification, including but not limited to pseudouridine, N1-methyl pseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thiol-1-methyl-1-deaza-pseudouridine, 2-thiol-1-methyl-pseudouridine, 2-thiol-5-aza-uridine, 2-thiol-dihydropseudouridine, 2-thiol-dihydrouridine, 2-thiol-pseudouridine, 4-methoxy-2-thiol-pseudouridine, 4-methoxy-pseudouridine, 4-thiol-1-methyl-pseudouridine, 4-thiol-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine and 2'-O-methyluridine. In some embodiments, the chemical modification is selected from pseudouridine, N1-methyl pseudouridine, 5-methylcytosine, 5-methoxyuridine and any combination thereof. In some embodiments, the chemical modification comprises N1-methylpseudouridine.

[0052] In some embodiments, 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. In some embodiments, 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.

[0053] Where appropriate, e.g., in the case of chemically synthesized molecules, the mRNA may comprise nucleoside analogs, e.g., analogs with chemically modified bases or sugars, backbone modifications, etc. In some embodiments, the mRNA is or comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-amino 5-methylcytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); inserted bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite bonds).

[0054] In addition to messenger RNA, there are also several non-coding types of RNA, which may be involved in the regulation of transcription and / or translation and immune stimulation. In the present invention, the term "RNA" further encompasses any type of single-stranded (ssRNA) or double-stranded RNA (dsRNA) molecules 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), asymmetric interfering RNA (aiRNA), CRISPR / Cas9 guide RNA (gRNA), Piwi interacting RNA (piRNA), dicer substrate RNA, ribozymes, aptamers, riboswitches, immunostimulatory RNA and long non-coding RNA (IncRNA) and any combination thereof.

[0055] In one embodiment, the RNA is double-stranded RNA. In another embodiment, the RNA is single-stranded RNA. In the case where the RNA is single-stranded, it may further include one or more secondary structures, such as hairpins. In one embodiment, the RNA is circular RNA (circRNA). In one embodiment, the RNA is linear RNA. In one embodiment, the RNA can 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.

[0056] As used herein, "lipid nanoparticle (LNP)" is a composition comprising one or more lipids. The lipid present in the LNP can include one or more cationic / ionizable, PEGylated, auxiliary or other lipids, such as phospholipids. The size of the LNP is typically on the order of micrometer or less, and can include a lipid bilayer. Unless otherwise specified, lipid nanoparticles as used herein encompass lipid nanoparticles (LNP), liposomes (e.g., lipid vesicles) and lipid complexes. In certain embodiments, the LNP can be a liposome with a lipid bilayer having a diameter of 500nm or less. Typically, the LNP is in the shape of a hollow sphere, encapsulating an aqueous compartment. "Lipid component" is a component of the LNP comprising one or more lipids. Typically, the LNP can be formed by mixing one or more lipids or by mixing one or more lipids and one or more polymers. The LNP can contain one or more ionizable / cationic lipids and optionally one or more non-cationic lipids, optionally one or more cholesterol-based lipids, and / or optionally one or more PEGylated lipids. "Non-cationic lipid" or "auxiliary lipid" refers to any neutral, zwitterionic or anionic lipid. "PEG lipid" or "PEGylated lipid" refers to a lipid that comprises a polyethylene glycol component.

[0057] As used herein, "encapsulation" and its grammatical equivalents refer to the process of confining nucleic acid in nanoparticles. Encapsulation can be particularly complete, basic or partial. The nucleic acid can be located in the aqueous phase within the liposome or integrated into the lipid layer. As used herein, "empty" LNP can refer to a nanoparticle that is substantially free of nucleic acid. As used herein, "empty" LNP can refer to a nanoparticle that is substantially only composed of lipid components. Nucleic acids that are not encapsulated in LNP are referred to herein as "unencapsulated", "non-encapsulated" or "free" nucleic acids.

[0058] As used herein, "encapsulation efficiency" and "encapsulated efficiency" refer to the amount of nucleic acid integrated into the LNP internal structure (i.e., when the LNP is complete, the hydrophilic solvent or molecule cannot access) relative to the initial total amount of nucleic acid used in the LNP preparation. As an example, if 95 mg of nucleic acid is encapsulated in the LNP out of a total of 100 mg of nucleic acid initially provided to the composition, the encapsulation efficiency can be given as 95%. It should be noted that the total initial nucleic acid can be determined by adding the amount of the encapsulated nucleic acid in a given sample to the amount of the non-encapsulated nucleic acid.

[0059] As used herein, the term "sample comprising nucleic acids encapsulated in LNPs" means a sample comprising nucleic acids (e.g., RNA or mRNA) encapsulated in LNPs. In certain embodiments, the sample comprising nucleic acids encapsulated in LNPs comprises a mixture of nucleic acids encapsulated in LNPs and unencapsulated nucleic acids. The sample comprising nucleic acids (e.g., RNA or mRNA) encapsulated in LNPs is, for example, a batch of RNA-LNPs or mRNA-LNPs obtained by a method for making LNPs encapsulating RNA or mRNA.

[0060] As used herein, the term "RNA-LNP" refers to RNA encapsulated in LNP. Similarly, "mRNA-LNP" refers to mRNA encapsulated in LNP.

[0061] As used herein, the term "labeled" refers to the attachment of a detectable signal, agent, or moiety (eg, a fluorophore) to a molecule (eg, a nucleic acid molecule).

[0062] As used herein, "fluorophore" or "fluorescent dye" refers to a chemical group that absorbs light of a specific wavelength (excitation frequency) and subsequently emits light of a longer wavelength (emission frequency) (i.e., it fluoresces). A fluorophore 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 is bound to its target compared to the level of fluorescence when the fluorophore is in its unbound form. Many fluorophores are known to those skilled in the art and include, but are not limited to, coumarins, cyanine dyes, phenanthridinium dyes, bisbenzimide dyes, bisbenzimidazole dyes, acridine dyes, chromomycinone dyes, benzofuran dyes, quinoline dyes, quinazolinone dyes, indole dyes, pyrene dyes, merocyanine dyes, benzocyanine dyes, penzopyrilium dyes, benzoxazole dyes, borapolyazaindacene dyes, xanthene dyes (including fluorescein, rhodamine or rhodol), and the fluorophores described in The Molecular Handbook:A Guide to FluorescentProbes and LabelingTechnologies[Molecular Handbook: A Guide to Fluorescent Probes and Labeling Technologies] (11th ed., 2010) and other fluorophores described in US 2005 / 0208534.

[0063] As used herein, the term "detectably different" refers to signals that are distinguishable or separable by physical properties by observation or instrumentation. For example, a fluorophore is easily distinguishable from another fluorophore in a sample and optionally additional materials present by spectral characteristics, i.e., excitation and emission spectra.

[0064] As used herein, the term "sensitivity range" or "sensitivity scale" refers to the range of RNA concentrations over which a given labeled fluorophore gives a linear emission curve.

[0065] As used herein, "permeability" refers to a material property that enables one or more substances to pass through a material. "Selectively permeable" refers to a material property that allows a specific substance (e.g., a fluorophore) to pass through a material while preventing other substances from passing through the material. In the context of the present invention, the term "permeation" 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). By comparing the fluorescence level detected between two samples, the ability of a fluorophore to penetrate (or not penetrate) LNP can be easily determined: a first sample, comprising mRNA (mRNA-LNP) encapsulated by free mRNA and LNP, free mRNA / mRNA-LNP (e.g., as can be determined by RiboGreen assay), and a second sample, having the same free mRNA concentration as the free mRNA in the first sample or as the total concentration of mRNA (free mRNA+mRNA-LNP) in the first sample. If the first sample and the second sample with the same free mRNA concentration as the total concentration of mRNA in the first sample have the same fluorescence level, then the fluorophore penetrates. If the fluorescence levels of a first sample and a second sample having the same free mRNA concentration as the free mRNA concentration in the first sample are the same, then the fluorophore is non-permeable.

[0066] As used herein, the term "contacting" refers to mixing two or more components so that those components can interact (e.g., contacting RNA-LNP with a fluorophore). The two or more components can be incubated for any time sufficient to produce the desired effect (e.g., so that they form a complex).

[0067] As used herein, the term "control" refers to a standard with which a result can be compared. Typically, control is used to enhance integrity in an experiment by separating variables, so as to draw conclusions about such variables. In certain embodiments, control is a reaction or determination carried out simultaneously with a test reaction or determination to provide a comparator. In an experiment, "test" (i.e., the variable tested) was applied. In a second experiment, "control", i.e., the variable tested, was not applied. In certain embodiments, control is a historical control (i.e., a control of a previously performed test or determination, or a previously known amount or result). In certain embodiments, control is or includes a record printed or otherwise preserved. Control can be a positive control or a negative control.

[0068] As used herein, the term "kit" refers to any delivery system for delivering materials. Such delivery systems can include systems that allow various diagnostic or therapeutic reagents (e.g., oligonucleotides, antibodies, enzymes, etc. in appropriate containers) and / or support materials (e.g., buffers, written instructions for measuring, etc.) to be stored, transported or delivered from one location to another. For example, the kit includes one or more housings (e.g., boxes) containing relevant reaction reagents and / or support materials. As used herein, the term "fragmentation kit" refers to a delivery system comprising two or more separate containers, each of which contains a sub-portion of the total kit components. These containers can be delivered to the intended recipient together or individually. For example, a first container can contain an enzyme for measuring, and a second container contains oligonucleotides. The term "fragmentation kit" is intended to cover a kit containing an analyte-specific reagent (ASR) regulated by Section 520 (e) of the Federal Food, Drug, and Cosmetic Act, but is not limited thereto. In fact, any delivery system comprising two or more separate containers is included in the term "fragmentation kit", each of which contains a sub-portion of the total kit components. In contrast, a "combination kit" refers to a delivery system that contains all of the components in a single container (eg, in a single box containing each of the desired components). The term "kit" includes both fragmented and combination kits.

[0069] As described in detail below, the present invention is based on the use of two kinds of fluorophores, which have different permeability spectra and in combination with nucleic acids to LNP. Specifically, the first fluorophore permeates the LNP (e.g., mRNA-LNP) of the encapsulated nucleic acid and the second fluorophore does not permeate the LNP. The first fluorophore forms a complex with the encapsulated and unencapsulated nucleic acid that may be present in the sample, while the second fluorophore forms a complex with the unencapsulated nucleic acid that may be present in the sample. Detecting the fluorescence emission of the two fluorophores ultimately allows the determination of encapsulation efficiency. Advantageously, the method of the present invention reduces the sample quantity required for the determination of encapsulation efficiency by at least two times, because both fluorescence measurements can be carried out on a single sample. In addition, the method can be used for high-throughput screening, because in the absence of detergent, foaming is no longer a problem. Therefore, the present invention provides a simple, reliable and effective quantitative or semi-quantitative method for assessing RNA encapsulation efficiency. The present invention is particularly useful for quality control during manufacturing and in the final therapeutic product as the characterization of the encapsulated nucleic acid (e.g., mRNA) of a pharmaceutical ingredient.

[0070] In one aspect, the present invention provides a method for determining nucleic acid encapsulation efficiency in LNPs, the method comprising the steps of: a) contacting a sample comprising a nucleic acid encapsulated in an LNP with a first fluorophore and a second fluorophore, thereby forming a fluorophore-nucleic acid complex, and b) detecting the fluorescent signal of the composite first and second fluorophores, Wherein the first fluorophore permeates the LNPs, and wherein the second fluorophore does not permeate the LNPs.

[0071] Although the encapsulation efficiency is desirably high (e.g., close to 100%), for example, for the nucleic acid to be used for the encapsulation of a pharmaceutical product, when using the method of the present invention, it is possible to determine an encapsulation efficiency ranging from 0% to 100%. Therefore, in some embodiments, the determined encapsulation efficiency is 0% to 100%. In some embodiments, the encapsulation efficiency is at least about 50%, such as 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the encapsulation efficiency is at least 80%. In some embodiments, 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 80% to 100%.

[0072] The total concentration of nucleic acid (that is, both unencapsulated and encapsulated) is such that it falls within the sensitivity range of the fluorophore used in the method of the present invention. In one embodiment, nucleic acid is present at a final concentration of at least 0.25 μg / mL. In one embodiment, nucleic acid is present at a final concentration comprised between 0.25 and 10 μg / mL. In one embodiment, nucleic acid is present at a final concentration comprised between 0.25 and 5 μg / mL. In one embodiment, nucleic acid is present at a final concentration comprised between 0.25 and 1.25 μg / mL. In one embodiment, nucleic acid is present at a final concentration comprised between 0.25 and 1 μg / mL. Samples comprising nucleic acids encapsulated in LNPs can be subjected to one or more dilutions to provide a nucleic acid concentration falling within the sensitivity range of the fluorophore.

[0073] The nucleic acid encapsulated in LNP can be any type of nucleic acid as provided herein. Encapsulated nucleic acid molecules (e.g., RNA) can have any length. In one embodiment, the length of nucleic acid molecules is at least about 10 nucleotides (nt). In one embodiment, the length of nucleic acid molecules is at least about 20 nt. In one embodiment, the length of nucleic acid molecules is at least about 50 nt. In one embodiment, the length of nucleic acid molecules is at least about 100 nt. In one embodiment, the length of nucleic acid molecules is at least about 500 nt. In one embodiment, the length of nucleic acid molecules is about 10 to 50000 nt. In one embodiment, the length of nucleic acid molecules is about 20 to 25000 nt. In one embodiment, the length of nucleic acid molecules is about 300 to 10000 nt. In one embodiment, the length of nucleic acid molecules is about 300 to 10000 nt. In one embodiment, the length of nucleic acid molecules is about 400 to 8000 nt. In one embodiment, the length of nucleic acid molecules is about 500 to 5000 nt. In certain embodiments, LNP comprises 1-20, optionally 5-10 or 6-8 nucleic acid molecules.

[0074] In the context of this method, the first fluorophore provided is permeated with the LNP. In one embodiment, the first fluorophore is Quant-iT TM High Sensitivity Reagent, also referred to herein as Quant-iT TM HS.Quant-iT TM The HS reagent is composed of the RiboRed fluorophore, and thus may alternatively be referred to herein as "RiboRed". In one embodiment, Quant-iT TM The excitation spectrum of HS is included in the range of 640nm to 648nm. In one embodiment, Quant-iT TM The excitation spectrum of HS is 644 nm. In one embodiment, the excitation is performed with a bandwidth of 9 nm (e.g., 644 nm + / - 4.5 nm). In one embodiment, Quant-iT TM The emission spectrum of HS is included in the range of 666nm to 680nm. In one embodiment, Quant-iT TM The emission spectrum of HS is 673 nm. In one embodiment, the emission spectrum is collected with a bandwidth of 15 nm (e.g., 673 nm + / - 7.5 nm). In one embodiment, Quant-iT TM The excitation and emission spectra of HS are 644 and 673 nm, respectively. In another embodiment, the first fluorophore is Green II fluorophore. In one embodiment, The excitation spectrum of Green II is included in the range of 491nm to 499nm. In one embodiment, The excitation spectrum of Green II is 495 nm. In one embodiment, the excitation is performed with a bandwidth of 9 nm (e.g., 495 nm + / - 4.5 nm). In one embodiment, The emission spectrum of Green II is included in the range of 513nm to 527nm. In one embodiment, The emission spectrum of Green II is 520 nm. In one embodiment, the emission spectrum is collected with a bandwidth of 15 nm (e.g., 520 nm + / - 7.5 nm). In one embodiment, The excitation and emission spectra of Green II are 495 and 520 nm, respectively.

[0075] The second fluorophore provided in the context of this method does not permeate the LNP. Furthermore, the skilled artisan will readily appreciate that the second fluorophore should be detectably different from the first fluorophore to ensure that the fluorescence of each fluorophore can be measured from a single sample. In one embodiment, the first fluorophore is a cyanine dye. In one embodiment, the second fluorophore is a cyanine dye. In one embodiment, the second fluorophore is (See, eg, Jones et al., Analytical Biochemistry. (1998) 265:368-374). Detectably different from Quant-iT TM HS. In one embodiment, it can be carried out at 485±10nm In one embodiment, the excitation can be carried out at 486 ± 5 nm. In one embodiment, The excitation spectrum of is included in the range of 475nm to 495nm. In one embodiment, The excitation spectrum of is included in the range of 470nm to 491nm. In one embodiment, The excitation spectrum is 485 nm. In one embodiment, the excitation is performed with a bandwidth of 9 nm (e.g., 485 nm + / - 4.5 nm). In one embodiment, the collection can be performed at 530 ± 15 nm. Fluorescence emission. In one embodiment, The emission spectrum of is included in the range of 515nm to 545nm. In one embodiment, The emission spectrum is 525 nm. In one embodiment, the emission spectrum is collected with a bandwidth of 15 nm (e.g., 525 nm + / - 7.5 nm). In one embodiment, The excitation and emission spectra of are 485 nm and 525 nm, respectively. In another embodiment, the second fluorophore is Gold. Gold is also known as [2-(4-{[diethyl(methyl)ammonio]methyl}phenyl)-6-methoxy-1-methyl-4-{[(2Z)-3-methyl-1,3-benzoxazol-2-ylidene]methyl}quinolin-1-ium]. In one embodiment, The excitation spectrum of Gold is included in the range of 491 to 499 nm. In one embodiment, The excitation spectrum of Gold is 495 nm. In one embodiment, the excitation is performed with a bandwidth of 9 nm (e.g., 495 nm + / - 4.5 nm). In one embodiment, The emission spectrum of Gold is 537 nm. In one embodiment, the emission spectrum is collected with a bandwidth of 15 nm (e.g., 537 nm + / - 7.5 nm). In one embodiment, The emission spectrum of Gold is included in the range of 530 to 544 nm. In one embodiment, The excitation and emission spectra of Gold are 495 and 537 nm, respectively.

[0076] As the technician will readily appreciate, in order to accurately determine encapsulation efficiency, the fluorophore should not produce non-specific fluorescence, for example, produced by the interaction of the fluorophore with the lipid components of the LNP. In certain embodiments, a control sample that does not comprise nucleic acid can be used to determine baseline fluorescence. This baseline can be subtracted from the fluorescence emission detected in one or more corresponding samples of the LNP comprising the encapsulated nucleic acid.

[0077] In some embodiments, one or both fluorophores are conditionally fluorescent when bound to a nucleic acid.

[0078] To ensure that the fluorescent signals from the composite first and second fluorophores can be individually identified, the fluorescent signals of the composite first and second fluorophores should be detectably different. In one embodiment, the fluorophores used in the method do not have overlapping excitation and emission spectra. In cases where the spectra may overlap, an appropriate cutoff value can be used to distinguish the signals.

[0079] To ensure that the fluorescent signal from the complexed first and second fluorophores can be determined without interference, the first and second fluorophores should not compete for binding to the nucleic acid.

[0080] In order to accurately determine encapsulation efficiency, the sensitivity range of the first and second fluorophores should cover the total concentration of nucleic acids present in the sample (ie, both unencapsulated and encapsulated).

[0081] Fluorophore concentrations can be expressed in units (e.g., μg / mL), or as a dilution factor relative to an initial concentration provided by the manufacturer. For example, a 200X concentrated solution diluted to a 1X concentration can be expressed as a dilution of 1:200. Alternatively, fluorophore concentrations can be expressed as a final concentration used (e.g., 0.5X, 1X, etc.). In one embodiment, the first fluorophore is present at a final concentration of 0.05X to 10X relative 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. In one embodiment, the second fluorophore is present at a final concentration of 0.1X 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 in the range of 1:1 to 20:1. In one embodiment, the ratio of the first fluorophore to the second fluorophore is comprised in the range of 1.5:1 to 10:1. In one embodiment, the ratio of the first fluorophore to the second fluorophore is comprised in 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.

[0082] In one embodiment, the first and second fluorophores are Quant-iT TM HS and In one embodiment, Quant-iT TM HS and The ratio is 2:1. In one embodiment, Quant-iT TM HS is provided at a final concentration of 0.5X, and Supplied at 0.25X final concentration.

[0083] Prior to measuring the fluorescence emission, the sample is contacted with the fluorophore for a sufficient amount of time to allow the fluorophore to bind to the nucleic acid so that they form a complex. In one embodiment, the fluorophore is incubated with the sample for more than 5 min. In one embodiment, the fluorophore is incubated with the sample for more than 10 min. In one embodiment, the fluorophore is incubated with the sample for 15-30 min. In one embodiment, the fluorophore is incubated with the sample for 20 min. In one embodiment, the fluorophore is incubated with the sample for 25 min. In one embodiment, the incubation occurs in the dark. In one embodiment, the incubation occurs at room temperature. In one embodiment, the fluorophore is incubated with the sample in the dark for 15-30 min at room temperature.

[0084] The above fluorophores bind to RNA. When the nucleic acid is not RNA, alternative fluorophores may be particularly useful. As an example, when the nucleic acid is dsDNA, it is conceivable that Fluorophore.

[0085] In one embodiment, the first and second fluorophores are added to the sample simultaneously, i.e., in a premixed solution. In another embodiment, the first and second fluorophores are added to the sample separately. In one embodiment, both fluorophores are added to the sample before measuring fluorescence. In another embodiment, a first fluorophore is added and a first fluorescence signal of the first fluorophore is measured, followed by the addition of a second fluorophore and the measurement of a second fluorescence signal, or vice versa.

[0086] The control can be used to quantify the amount of encapsulated nucleic acid. In some embodiments, the control includes a sample with a predetermined amount of nucleic acid. In some embodiments, the control includes a predetermined amount of free nucleic acid. In some embodiments, the control includes a predetermined amount of encapsulated nucleic acid. In some embodiments, the control includes a predetermined amount of total nucleic acid. In some embodiments, the control includes a predetermined amount of free and encapsulated nucleic acid. In some embodiments, the control can be a predetermined amount of free RNA. In some embodiments, the control can be an RNA-LNP containing a predetermined amount of encapsulated RNA.

[0087] The efficiency of nucleic acid (eg, RNA or mRNA) encapsulation in the lipid nanoparticles is determined based on the detected fluorescence signals of the composited first and second fluorophores.

[0088] In certain embodiments, the determination is carried out quantitatively by establishing a calibration curve (also referred to as a standard curve). In other words, the encapsulation efficiency can be determined qualitatively or semi-quantitatively by comparing the fluorescent signal detected in the unknown sample with a known standard, or by comparing the encapsulation efficiency with a standard curve prepared using a plurality of samples of known nucleic acid concentrations. For example, quantitatively, a group of standards or calibrators with a known amount of total nucleic acid and / or a known amount of free nucleic acid can be prepared. These standards or calibrators can be serially diluted, and the resulting signal value from each test concentration of the standards or calibrators is used to generate a standard curve; a relationship diagram of the standard concentration compared to the resulting signal value is drawn. Once a standard quantitative curve is established, the level of total nucleic acid and free nucleic acid in the sample can be determined by drawing the resulting signal on the corresponding standard curve.

[0089] Therefore, in one embodiment, the method further includes generating a standard curve of the first and second fluorescent signals. In one embodiment, the method further includes determining the absolute amount of the RNA of encapsulation by mapping each fluorescent signal detected in step b) to a corresponding standard curve. The amount of the nucleic acid detected by the second fluorophore (i.e., the fluorophore that does not permeate LNP) can then be determined by dividing the amount of the nucleic acid detected by the first fluorophore (i.e., the fluorophore that permeates LNP) by the amount of the non-encapsulated nucleic acid (in percentage). Corresponding equation is shown below: The encapsulation efficiency (i.e., the relative amount of encapsulated nucleic acid as a percentage) can then be determined by subtracting the amount of free (unencapsulated) nucleic acid from 100, as shown in the following equation: Encapsulation efficiency (%) = 100 - free nucleic acid (%)

[0090] In some embodiments, the fluorescence ratio between the two fluorophores can be used to directly determine the encapsulation efficiency (i.e., without the need to generate a standard curve). In some embodiments, a fluorescence ratio is first established between the two fluorophores over a range of nucleic acid concentrations. The fluorescence coefficient (C) can be obtained by dividing the fluorescence signal of the second fluorophore by the fluorescence signal of the first fluorophore at each nucleic acid concentration. f ). In some embodiments, the C f Is 0.01. In some embodiments, the fluorescent signal provided by the second fluorophore (i.e., the fluorophore that does not penetrate the LNP and thus binds to the unencapsulated nucleic acid) is divided by the fluorescent signal provided by the first fluorophore (i.e., the fluorophore that penetrates the LNP and thus binds to the total nucleic acid). This allows the relative amount of free nucleic acid to be determined (in percentage). An exemplary equation is shown below:

[0091] This equation can be alternatively expressed as:

[0092] The encapsulation efficiency (as a percentage) can then be determined by subtracting the amount of free nucleic acid from 100, as described above.

[0093] In some embodiments, a sample that does not contain any nucleic acid (ie, a blank) can be measured and the corresponding fluorescent signal subtracted from one or more signals measured in a corresponding sample containing nucleic acid. In some embodiments, the blank can contain empty LNPs.

[0094] In some embodiments, the fluorescence ratio is determined at two RNA-LNP dilutions and then averaged to provide the amount of free nucleic acid (in percent). In some embodiments, the estimated final concentration of RNA in the dilution (i.e., total RNA) is comprised between 0.25 and 10 μg / mL. In some embodiments, the estimated final concentration of RNA in the dilution is comprised between 0.25 and 5 μg / mL. In some embodiments, the estimated final concentration of RNA in the two RNA-LNP dilutions is about 1 and about 1.25 μg / mL, respectively.

[0095] In certain embodiments, the method provided herein is used to characterize the encapsulation of a batch of RNA-LNP. In certain embodiments, the method provided herein is carried out before releasing a batch of LNPs of encapsulated nucleic acids. In certain embodiments, the encapsulation efficiency determined according to the method provided herein is at least 80% for batch release. Therefore, in one embodiment, a method for batch release includes: a) determining the efficiency of RNA encapsulation in LNP according to the method provided herein, and b) releasing a batch of RNA-LNP when the encapsulation efficiency is at least 80%.

[0096] The present invention further relates to a method for making a LNP encapsulating a nucleic acid, the method comprising step a) encapsulating the nucleic acid in the LNP, and step b) determining the nucleic acid encapsulation efficiency in the LNP according to the method provided herein. In one embodiment, the nucleic acid is RNA, such as mRNA, as described herein. Therefore, a method for making a LNP encapsulating RNA is further disclosed, the method comprising step a) encapsulating the RNA in the LNP, and step b) determining the RNA encapsulation efficiency in the LNP according to the method provided herein.

[0097] In one embodiment, the LNP comprises one or more ionizable lipids, one or more helper lipids, and one or more PEG-modified lipids.

[0098] In one embodiment, the nucleic acid molecule and / or LNP corresponds to a nucleic acid molecule and / or LNP disclosed in US2022 / 0142923 (incorporated herein in its entirety by reference). In particular, the LNP may comprise four types of lipids: (i) ionizable lipids; (ii) PEGylated lipids; (iii) cholesterol-based lipids; and (iv) helper lipids.

[0099] Ionizable lipids.

[0100] The ionizable lipid facilitates mRNA encapsulation and can be a cationic lipid. Cationic lipids provide a positively charged environment at low pH to facilitate efficient encapsulation of negatively charged mRNA drug substances.

[0101] In some embodiments, the cationic lipid is OF-02. OF-02 is a non-degradable structural analog of OF-Deg-Lin. OF-Deg-Lin contains a degradable ester bond to connect the diketopiperazine core and the diunsaturated tail, while OF-02 contains a non-degradable 1,2-amino-alcohol bond to connect the same diketopiperazine core and the diunsaturated tail (Fenton et al., Adv Mater. [Advanced Materials] (2016) 28: 2939; U.S. Patent No. 10,201,618).

[0102] In some embodiments, the cationic lipid is cKK-E10 (Dong et al., PNAS (2014) 111(11):3955-60; U.S. Pat. No. 9,512,073).

[0103] In some embodiments, 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)disulfanyl)ethyl)piperazin-1-yl)ethyl 4-(bis(2-hydroxydecyl)amino)butyrate), GL-HEPES-E3-E12-DS-4-E10(2-(4-(2-((3-(bis(2-hydroxydecyl)amino)propyl)disulfanyl)ethyl)piperazin-1-yl)ethyl 4-(bis(2-hydroxydecyl)amino)butyrate. )amino)butyl)disulfanyl)ethyl)piperazin-1-yl)ethyl 4-(bis(2-hydroxydodecyl)amino)butyrate) or GL-HEPES-E3-E12-DS-3-E14 (2-(4-(2-((3-(bis(2-hydroxytetradecyl)amino)propyl)disulfanyl)ethyl)piperazin-1-yl)ethyl 4-(bis(2-hydroxydodecyl)amino)butyrate), which is a HEPES-based disulfide cationic lipid with a piperazine core.

[0104] Other cationic lipids that can be used include those described in Dong, supra; and in US Pat. No. 10,201,618.

[0105] PEGylated lipids

[0106] The PEGylated lipid component provides control of the particle size and stability of nanoparticles. The addition of such components can prevent complex aggregation and provide for increasing the cycle life and increasing the mode of delivery of lipid-nucleic acid pharmaceutical compositions to target tissues (Klibanov et al., FEBS Letters [European Biochemical Society Federation Express] (1990) 268 (1): 235-7). These components can be selected to quickly exchange pharmaceutical compositions in vivo (see, e.g., U.S. Patent No. 5,885,613).

[0107] Contemplated PEGylated lipids include, but are not limited to

[0108] Polyethylene glycol (PEG) chains up to 5 kDa in length are covalently attached to C6-C 20 (e.g., C8, C 10 , C 12 , C 14 , C 15 or C 18 ) length, such as a derivatized ceramide (e.g., N-octanoyl-sphingosine-1-[succinyl(methoxypolyethylene glycol)] (C8 PEG ceramide)). In some embodiments, 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-polyethylene glycol (DSG-PEG).

[0109] In a particular exemplary embodiment, PEG has a high molecular weight, such as 2000-2400 g / mol. In certain embodiments, PEG is PEG2000 (or PEG-2K). In a specific embodiment, the PEGylated lipid herein is DMG-PEG2000, DSPE-PEG2000, DLPE-PEG2000, DSG-PEG2000 or C8PEG2000.

[0110] Cholesterol-based lipids

[0111] The cholesterol component provides stability for the lipid bilayer structure in the nanoparticle. In some embodiments, LNP comprises one or more cholesterol-based lipids. Suitable cholesterol-based lipids include, for example: DC-Choi (N, N-dimethyl-N-ethylformamido cholesterol), 1,4-bis (3-N-oleylamino-propyl) piperazine (Gao et al., BiochemBiophys Res Comm. [Biochemistry and Biophysics Research Communications] (1991) 179: 280; Wolf et al., BioTechniques [Biotechnology] (1997) 23: 139; U.S. Patent 5,744,335), imidazole cholesterol ester ("ICE"; WO2011 / 068810), [3-sitosterol, fucosterol, stigmasterol and other modified forms of cholesterol. In some embodiments, the cholesterol-based lipid used in LNP is cholesterol.

[0112] Helper lipids

[0113] The helper lipid enhances the structural stability of LNP and illustrates that LNP escapes in endosomes. It improves the uptake and release of mRNA drug payload. In certain embodiments, the helper lipid is a neutral lipid, i.e., a lipid that does not carry a net charge under the conditions of preparing and / or applying the composition. In certain embodiments, the helper lipid is an "anionic lipid", i.e., a lipid that carries a net negative charge under a selected pH (such as physiological pH). In certain embodiments, the helper lipid is a zwitterionic lipid, which has a fusogenic property for enhancing the uptake and release of drug payload. Examples of 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-dioleoyl-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).

[0114] Other exemplary helper lipids are dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), phosphatidylserine, sphingolipids, cerebrosides, gangliosides, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), or a combination thereof.

[0115] In certain embodiments, the helper lipid is DOPE. In other embodiments, the LNP comprises (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.

[0116] Molar ratio of lipid components

[0117] When cationic lipids, PEGylated lipids, cholesterol-based lipids and auxiliary lipids are present, the molar ratio is provided as A: B: C: D, wherein A + B + C + D = 100%. In some embodiments, the molar ratio of cationic lipids relative to total lipids in LNP (i.e., A) is 35%-45% (e.g., 38%-42%, such as 40%). In some embodiments, the molar ratio of the PEGylated lipid component relative to total lipids (i.e., B) is 0.25%-2.75% (e.g., 1%-2%, such as 1.5%). In some embodiments, the molar ratio of cholesterol-based lipids relative to total lipids (i.e., C) is 20%-35% (e.g., 27%-30%, such as 28.5%). In some embodiments, the molar ratio of auxiliary lipids relative to total lipids (i.e., D) is 25%-35% (e.g., 28%-32%, such as 30%). In some embodiments, the (PEGylated lipid + cholesterol) component has the same molar amount as the auxiliary lipid. In some embodiments, the molar ratio of the cationic lipids to the auxiliary lipids contained in the LNP is greater than 1.

[0118] In a specific embodiment, LNP contains cationic lipids, PEGylated lipids, cholesterol-based lipids and auxiliary lipids in a molar ratio of 40:1.5:28.5:30. In another specific embodiment, LNP contains 40:1.5:28.5:30 of (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.

[0119] In order to calculate the actual amount of each lipid to be placed in the LNP formulation, the molar amount of the cationic lipid is first determined based on the 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. Next, the molar amount of each other lipid is calculated based on the molar amount of the cationic lipid and the selected molar ratio. These molar amounts are then converted to weight using the molecular weight of each lipid.

[0120] In one embodiment, nucleic acid is encapsulated in LNP, and this LNP comprises: the cationic lipid of molar ratio between 35% and 45%, the polyethylene glycol (PEG) conjugated (PEGization) lipid of molar ratio between 0.25% and 2.75%, the lipid based on cholesterol of molar ratio between 20% and 35% and the auxiliary lipid of molar ratio between 25% and 35%, wherein all molar ratios are relative to the total lipid content of LNP.In certain embodiments, 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.In certain embodiments, this LNP comprises the cationic lipid of molar ratio being 40%, the PEGization lipid of molar ratio being 1.5%, the lipid based on cholesterol of molar ratio being 28.5% and the auxiliary lipid of molar ratio being 30%.

[0121] In some embodiments, 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, and / or the helper lipid is 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE). In a specific embodiment, the LNP comprises 40% 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, 1.5% DMG-PEG2000 at a molar ratio, 28.5% cholesterol at a molar ratio, and 30% DOPE at a molar ratio.

[0122] In certain embodiments, LNP can have the average diameter of about 30nm to about 200nm, about 80nm to about 150nm or about 30nm, 35nm, 40nm, 45nm, 50nm, 55nm, 60nm, 65nm, 70nm, 75nm, 80nm, 85nm, 90nm, 95nm, 100nm, 105nm, 110nm, 115nm, 120nm, 125nm, 130nm, 135nm, 140nm, 145nm, 150nm, 155nm, 160nm, 165nm, 170nm, 175nm, 180nm, 185nm, 190nm, 195nm or 200nm. In certain embodiments, LNP is substantially nontoxic. When present in one or more LNPs, the one or more nucleic acid molecules typically have resistance to the degradation of nuclease in aqueous solution. In certain embodiments, RNA-LNP has the N / P-ratio of 1-20,1-15,1-10,2-8,2-6 or 2-4.Term " N / P ratio " refers to the molar ratio of the positively charged molecular unit in the cationic lipid in LNP relative to the negatively charged molecular unit in the RNA encapsulated in the LNP.Accordingly, the N / P ratio is typically calculated as the mole of the amine group in the cationic lipid in LNP relative to the mole of the phosphate group in the RNA encapsulated in the LNP.In certain embodiments, the N / P ratio is about 1 to about 20, about 1 to about 18, about 1 to about 16, about 1 to about 14, about 1 to about 12, about 1 to about 10, about 1 to about 8 or about 1 to about 6.In certain embodiments, the N / P ratio is about 2 to about 20, about 2 to about 16, about 2 to about 12, about 2 to about 8 or about 2 to about 4.In certain embodiments, the N / P ratio is about 4 to about 20, about 4 to about 16 or about 4 to 8. In some embodiments, the N / P-ratio is higher than 1, about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8. In certain embodiments, the RNA-LNP has an N / P ratio of 8. In certain embodiments, the RNA-LNP has an N / P ratio of 4. In certain embodiments, the RNA-LNP has an N / P ratio of 2.

[0123] In some embodiments, the LNP comprises one or more mRNA molecules encoding an antigen (eg, a viral antigen such as an influenza virus antigen or a bacterial antigen).

[0124] LNPs can be prepared by a variety of techniques currently known in the art. For example, multilamellar vesicles (MLVs) can be prepared according to conventional techniques, such as by depositing the selected lipids on the inner wall of a suitable container or vessel (by dissolving the lipids in an appropriate solvent and then evaporating the solvent to leave a thin film inside the vessel) or by spray drying. The aqueous phase can then be added to the vessel with a vortex motion, which allows the MLV to form. Unilamellar vesicles (ULVs) can then be formed by homogenizing, sonicating or extruding the multilamellar vesicles. In addition, unilamellar vesicles can be formed by detergent removal techniques.

[0125] Various methods are described in US2011 / 0244026, US2016 / 0038432, US2018 / 0153822, US2018 / 0125989 and US2021 / 0046192, and can be used to practice this disclosure. An exemplary method needs to be encapsulated mRNA by mixing it with a mixture of lipids, without first preforming lipid nanoparticles, as described in US2016 / 0038432. Another exemplary method needs to be encapsulated mRNA by mixing preformed LNP with mRNA, as described in US2018 / 0153822.

[0126] In one embodiment, nucleic acid is prepared in aqueous buffer, and it is mixed with the amphipathic solution of the lipid component containing LNP. The amphipathic solution for dissolving four kinds of lipid components of LNP can be an alcoholic solution. In certain embodiments, alcohol is ethanol. Aqueous buffer can be, for example, citrate, phosphate, acetate or succinate buffer, and can have a pH of about 3.0-7.0 (for example, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0 or about 6.5). Buffer can contain other components, such as salt (for example, sodium, potassium and / or calcium salt). In a particular embodiment, aqueous buffer has 1mM citrate, 150mM NaCl, pH 4.5.

[0127] In another embodiment, a preformed LNP is mixed with the nucleic acid under conditions that allow the formation of the nucleic acid-LNP in step a).

[0128] In one embodiment, the method comprises the step of synthesizing mRNA in vitro prior to step a). In one embodiment, the mRNA synthesized in vitro is purified prior to encapsulation in LNP.

[0129] In one embodiment, the encapsulation efficiency is at least 80%.

[0130] In certain embodiments, the method provided herein may further include the step of removing nucleic acid molecules, which are not properly encapsulated in LNP, but are combined with the outer surface of LNP. In particular, RNA molecules combined with the outer surface of LNP can be removed by contacting RNA-LNP with high salt. Therefore, the method provided herein may further include the step of dissociating RNA combined with the outer surface of LNP. Specifically, the step of dissociating RNA can include contacting RNA-LNP with high salt before determining the encapsulation efficiency. "High salt" as used herein can refer to the salt provided with a final concentration ranging from 500mM to 5M. In certain embodiments, salt is provided with a final concentration of 1M to 5M. In certain embodiments, salt is provided with a final concentration of 0.75M to 3M. The salt may be NaCl. In certain embodiments, NaCl is used with a final concentration ranging from about 500mM to 5M, optionally 1 to 2M.

[0131] In some embodiments, the step of dissociating the RNA bound to the outer surface of the LNP may additionally require heating. A suitable temperature for the dissociation step may be about 60°C to 95°C. In some embodiments, the dissociation step is performed at a temperature of about 70°C to about 90°C. In some embodiments, the dissociation step is performed at a temperature of about 80°C to 90°C. In some embodiments, the dissociation step is performed at a temperature of about 85°C.

[0132] The present invention further provides test kits, which comprise various reagents and materials that can be used for carrying out the inventive method according to the present invention. The quantitative procedure described herein can be performed by a diagnostic laboratory, an experimental laboratory or a commercial laboratory. The present invention provides test kits that can be used in these different environments.

[0133] For example, the material and reagent for RNA encapsulation efficiency in the quantitative sample according to the method provided herein can be assembled together in a test kit. Each test kit preferably includes a reagent that makes program specific. In certain embodiments, the test kit includes two fluorophores, wherein the first fluorophore permeates LNP and the second fluorophore does not permeate LNP, such as those described herein. The test kit optionally includes other reagents (such as buffer) and uses the instructions for the test kit according to the method of the present invention.

[0134] The present disclosure further provides a kit for determining RNA encapsulation efficiency in LNPs, the kit comprising: - a first fluorophore that permeates the LNP; - a second fluorophore that does not permeate the LNP; and -Optionally, instructions for use according to the methods provided herein.

[0135] Test kits according to the present invention or other articles may include one or more containers to hold various reagents. Suitable containers include, for example, bottles, vials, syringes (e.g., pre-filled syringes), ampoules. The container may be formed from various materials (e.g., glass or plastic).

[0136] In certain embodiments, the kit of the present invention may include a suitable control level or control sample for determining a control level as described herein. For example, the kit may include RNA of known concentration and / or LNPs with RNA encapsulation of known levels. In certain embodiments, the kit of the present invention may include instructions for using the kit according to the method provided herein. In certain embodiments, the kit of the present invention may further include instructions for RNA encapsulation in LNP.

[0137] The present invention further relates to Quant-iT TM The present invention further relates to the use of Quant-iT TM HS and Use in quantifying LNP-encapsulated RNA. In one embodiment, the RNA is present at a final concentration of at least 0.25 μg / mL. In one embodiment, the RNA is present at a final concentration comprised between 0.25 μg / mL and 10 μg / mL.

[0138] The provided method can be used for quality control of LNP-encapsulated RNA and batch release of RNA-LNP compositions. In fact, the present invention is particularly useful for quality control during the manufacture of LNP-encapsulated mRNA and the characterization of LNP-encapsulated mRNA as an active pharmaceutical ingredient (API) in the final therapeutic product.

[0139] The present invention includes the following embodiments.

[0140] Example 1. A method for determining RNA encapsulation efficiency in lipid nanoparticles (LNPs), the method comprising: a) contacting a sample comprising RNA encapsulated in LNPs with a first fluorophore and a second fluorophore, thereby forming a fluorophore-RNA complex, and b) detecting the fluorescent signal of the composite first and second fluorophores, Wherein the first fluorophore permeates the LNPs, and wherein the second fluorophore does not permeate the LNPs.

[0141] Embodiment 2. The method of embodiment 1, wherein the first fluorophore is Quant-iT TM HS or Green II.

[0142] Embodiment 3. The method of embodiment 1 or 2, wherein the second fluorophore is or Gold.

[0143] Embodiment 4. The method as described in any of the preceding embodiments, wherein the length of the RNA is 10 to 50,000 nucleotides.

[0144] Embodiment 5. The method as described in any of the preceding embodiments, wherein the length of the RNA is 300 to 10,000 nucleotides.

[0145] Embodiment 6. The method as described in any of the preceding embodiments, wherein the RNA is 500 to 5000 nucleotides in length.

[0146] Embodiment 7. The method as described in any one of the preceding embodiments, wherein the RNA is double-stranded RNA or single-stranded RNA.

[0147] Embodiment 8. The method of any of the preceding embodiments, wherein the RNA comprises mRNA, miRNA, shRNA, rRNA, tRNA, snRNA, snoRNA, asRNA, siRNA, aiRNA, gRNA and / or piRNA.

[0148] 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.

[0149] 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.

[0150] Embodiment 11. The method of any one of the preceding embodiments, wherein the RNA is present at a final concentration of at least 0.25 μg / mL, optionally at a final concentration comprised between 0.25 and 10 μg / mL.

[0151] 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.

[0152] Embodiment 13. The method of any one of the preceding embodiments, wherein the first fluorophore is present at 0.5X the final concentration.

[0153] Embodiment 14. The method of any of the preceding embodiments, wherein the second fluorophore is present at 0.25X the final concentration.

[0154] Embodiment 15. The method of any of the preceding embodiments, wherein the method further comprises generating a standard curve of the first and second fluorescent signals.

[0155] 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 a corresponding standard curve.

[0156] Embodiment 17. The method of any one of embodiments 1-14, wherein the method further comprises determining a ratio of the second fluorescent signal to the first fluorescent signal.

[0157] Example 18. A method for making LNPs encapsulating RNA, the method comprising: a) encapsulating RNA in LNPs, and b) Determining the efficiency of RNA encapsulation in LNPs according to the method of any one of Examples 1-17.

[0158] Embodiment 19. The method of embodiment 18, wherein the RNA is mRNA.

[0159] Embodiment 20. The method of embodiment 19, wherein the method further comprises the step of synthesizing mRNA in vitro before step a).

[0160] Embodiment 21. The method of embodiment 20, wherein the in vitro synthesized mRNA is purified prior to encapsulation in LNPs.

[0161] Embodiment 22. The method of any one of embodiments 18-21, wherein the lipids are mixed with the RNA under conditions that allow for the formation of LNPs encapsulating the RNA.

[0162] Embodiment 23. The method of any one of embodiments 18-22, wherein the encapsulation efficiency is at least 80%.

[0163] Embodiment 24. The method of any one of embodiments 18-23, wherein the method is performed before releasing a batch of LNPs encapsulating RNA.

[0164] Embodiment 25. The method of any 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.

[0165] Embodiment 26. A kit for determining RNA encapsulation efficiency in LNP, the kit comprising: - a first fluorophore that permeates the LNP; - a second fluorophore that does not permeate the LNP; and - Instructions for use according to the methods provided herein.

[0166] Example 27. Quant-iTTM Use of HS for quantification of LNP-encapsulated RNA.

[0167] In order to better understand the present invention, the following examples are described. These examples are for illustrative purposes only and are not to be construed as limiting the scope of the present disclosure in any way. Examples

[0168] Example 1: Materials and Methods

[0169] mRNA production

[0170] mRNA was generated as previously described (see Kalnin et al. (2021), NPJ Vaccines 6(1):61 and US2022 / 0142923). Briefly, mRNA incorporating the coding sequence was synthesized using unmodified or modified nucleotides by in vitro transcription using RNA polymerase and a plasmid DNA template encoding the desired gene. The length of the exemplary mRNA (mRNA1) was approximately 2000 nucleotides. The resulting purified pre-mRNA was further reacted 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 purification. All mRNA preparations were analyzed for purity, integrity, and percentage of cap 1 before storage at -80°C.

[0171] mRNA encapsulation in LNPs

[0172] For mRNA encapsulation in LNPs, an ethanolic solution of a mixture of lipids (cationic / ionizable lipids, helper lipids, cholesterol, and polyethylene glycol-lipids) was combined with an aqueous buffer solution of the target mRNA at a fixed lipid to mRNA ratio under controlled conditions at acidic pH to obtain a suspension of uniform LNPs. After ultrafiltration and diafiltration into a suitable dilution system, the resulting nanoparticle suspension was diluted to the final concentration, filtered, and stored frozen at -80°C until use. The size of the mRNA-LNP formulations was characterized by dynamic light scattering and encapsulation efficiency using the RiboGreen assay. The LNPs consisted of cationic lipids (40%), 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE; 30%), cholesterol (28.5%), and dimyristoyl-PEG2000 (DMG-PEG; 1.5%). Three types of LNPs were evaluated, each containing a different cationic lipid (OF-02, cKK-E10, or GL-HEPES-E3-E12-DS-3-E14). Unless otherwise stated, the LNPs contained OF-02 cationic lipid.

[0173] Fluorescence measurement

[0174] Unless otherwise indicated, the fluorescence emission (also referred to herein as fluorescence) was measured after incubating the sample for 20 min in the presence of one or more fluorophores at room temperature in the dark. The measurements were performed using a Spectramax I3 microplate reader (Molecular Devices) under the following settings: · : Excitation 485nm and emission 525nm, Quant-iT TM HS: excitation 644nm and emission 673nm, · Gold: excitation 495nm and emission 537nm, · Green II: excitation 495nm and emission 520nm, · 17: Excitation 621nm and emission 646nm.

[0175] In each case the excitation bandwidth was 9 nm and the emission bandwidth was 15 nm.

[0176] RiboGreen assay (comparative example)

[0177] Encapsulation efficiency was determined using a standard RiboGreen assay, with samples tested in duplicate. First, free mRNA and mRNA encapsulated in LNP (mRNA-LNP) were diluted to 2ng / μL and 100ng / μL mRNA in TE buffer (10mM Tris-HCl, 1mM EDTA, pH 7.5), respectively. In parallel, mRNA-LNP was diluted to a final concentration of 2ng / μL mRNA in TET buffer (TE buffer containing 0.5% Triton X-100). Free mRNA and mRNA-LNP dilutions were performed in black 96-well microplates with flat transparent bottoms using 100μL as final volume. A standard scale of 8 independent dilutions of free mRNA in TE or TET buffer (0 to 1 ng / μL) was performed, with 4 independent dilutions of 20 ng / μL mRNA-LNPs in TE (0.5 to 20 ng / μL) and 4 independent dilutions of 2 ng / μL mRNA-LNPs in TET (0.05 to 0.4 ng / μL).

[0178] Will The reagents were diluted extemporaneously to 1X in TE buffer and 100 μL of the 1X solution was added to each well. At a final concentration of 0.5X. After incubation at room temperature in the dark for 10 min, the concentrations were measured as described in Example 1. Fluorescence. RNA quantification was performed based on a standard simple linear regression curve consisting of the fluorescence intensity of mRNA-LNPs in TE or TET buffer. Fluorescence emission was calculated by the mRNA-LNP in TE buffer. The concentration of non-encapsulated mRNA was determined by fluorescence and by the concentration of LNPs in TET buffer. The total mRNA concentration was determined by emission. The encapsulation efficiency was then calculated using the following equation: Encapsulation efficiency (%) = 100 - free mRNA (%)

[0179] Evaluation of LNP permeability to fluorophores

[0180] To determine the permeability of LNPs to fluorophores, mRNA-LNPs and corresponding free mRNA were diluted in TE at different total concentrations in a final volume of 100 μL in a black 96-well microplate with a flat transparent bottom. 100 μL of Quant-iT TM HS (2X) or 100 μL 17 (2 μM), and the samples were incubated at room temperature in the dark for 20 min. Fluorescence emission was measured as described above. To determine whether the fluorophore can penetrate LNP and specifically bind to RNA, a fluorescence standard curve was obtained and compared between free mRNA and mRNA-LNP ranges. 17 is provided at a final concentration of 1 μM and Quant-iT TM HS is provided at a final concentration of 1X.

[0181] Evaluation of a dual fluorescence assay for measuring encapsulation

[0182] Characteristics of the two selected fluorophores are provided in Table 1 below. Table 1: Characteristics of fluorophores used in the assay. Quant-iT TM HS and Combine.

[0183] Each point was assayed in duplicate starting with an initial concentration of 2.5 ng / μL of free mRNA and 100 ng / μL of mRNA-LNP in TE buffer. Eight independent dilutions of free mRNA (0 to 2.5 ng / μL) and eight independent dilutions of mRNA-LNP (0.5 to 20 ng / μL) were performed in a black 96-well microplate with a flat transparent bottom using 100 μL as a final volume. and Quant-iT TM HS reagent was diluted temporarily to 0.5X and 1X, respectively, and 100 μL of the fluorophore mixture was added to each well. and Quant-iT TM HS was present at 0.25X and 0.5X final concentrations, respectively. Fluorescence was measured as described in Example 1. A standard simple linear regression curve was established for each fluorophore ( Figure 3 ).

[0184] Using free mRNA in TE, based on and Quant-iT TM RNA quantification was performed using two standard curves calculated using HS fluorescence (see Figure 3 Then, the mRNA-LNP-containing samples were measured. The free mRNA concentration was determined by fluorescence and the Quant-iT TM HS fluorescence was used to determine the total mRNA concentration.

[0185] A summary of fluorophore concentrations, final mRNA concentration ranges, and number of spots measured for either the RiboGreen assay or the assay of the invention is provided in Table 2 below. Table 2: Summary of fluorophore concentrations and the range of free and encapsulated mRNA, with the number of diluted data points analyzed. Dilutions of free mRNA of known concentration were used to establish the standard curve required for quantification of free and total mRNA concentrations in samples. Conc.: concentration, TE: Tris-EDTA, TET: Tris-EDTA-Triton.

[0186] Use of Dual RNA Encapsulation Assays for High-Throughput Screening

[0187] Each point was assayed in triplicate in TE buffer using mRNA-LNP samples at an initial concentration of 20 ng / μL. Two independent dilutions (to 2 and 2.5 ng / μL) were made in TE buffer in a black 96-well microplate with a flat transparent bottom, with a final volume of 100 μL. A blank without mRNA-LNP was also included to remove background fluorescence. and Quant-iT TM HS reagents were extemporaneously diluted to 0.5X and 1X in TE buffer, respectively, and 100 μL of the fluorophore mixture was added to each well. and Quant-iT TM HS launch.

[0188] Determine the encapsulation efficiency using the following equation: Encapsulation efficiency (%) = 100 - free mRNA (%)

[0189] Example 2: Fluorophore selection

[0190] The use of two fluorophores in a single well to determine RNA encapsulation efficiency (also referred to herein as a "dual RNA encapsulation assay") was evaluated. An illustration of the principle of this method, compared to the RiboGreen assay, is provided in Figure 1 middle.

[0191] Initially, compatible fluorophores are selected for the assay. To be used in the same well, the two selected fluorophores should not show spectral overlap (i.e., in excitation and emission wavelengths). The two selected fluorophores should also detect a similar mRNA concentration range (i.e., have a similar sensitivity scale). and Quant-iT TM This is particularly the case for HS (see Table 1 above).

[0192] The second fluorophore should not be permeable to the LNPs because in the RiboGreen assay, detergent must be added to lyse the LNPs in order to measure the fluorescence emitted by the total RNA.

[0193] Quant-iT was measured in the presence of free mRNA or encapsulated mRNA (i.e., mRNA-LNP). TM The emission of HS was determined to determine its ability to label total mRNA in LNPs. TM The HS emission was identical between free and encapsulated mRNA, demonstrating the permeability properties of this fluorophore for LNPs (see Figure 2These results further indicate that Quant-iT TM HS did not interact with the lipids comprising the LNPs.

[0194] The two fluorophores should also not show any binding to the lipids or compete with each other. Quantification of non-encapsulated or total mRNA concentration is based on the standard free mRNA curve, which must be fit to a standard linear regression. and Quant-iT TM The ability of HS to concomitantly label free mRNA at different concentrations (see Figure 3 ). The results show that the emission from both fluorophores results in a linear regression that can be used as a standard curve. In addition, and Quant-iT TM Neither HS loses their sensitivity range in the presence of the other. and Quant-iT TM HS were the two fluorophores chosen for further evaluation of the dual labeling assay.

[0195] Example 3: Evaluation of dual RNA encapsulation assay

[0196] In order to verify the determination according to the present invention, the fluorophore concentration and standard mRNA and LNP range provided in Table 2 were used, and the RiboGreen determination was performed in parallel. As shown in Table 3 below, the encapsulation efficiency between the two methods is highly similar. Therefore, the method of the present invention can be successfully used to determine the encapsulation efficiency. In addition, the present invention is advantageous because compared with the RiboGreen determination, the variability is reduced, as shown in Table 3. Table 3: Encapsulation efficiency as determined by RiboGreen assay or assay of the present invention. RiboGreen assay and dual RNA encapsulation assay were performed as described above using unmodified free mRNA and mRNA encapsulated in LNPs containing OF-02 cationic lipids. mRNA concentrations were determined based on the standard linear regression line established for each assay. The standard deviation (%) of the two mRNA concentrations from 4 independent dilution points of mRNA-LNPs was calculated in triplicate.

[0197] Example 4: Determining the range of encapsulation efficiencies 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 shown in Table 4 below, contrary to what is required in the classical RiboGreen assay, the assay of the present invention can accurately quantify free and total mRNA concentrations in samples containing LNPs without adding detergent. The determination of encapsulation efficiency is accurate (here, as low as 50%) at various encapsulation rates. Table 4: Different encapsulation efficiencies as determined by dual RNA encapsulation assay. Dual RNA encapsulation assay was performed as described above using unmodified free mRNA encapsulated in LNPs containing OF-02 cationic lipids. A source sample known to have a total mRNA concentration of 1000 μg / mL and an encapsulation efficiency of 95% (as determined by RiboGreen assay) was used as a baseline. Free mRNA was added to the sample to artificially adjust the encapsulation efficiency to 70% and 50%. The dual RNA encapsulation assay used a standard linear regression line, which was calculated by and Quant-iT TM The labeling of free mRNA by both HS fluorophores is calculated. and Quant-iT TM HS fluorescence was used to measure the concentration of non-encapsulated (free) and total mRNA, respectively. Finally, the encapsulation efficiency was determined by the ratio between the concentration of free mRNA and total mRNA. Four independent dilution points of mRNA-LNPs at each concentration were measured in triplicate.

[0198] Example 5: Establishment of a high-throughput dual RNA encapsulation assay

[0199] A protocol for high-throughput determination of encapsulation efficiency that does not require measurement of mRNA concentration was developed. The method compares the ratio of fluorophore emissions in samples to directly determine the encapsulation ratio. First, for the same concentration of labeled mRNA, the ratio of With Quant-iT TM The fluorescence coefficient between HS (C f )( Figure 4 ). Different concentrations of non-encapsulated mRNA and their corresponding same concentrations of total mRNA were labeled with fluorophores and the emission ratio was calculated (Table 5). Regardless of the mRNA concentration, the ratio was always 0.01. f The consistency of the results allows its integration into a new encapsulation efficiency equation, in which the percentage of non-encapsulated mRNA is calculated from the mRNA in the same well. The simple ratio of the emission to the Quant-iT HS emission gives ( Figure 5 ). Table 5: For the same amount of labeled mRNA, and Quant-iT TM The fluorescence coefficient between HS fluorophores. LNPs encapsulating unmodified mRNA were diluted at different rates and mRNA was diluted with and Quant-iT TM HS two fluorophore labels. fluorescence) and total mRNA concentration (Quant-iT TM HS fluorescence) (N=3) was measured under the same conditions. and Quant-iT TM The fluorescence coefficient (C f ).

[0200] The protocol was then designed to use two dilution points of mRNA-LNPs that corresponded to a more precise range of sensitivity for the two fluorophores (final mRNA concentrations were 1000 and 1250 ng / mL, respectively, in the diluted samples; see Table 6). The accuracy of this high-throughput assay was validated for different levels of encapsulation efficiency (see Table 7). Table 6: Biomaterials for standard and dual RNA encapsulation assays. This table summarizes the fluorophore concentrations used to quantify free and total mRNA concentrations in mRNA-LNP samples and the range of free and encapsulated mRNA (LNP). A high throughput (HT) screen based on the dual RNA encapsulation scheme provided in Example 4 was performed, but without absolute quantification of mRNA concentrations. Table 7: Encapsulation efficiency of mRNA-LNP using simplified assay. The mRNA-LNP source sample was known to have an encapsulation efficiency of 95%. Free mRNA was added to the sample to artificially adjust the encapsulation efficiency to 50%. and Quant-iT TM Prior to labeling free and total mRNA with HS fluorophores, LNPs were diluted to 2000 and 2500 ng / mL of total RNA. With Quant-iT TM The ratio between HS fluorescence (N=3) determined the percentage of free mRNA. The experiment was performed with two independent dilutions and the standard deviation of the encapsulation efficiency was calculated from the two dilutions. Em: Fluorescence emission.

[0201] As demonstrated herein, determination of encapsulation efficiency is highly accurate even when only two dilution points are used.Thus, the methods of the present invention can be successfully used in high throughput applications, such as screening, where a completely quantitative approach is not required.

[0202] Example 6: Evaluation of encapsulation efficiency using various mRNA-LNPs

[0203] Various mRNAs (comprising unmodified or modified nucleotides) and LNPs comprising different cationic lipids were evaluated using the high throughput method described in Example 5. The results obtained with this method were similar to those obtained with the RiboGreen assay and indicate that this method can be used regardless of LNP composition, mRNA sequence, or the presence of modified nucleosides in the mRNA (see Table 8). Table 8: Encapsulation efficiency as determined by RiboGreen assay and dual RNA encapsulation assay of the present 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) was determined in parallel. The dual RNA encapsulation assay used 100 mRNAs in the same well. (non-encapsulated mRNA) and Quant-iT TM Ratio of HS (total mRNA) emission (N=3). As described in Example 5, the standard deviation of the encapsulation efficiency was calculated from two different LNP dilutions (see Table 6).

[0204] Example 7: Evaluation of alternative fluorophores

[0205] Rated 17 to determine whether it can be used as an alternative fluorophore in the methods of the present invention. A linear standard curve was obtained, but when and 17 is no longer the case when combined ( Figure 6 , compare sub-figures A and B). This shows that The existence of 17 modifies Fluorescence. In addition, Figure 6 As shown in C, for The standard curves established by 17 were different for free mRNA and mRNA-LNP. In particular, the fluorescence of mRNA-LNP was higher than that of free mRNA. Without being bound by theory, this suggests that 17 can interact with the lipid component of LNPs.

[0206] Also rated Gold and Green II to determine whether they could be used as alternative fluorophores. Figure 7 As shown, when using Green II, the standard curves obtained for free mRNA and encapsulated mRNA were identical, indicating that this fluorophore permeates the LNPs. The standard curve obtained by Gold varied depending on the sample tested (i.e., free mRNA alone or mRNA-LNP). When exposed to gold, almost no fluorescence emission was detected, indicating that this fluorophore does not permeate the LNP.

[0207] In general, using Green II or A simple linear regression line determined when Gold labeled mRNA indicated that any of these fluorophores could be used in dual RNA encapsulation assays in combination with compatible fluorophores with detectably different fluorescence emissions.

[0208] Example 8: Automation of the Dual RNA Encapsulation Assay and Validation of the Method Using Two Different Spectrophotometers

[0209] Considering making this method automated, dual RNA encapsulation determination was implemented on the Starlet Hamilton robot platform.Used a source sample containing unmodified mRNA encapsulated in LNP containing OF-02 cationic lipids, known to have a total mRNA concentration of 1000 μg / mL and an encapsulation efficiency of 95% (as determined by the RiboGreen assay described in Example 1 above).Add unmodified free mRNA to the source sample to manually adjust the encapsulation efficiency to 70% and 50%.Using 40 μL as the final volume, various concentrations of these mRNA-LNP (ranging from 20 to 200 ng / μL) are loaded into PCR 96-well plates.As needed, the sample is diluted to 10 ng / μL with TE buffer in another PCR 96-well plate.Finally, the sample is diluted in a black 384-well microplate with a flat transparent bottom to provide two data points for each sample, loaded in duplicate (i.e., a total of four repeats for each condition).In TE buffer, the robot is loaded with and Quant-iT TM HS reagent was temporarily diluted to 0.5X and 1X, respectively, and 40 μL of the fluorophore mixture was added to each well. and Quant-iT TMHS was present at final concentrations of 0.25X and 0.5X, respectively. Fluorescence was measured as described in Example 1 using a Cytation 7 (Agilent Biotek) or Spectramax i3 spectrophotometer, and encapsulation efficiency was then determined using the corresponding equation provided in Example 1. All steps were performed on a robotic platform, from initial dilution of the sample to measurement of fluorescence by an appropriate spectrophotometer.

[0210] Figure 8 The results shown demonstrate that the method is automatable. Furthermore, the measurements are accurate and independent of the spectrophotometer used.

[0211] in conclusion

[0212] In view of the above content, method of the present invention represents a kind of improvement method of determining encapsulation efficiency.Especially, the quantity of required sample has reduced by at least 2 times, because can determine dual measurement from single hole.The method can be used for both quantitative and high throughput screening methods, and shows high-level accuracy in determining the encapsulation efficiency of nucleic acid in LNP.Especially, by using the ratio method provided in example 5 to measure LNP-RNA encapsulation efficiency, many different RNAs can be evaluated, and it is not necessary to provide a standard curve for each sample for the absolute quantification of the mRNA free and encapsulated.In addition, using the automated dual RNA encapsulation of example 8 to measure, the scheme is fully automated, wherein up to 95 samples are processed in less than two hours, and only a limited amount of raw materials (such as mRNA-LNP samples, fluorophores) are required.

[0213] Other embodiments of the disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the disclosure disclosed herein. It is intended that the specification and examples be considered exemplary only, with the true scope and spirit of the disclosure being indicated by the following claims.

[0214] All patents and publications cited herein are incorporated by reference in their entirety.

Claims

1. A method for determining RNA encapsulation efficiency in lipid nanoparticles (LNPs), the method comprising: a) contacting a sample comprising RNA encapsulated in LNPs with a first fluorophore and a second fluorophore, thereby forming a fluorophore-RNA complex, and b) detecting the fluorescent signal of the composite first and second fluorophores, Wherein the first fluorophore permeates the LNPs, and wherein the second fluorophore does not permeate the LNPs.

2. The method of claim 1, wherein the first fluorophore is Quant-iT TM HS or Green II.

3. The method of claim 1 or 2, wherein the second fluorophore is or Gold.

4. The method according to any one of the preceding claims, wherein the RNA is 10 to 50,000 nucleotides in length.

5. The method of any one of the preceding claims, wherein the RNA is double-stranded RNA or single-stranded RNA.

6. The method of any of the preceding claims, wherein the RNA comprises mRNA, miRNA, shRNA, rRNA, tRNA, snRNA, snoRNA, asRNA, siRNA, aiRNA, gRNA and / or piRNA.

7. The method of any one of the preceding claims, wherein the first fluorophore and the second fluorophore are added to the sample simultaneously.

8. The method of any one of the preceding claims, wherein the ratio of the first fluorophore to the second fluorophore is 2:

1.

9. The method of any one of the preceding claims, further comprising determining a ratio of the second fluorescent signal to the first fluorescent signal.

10. The method of any preceding claim, further comprising generating a standard curve of the first and second fluorescent signals.

11. The method of claim 10, further comprising determining the absolute amount of encapsulated RNA by matching each fluorescent signal detected in step b) with a corresponding standard curve.

12. A method for making LNPs encapsulating RNA, the method comprising a) encapsulating RNA in LNPs, and b) determining the efficiency of RNA encapsulation in LNPs according to the method of any one of claims 1-11.

13. The method of claim 12, wherein the method further comprises a step of synthesizing mRNA in vitro before step a).

14. A kit for determining RNA encapsulation efficiency in lipid nanoparticles (LNPs), the kit comprising: - a first fluorophore that permeates the LNP; - A second fluorophore that does not permeate the LNP; as well as - Instructions for use of the method according to any one of claims 1 to 11. 15.Quant-iT TM Use of HS for quantification of LNP-encapsulated RNA.

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