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, the method according to the present invention comprises step a) contacting a sample containing RNA encapsulated in LNPs with a first fluorophore and a second fluorophore, thereby forming a fluorophore-RNA complex, and step b) detecting the fluorescent signal of the complexed first and second fluorophores, wherein the first fluorophore is permeable to the LNPs and the second fluorophore is impermeable to the LNPs.

Description

[Technical Field]

[0001] The present invention relates to the field of RNA analysis, more specifically to the determination of RNA encapsulation efficiency. [Background technology]

[0002] Messenger RNA therapy (MRT) is becoming increasingly important as an approach to treating various diseases. MRT involves administering messenger RNA (mRNA) to patients in need of treatment to produce the protein encoded by the mRNA in vivo. To ensure efficient delivery of RNA to host cells in vivo, RNA is commonly encapsulated in carriers such as lipid nanoparticles (LNPs). Therefore, accurate characterization of the efficiency of RNA encapsulation is particularly important for determining the quality of mRNA for therapeutic applications.

[0003] RNA encapsulation efficiency is typically determined using the RiboGreen assay, which involves dissolving LNPs with a detergent, typically Triton X-100, to release the encapsulated nucleic acid so that it can be detected by the RiboGreen® fluorophore. However, the use of detergent results in foaming, which is undesirable, especially in high-throughput assays where sample volumes are low. In addition, to determine encapsulation efficiency, two separate measurements must be performed on untreated and detergent-treated samples to determine the free and total mRNA content (which allows for calculation of the percentage of encapsulated mRNA payload).

[0004] Therefore, there remains a need for improved methods for determining RNA encapsulation efficiency, which should, among other things, be fast, robust, cost-effective, and easy to perform. Summary of the Invention [Means for solving the problem]

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

[0006] 1. A method for determining RNA encapsulation efficiency in lipid nanoparticles (LNPs), comprising: a) contacting a sample containing RNA encapsulated in LNPs with a first fluorophore and a second fluorophore, thereby forming a fluorophore-RNA complex; and b) detecting the fluorescent signals of the conjugated first and second fluorophores; Including, The first fluorophore is permeable to the LNP, and the second fluorophore is impermeable to the LNP. A method is provided herein.

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

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

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

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

[0011] In certain embodiments, the RNA is between 500 and 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 sequentially to the sample.

[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, hi 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 a final concentration of 0.5×.

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

[0020] In certain embodiments, the method further comprises determining a ratio of the second fluorescent signal to the first fluorescent signal (i.e., the 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 of the first fluorescent signal and the second fluorescent signal.

[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, there is provided a method for producing RNA-encapsulated LNPs, comprising: a) encapsulating RNA into LNPs; and b) determining the efficiency of RNA encapsulation into LNPs according to the methods provided herein A method is provided which includes

[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 into LNPs.

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

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

[0030] In another aspect, there is provided a kit for determining the encapsulation efficiency of RNA into LNPs, comprising: - a first fluorophore that is permeable to the LNP; a second fluorophore that is impermeable to the LNP; and -Instructions for use in accordance with the methods provided herein A kit is provided comprising:

[0031] In another embodiment, Quant-iT for quantification of LNP-encapsulated RNA TM The use of HS is provided. [Brief explanation of the drawings]

[0032] [Figure 1] Determination of mRNA encapsulation efficiency by the RiboGreen assay or the method of the present invention ("dual RNA encapsulation" assay). After encapsulation in LNPs, mRNA generally exists in two different states in a sample: unencapsulated or "free" mRNA and mRNA encapsulated in LNPs (mRNA-LNPs). "Total mRNA" refers to the combination of both free and encapsulated mRNA. The RiboGreen assay (left panel) uses the RiboGreen® fluorophore (filled circle), which labels RNA but does not permeate LNPs. Consequently, unencapsulated mRNA can be measured with RiboGreen® alone (i.e., in TE buffer), while total mRNA can be measured in the presence of RiboGreen® in combination with a detergent such as Triton, which dissolves LNPs. The principle of the present invention is based on the use of two fluorophore-labeled RNAs (right panel), one of which is non-permeable (here, RiboGreen®, filled circle) and the other is permeable (here, Quant-iTTMHS, open circle). Advantageously, both fluorophores can be used in the same well without overlap or detrimental competition for spectral detection. [Figure 2] Determination of LNP permeability to Quant-iTTMHS. Quant-iTTMHS fluorescence emission was measured for unencapsulated (free) or encapsulated mRNA at different total mRNA concentrations. [Figure 3]Establishing linear regression curves for RiboGreen® and Quant-iTTM fluorophores in the presence of each other. Eight different concentrations of unencapsulated mRNA were labeled with both RiboGreen® and Quant-iTTMHS. Simple linear regression curves and the corresponding equations (Y) and R2 were determined from these eight points for each fluorophore. (A) Emission at 525 nm of RiboGreen® in the presence of Quant-iTTMHS as a function of free mRNA concentration. (B) Emission at 673 nm of Quant-iTTMHS in the presence of RiboGreen® as a function of free mRNA concentration. [Figure 4] Determination of the emission ratio between RiboGreen® and Quant-iTTMHS for the same concentration of labeled mRNA. The RiboGreen® fluorophore does not permeate the LNP; therefore, only non-encapsulated mRNA was labeled. The Quant-iTTMHS (Q) fluorophore permeates the LNP; therefore, both encapsulated and non-encapsulated mRNA were labeled. The fluorescence coefficient (Cf) between RiboGreen® and Quant-iTTMHS was determined for various concentrations (x) of mRNA labeled with a mixture of RiboGreen® and Quant-iTTMHS. Using several factors of dilution of the mRNA-LNP, the xR- and xQ-labeled mRNAs were adjusted, allowing for the calculation of Cf at various x. [Figure 5] Determination of encapsulation efficiency without a standard RNA curve. The percentage of free mRNA, and therefore the encapsulation efficiency, can be calculated from the emission ratio of RiboGreen® and Quant-iTTMHS in the same well, as shown in the figure. The Cf between RiboGreen® and Quant-iTTMHS for the same concentration of labeled mRNA was experimentally determined to be constant (0.01). [Figure 6]Evaluation of the SYTO® 17 fluorophore. (A-B) A range of standard mRNA concentrations with the RiboGreen® fluorophore alone (A) or with SYTO® 17 in the same well (B). Five different concentrations of free mRNA were labeled with RiboGreen® (0.5x) in TE buffer with or without SYTO® 17 (1 μM). A simple linear regression curve and the corresponding equations (Y) and R2 were determined from five points of standard mRNA dilution. (C) Determination of LNP permeability to SYTO® 17. SYTO® 17 fluorescence emission was measured for free or encapsulated mRNA at various total mRNA concentrations. [Figure 7] Evaluation of SYBR® Green II and SYBR® Gold Fluorophores. The permeability of SYBR® Green II and SYBR® Gold through LNPs was evaluated to determine whether these fluorophores could be suitable for use in dual RNA encapsulation assays. (A) When SYBR® Green II was used, the emission curves obtained for free and encapsulated mRNA were identical, indicating that this fluorophore permeates LNPs. (B) When SYBR® Gold was used, an emission curve was obtained for free mRNA, and almost no fluorescence emission was detected for encapsulated mRNA, indicating that this fluorophore does not permeate LNPs. Simple linear regression curves determined by labeling mRNA with SYBR® Green II or SYBR® Gold indicate that these fluorophores can be utilized in dual RNA encapsulation assays in combination with compatible fluorophores. [Figure 8]Automation of the Dual RNA Encapsulation Assay: Validation of the Method with Two Different Spectrophotometers. Encapsulation efficiency was determined using an automated system and two different spectrophotometers: Cytation 7 and Spectramax i3. Results were shown to be accurate regardless of the spectrophotometer used and demonstrate that the method according to the present invention can be automated. DETAILED DESCRIPTION OF THE INVENTION

[0033] The present disclosure relates, inter alia, to methods for determining RNA encapsulation efficiency.

[0034] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by one of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein may also be used in the practice or testing of this disclosure. In the case of conflict, the present specification, including definitions, shall control. Generally, terminology used in connection with and in the arts of cell and tissue culture, molecular biology, virology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein is that well known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications as commonly accomplished in the art or as described herein. Furthermore, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Throughout this specification and the embodiments, the words "have" and "comprise" or variations thereof, such as "has," "having," "comprises," or "comprising," are understood to mean the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group 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 form part of the common general knowledge in the art.

[0035] It should be noted that the term "a" or "an" entity refers to one or more of that entity; for example, a "nucleotide sequence" is understood to refer to one or more nucleotide sequences. Thus, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein.

[0036] Furthermore, "and / or," as used herein, should be considered a specific disclosure of each of the two specified features or components, regardless of the presence or absence of the other. Thus, the term "and / or," as used in phrases such as "A and / or B," is intended herein 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 encompass each of the following embodiments: 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).

[0037] Whenever an embodiment is described herein in the language of "comprising," it is understood that otherwise similar embodiments described in the terms "consisting of" and / or "consisting essentially of" are also provided.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press can provide those skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0039] Units, prefixes, and symbols are denoted in the form accepted by the International System of Units (SI). Numerical ranges are inclusive of the numbers defining the range. The headings provided herein are not intended to limit the various aspects of this disclosure. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0040] The terms "approximately" or "about" are used herein to mean approximately, roughly, around, around, or within a range. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical values. In general, the term "about" may modify a numerical value above and below the stated value by, for example, a variance of 10 percent above or below (higher or lower). In some embodiments, the term indicates a deviation from the stated numerical value by only ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, or ±0.01%. In some embodiments, "about" indicates a deviation from the stated numerical value by only ±10%. In some embodiments, "about" indicates a deviation from the stated numerical value by only ±5%. In some embodiments, "about" indicates a deviation from the stated numerical value by only ±4%. In some embodiments, "about" indicates only a ±3% deviation from the indicated numerical value. In some embodiments, "about" indicates only a ±2% deviation from the indicated numerical value. In some embodiments, "about" indicates only a ±1% deviation from the indicated numerical value. In some embodiments, "about" indicates only a ±0.9% deviation from the indicated numerical value. In some embodiments, "about" indicates only a ±0.8% deviation from the indicated numerical value. In some embodiments, "about" indicates only a ±0.7% deviation from the indicated numerical value. In some embodiments, "about" indicates only a ±0.6% deviation from the indicated numerical value. In some embodiments, "about" indicates only a ±0.5% deviation from the indicated numerical value. In some embodiments, "about" indicates only a ±0.4% deviation from the indicated numerical value. In some embodiments, "about" indicates only a ±0.3% deviation from the indicated numerical value. In some embodiments, "about" indicates only a ±0.1% deviation from the indicated numerical value. In some embodiments, "about" indicates only a ±0.05% deviation from the indicated numerical value. In some embodiments, "about" indicates only a ±0.01% deviation from the indicated numerical value.

[0041] As used herein, the term "nucleic acid" or "nucleic acid molecule" refers to a polynucleotide chain comprising individual nucleic acid residues. "Nucleic acid" encompasses single- and / or double-stranded DNA and / or cDNA, as well as single- and / or double-stranded RNA. Furthermore, "nucleic acid," "DNA," "RNA," and / or similar terms include nucleic acid analogs, i.e., analogs having other than a phosphodiester backbone. For example, so-called "peptide nucleic acids," which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention. Nucleic acid sequences encoding proteins and / or RNA may contain introns. Nucleic acids can be of any origin, e.g., viral, bacterial, archaeal, fungal, ribosomal, eukaryotic, or prokaryotic. Nucleic acids can be purified from natural sources (e.g., any biological sample and any organism, tissue, cell, or subcellular compartment), produced using recombinant expression systems, optionally purified, chemically synthesized, etc. Where appropriate, for example, in the case of chemically synthesized molecules, nucleic acids can include nucleoside analogs, such as analogs having chemically modified bases or sugars, backbone modifications, and the like.In some embodiments, nucleic acids are selected from natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5- The nucleic acid may comprise propynylcytidine, 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); intercalated 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 linkages). In some embodiments, the nucleic acid is comprised of "unmodified nucleic acid," meaning a nucleic acid (e.g., a polynucleotide and residues, e.g., 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 certain embodiments, the nucleic acid is mRNA.

[0042] As used herein, the term "messenger RNA ("mRNA") refers to at least one peptide, polypeptide, or protein. An mRNA can contain one or more coding and non-coding regions. The coding region is alternatively referred to as an open reading frame (ORF). The non-coding regions of an mRNA include the 5' cap, 5' untranslated region (UTR), 3' UTR, and poly A tail.

[0043] As used herein, mRNA encompasses both modified and unmodified RNA. In some embodiments, the mRNA disclosed herein may contain one or more modifications that typically improve RNA stability. Exemplary modifications may include backbone modifications, sugar modifications, or base modifications. In some embodiments, the disclosed mRNA may be synthesized from 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 mRNAs may contain modified nucleotide analogs or derivatives of purines and pyrimidines, such as 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxymethylaminomethyl) ...

[0033] Uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, queosine, pD-mannosyl-queosine, phosphoramidate, phosphorothioate, peptide nucleotide, methylphosphonate, 7-deazaguanosine, 5-methylcytosine, and inosine.

[0044] In some embodiments, the mRNA may comprise at least one chemical modification including, but not limited to, pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-l-methyl-l-deaza-pseudouridine, 2-thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine. In some embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and any combination thereof, hi some embodiments, the chemical modification comprises N1-methylpseudouridine.

[0045] 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.

[0046] Where appropriate, e.g., in the case of chemically synthesized molecules, mRNA may include nucleoside analogs, such as analogs having chemically modified bases or sugars, backbone modifications, etc. In some embodiments, mRNA may include natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5-propynyl-cytidine, C-5-propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-amino-2-methyl ... -deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, pseudouridine, and 5-methylcytidine; chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated 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 linkages).

[0047] In addition to messenger RNA, there are several non-coding RNAs that may be involved in regulating transcription and / or translation and immune stimulation. Within the present invention, the term "RNA" further encompasses any type of single-stranded RNA (ssRNA) or double-stranded RNA (dsRNA) molecule known in the art, such as viral RNA, retroviral RNA and replicon RNA, messenger RNA (mRNA), microRNA (miRNA), small hairpin RNA (shRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), antisense RNA (asRNA), small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), CRISPR / Cas9 guide RNA (gRNA), Piwi-interacting RNA (piRNA), dicer substrate RNA, ribozyme, aptamer, riboswitch, immune stimulatory RNA, and long non-coding RNA (IncRNA), and any combination thereof.

[0048] In one embodiment, the RNA is double-stranded RNA. In another embodiment, the RNA is single-stranded RNA. If the RNA is single-stranded, the RNA may further comprise one or more secondary structures, such as a hairpin. 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.

[0049] As used herein, a "lipid nanoparticle (LNP)" is a composition comprising one or more lipids. The lipids present in an LNP may include one or more cationic / ionized, PEGylated, helper, or other lipids, such as phospholipids. LNPs are typically on the order of micrometers or less in size and may comprise a lipid bilayer. As used herein, lipid nanoparticles encompass lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and lipoplexes, unless otherwise specified. In some embodiments, an LNP may be a liposome having a lipid bilayer with a diameter of 500 nm or less. Typically, an LNP is in the shape of a hollow sphere containing an aqueous compartment. A "lipid component" is a component of an LNP that comprises one or more lipids. Typically, liposomes, as used herein, may be formed by mixing one or more lipids or by mixing one or more lipids with a polymer. LNPs may contain, inter alia, an ionized / cationic lipid, as well as optional non-cationic lipids, optional cholesterol-based lipids, and / or optional PEGylated lipids. "Non-cationic lipid" or "helper lipid" refers to any neutral, zwitterionic, or anionic lipid. "PEG lipid" or "PEGylated lipid" refers to a lipid that includes a polyethylene glycol moiety.

[0050] As used herein, "encapsulation" and its grammatical equivalents refer to the process of enclosing nucleic acid within a nanoparticle. Obviously, encapsulation can be complete, substantial, or partial. The nucleic acid can be present in the aqueous phase within a liposome or incorporated into the lipid layer. As used herein, "empty" LNPs can refer to nanoparticles that are substantially free of nucleic acid. As used herein, "empty" LNPs can refer to nanoparticles that consist essentially only of lipid components. Nucleic acid that is not encapsulated in an LNP is referred to herein as "unencapsulated," "non-encapsulated," or "free" nucleic acid.

[0051] As used herein, "encapsulation efficiency" and "encapsulation efficiency" refer to the amount of nucleic acid incorporated into the internal structure of the LNP (i.e., inaccessible to hydrophilic solvents or molecules when the LNP is intact) relative to the initial total amount of nucleic acid used to prepare the LNP. As an example, if 95 mg of nucleic acid is encapsulated in the LNP out of a total amount of 100 mg of nucleic acid initially provided in the composition, the encapsulation efficiency may be indicated as 95%. The initial total amount of nucleic acid may be determined, inter alia, by adding the amount of encapsulated nucleic acid to the amount of unencapsulated nucleic acid in a given sample.

[0052] As used herein, the term "sample containing nucleic acids encapsulated in LNPs" refers to a sample containing nucleic acids encapsulated in LNPs, such as RNA or mRNA. In some embodiments, a sample containing nucleic acids encapsulated in LNPs comprises a mixture of nucleic acids encapsulated in LNPs and unencapsulated nucleic acids. A sample containing nucleic acids encapsulated in LNPs (e.g., RNA or mRNA) is, for example, a batch of RNA-LNPs or mRNA-LNPs obtained by a method for producing LNPs that encapsulate RNA or mRNA.

[0053] As used herein, the term "RNA-LNP" refers to RNA encapsulated in LNPs. Similarly, "mRNA-LNP" refers to mRNA encapsulated in LNPs.

[0054] As used herein, the term "labeled" refers to the attachment of a detectable signal, agent or moiety, such as a fluorophore, to a molecule, such as a nucleic acid molecule.

[0055] As used herein, "fluorophore" or "fluorochrome" refers to a chemical group that absorbs light of a particular wavelength (excitation frequency) and subsequently emits light at a longer wavelength (emission frequency), i.e., it fluoresces. Fluorophores may contain substituents that alter the solubility, spectral properties, or physical properties of the fluorophore. Fluorophores 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, including, but 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, benzopyrylium dyes, benzazole dyes, borapolyazaindacene dyes, such as fluorescein, rhodamine, or rhodol, as well as those described in Molecular Probes® Handbook: A Guide to Fluorescent Probes and Labeling Technologies (11 th edition, 2010), and other fluorophores described in U.S. Patent Application Publication No. 2005 / 0208534.

[0056] As used herein, the term "detectably distinct" refers to signals that are distinguishable or separable by observational or instrumental physical properties. For example, a fluorophore is readily distinguishable by its spectral properties, i.e., excitation and emission spectra, from other fluorophores in a sample, and optionally from additional materials that are present.

[0057] As used herein, the term "sensitivity range" or "sensitivity scale" refers to the range of RNA concentrations over which a given labeling fluorophore gives a linear curve of emission.

[0058] As used herein, "permeability" refers to a material property that allows one or more substances to pass through the material. "Selective permeability" refers to a material property that allows certain substances (e.g., fluorophores) to pass through the material while preventing other substances from passing through the material. In this context, the term "permeating" refers to the ability of a substance (i.e., a fluorophore) to penetrate or pass through a lipid structure, such as the lipid component of an LNP. The ability of a fluorophore to permeate (or not permeate) an LNP can be readily determined by comparing the fluorescence levels detected between two samples: a first sample containing free mRNA and LNP-encapsulated mRNA (mRNA-LNP) with a known concentration of free mRNA / mRNA-LNP (e.g., as can be determined by RiboGreen assay), and a second sample with a free mRNA concentration identical to the concentration of free mRNA in the first sample or the total concentration of mRNA (free mRNA + mRNA-LNP) in the first sample. A fluorophore is permeable if the fluorescence level is the same in a first sample and a second sample with a concentration of free mRNA that is the same as the total concentration of mRNA in the first sample. A fluorophore is non-permeable if the fluorescence level is the same in a first sample and a second sample with a concentration of free mRNA that is the same as the concentration of free mRNA in the first sample.

[0059] As used herein, the term "contacting" refers to mixing two or more components so that they can interact (e.g., contacting an RNA-LNP with a fluorophore). The two or more components can be incubated for a time sufficient to produce a desired effect (e.g., to form a complex).

[0060] As used herein, the term "control" refers to a standard against which results can be compared. Typically, controls are used to enhance the integrity of an experiment by isolating a variable in order to draw conclusions about that variable. In some embodiments, a control is a reaction or assay performed simultaneously with a test reaction or assay to provide a basis for comparison. In one experiment, the "test" (i.e., the variable being tested) is applied. In a second experiment, the "control," the variable being tested, is not applied. In some embodiments, a control is a historical control (i.e., a previously performed test or assay, or a previously known amount or result). In some embodiments, a control is or includes a printed or otherwise kept record. A control can be a positive control or a negative control.

[0061] As used herein, the term "kit" refers to any delivery system for delivering materials. Such delivery systems may include systems that allow for the storage, transport, or delivery of various diagnostic or therapeutic reagents (e.g., oligonucleotides, antibodies, enzymes, etc. in appropriate containers) and / or supporting materials (e.g., buffers, instructions for performing an assay, etc.) from one location to another. For example, a kit may include one or more enclosures (e.g., boxes) containing relevant reaction reagents and / or supporting materials. As used herein, the term "fragmentation kit" refers to a delivery system that includes two or more separate containers, each housing a portion of the total kit components. The containers may be delivered to the intended recipient together or separately. For example, a first container may contain an enzyme for use in an assay, while a second container contains oligonucleotides. The term "fragmentation kit" is intended to encompass, but is not limited to, kits containing analyte-specific reagents (ASRs) regulated under Section 520(e) of the Federal Food, Drug, and Cosmetic Act. Indeed, any delivery system that includes two or more separate containers, each containing a portion of the total kit components, is encompassed by the term "fragmented kit." In contrast, a "combined kit" refers to a delivery system that includes all of the components in a single container (e.g., a single box containing each of the desired components). The term "kit" includes both fragmented kits and combined kits.

[0062] As described in detail below, the present invention is based on the use of two fluorophores that bind to nucleic acids and have different permeability profiles through LNPs. Specifically, the first fluorophore permeates LNPs that encapsulate nucleic acids (e.g., mRNA-LNPs), while the second fluorophore does not permeate LNPs. The first fluorophore forms complexes with encapsulated and unencapsulated nucleic acids that may be present in the sample, while the second fluorophore forms complexes with unencapsulated nucleic acids that may be present in the sample. Detecting the fluorescence emission of the two fluorophores ultimately establishes the encapsulation efficiency. Advantageously, the method of the present invention allows both fluorescence measurements to be performed on a single sample, thereby reducing the number of samples required to determine encapsulation efficiency by at least two-fold. Furthermore, the method can be used for high-throughput screening, since foaming is no longer an issue in the absence of detergent. Thus, the present invention provides a simple, reliable, and efficient quantitative or semi-quantitative approach for assessing RNA encapsulation efficiency. The present invention is particularly useful for quality control during manufacturing and for characterizing encapsulated nucleic acids, such as mRNA, as pharmaceutical components of final therapeutic products.

[0063] In one aspect, the present invention provides a method for determining the efficiency of nucleic acid encapsulation into LNPs, said method comprising: a) contacting a sample containing nucleic acids encapsulated in LNPs with a first fluorophore and a second fluorophore, thereby forming a fluorophore-nucleic acid complex; and b) detecting the fluorescent signals of the conjugated first and second fluorophores; Including, The first fluorophore is permeable to the LNP, and the second fluorophore is impermeable to the LNP.

[0064] While a high encapsulation efficiency is desirable (e.g., approaching 100%), for example, when encapsulating nucleic acids for pharmaceutical use, encapsulation efficiencies ranging from 0 to 100% can be determined using the methods of the present invention. Thus, in some embodiments, the determined encapsulation efficiency is 0 to 100%. In some embodiments, the encapsulation efficiency is at least about 50%, e.g., 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 80-100%.

[0065] The total concentration of nucleic acid (i.e., both unencapsulated and encapsulated nucleic acid) is such that it falls within the sensitivity range of the fluorophore used in the methods of the invention. In one embodiment, the nucleic acid is present at a final concentration of at least 0.25 μg / mL. In one embodiment, the nucleic acid is present at a final concentration comprised between 0.25 and 10 μg / mL. In one embodiment, the nucleic acid is present at a final concentration comprised between 0.25 and 5 μg / mL. In one embodiment, the nucleic acid is present at a final concentration comprised between 0.25 and 1.25 μg / mL. In one embodiment, the nucleic acid is present at a final concentration comprised between 0.25 and 1 μg / mL. A sample containing nucleic acid encapsulated in LNPs may be diluted one or more times to provide a nucleic acid concentration that falls within the sensitivity range of the fluorophore.

[0066] The nucleic acid encapsulated in the LNP can be any type of nucleic acid provided herein. The encapsulated nucleic acid molecule (e.g., RNA) can be of any length. In one embodiment, the length of the nucleic acid molecule is at least about 10 nucleotides (nt). In one embodiment, the length of the nucleic acid molecule is at least about 20 nt. In one embodiment, the length of the nucleic acid molecule is at least about 50 nt. In one embodiment, the length of the nucleic acid molecule is at least about 100 nt. In one embodiment, the length of the nucleic acid molecule is at least about 500 nt. In one embodiment, the length of the nucleic acid molecule is about 10 to 50,000 nt. In one embodiment, the length of the nucleic acid molecule is about 20 to 25,000 nt. In one embodiment, the length of the nucleic acid molecule is about 300 to 10,000 nt. In one embodiment, the length of the nucleic acid molecule is about 300 to 10,000 nt. In one embodiment, the length of the nucleic acid molecule is about 400 to 8,000 nt. In one embodiment, the length of the nucleic acid molecule is about 500 to 5,000 nt. In some embodiments, the LNP comprises between 1 and 20, optionally between 5 and 10 or between 6 and 8, nucleic acid molecules.

[0067] The first fluorophore provided in the context of this method is permeable to the LNP. In one embodiment, the first fluorophore is a fluorophore as described herein in Quant-iT TM Quant-iT, also known as HS TMHighly sensitive reagent. Quant-iT TM The HS reagent is comprised of the RiboRed fluorophore and therefore may alternatively be referred to herein as "RiboRed." TM The excitation spectrum of HS is in the range of 640 nm to 648 nm. TM The excitation spectrum of HS is 644 nm. In one embodiment, 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 in the range of 666 nm to 680 nm. 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, the Quant-iT TM The excitation and emission spectra of HS are 644 and 673 nm, respectively. In another embodiment, the first fluorophore is a SYBR® Green II fluorophore. In one embodiment, the excitation spectrum of SYBR® Green II is in the range of 491 nm to 499 nm. In one embodiment, the excitation spectrum of SYBR® Green II is 495 nm. In one embodiment, excitation is performed with a 9 nm bandwidth (e.g., 495 nm + / - 4.5 nm). In one embodiment, the emission spectrum of SYBR® Green II is in the range of 513 nm to 527 nm. In one embodiment, the emission spectrum of SYBR® Green II is 520 nm. In one embodiment, the emission spectrum is collected with a 15 nm bandwidth (e.g., 520 nm + / - 7.5 nm). In one embodiment, the excitation and emission spectra of SYBR® Green II are 495 and 520 nm, respectively.

[0068] The second fluorophore provided in the context of this method does not penetrate LNP. Those skilled in the art will further readily understand that the second fluorophore should be detectably distinct from the first fluorophore to ensure that the fluorescence of each fluorophore can be measured from a single sample. 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 RiboGreen® (see, e.g., Jones et al., Analytical Biochemistry. (1998) 265:368-374). RiboGreen® is a fluorophore that is used in Quant-iT TMIt is detectably distinguishable from HS. In one embodiment, excitation of RiboGreen® can be performed at 485±10 nm. In one embodiment, excitation of RiboGreen® can be performed at 486±5 nm. In one embodiment, the excitation spectrum of RiboGreen® is in the range of 475 nm to 495 nm. In one embodiment, the excitation spectrum of RiboGreen® is in the range of 470 nm to 491 nm. In one embodiment, the excitation spectrum of RiboGreen® is 485 nm. In one embodiment, excitation is performed with a 9 nm bandwidth (e.g., 485 nm + / - 4.5 nm). In one embodiment, the fluorescence emission of RiboGreen® can be collected at 530±15 nm. In one embodiment, the emission spectrum of RiboGreen® is in the range of 515 nm to 545 nm. In one embodiment, the emission spectrum of RiboGreen® is 525 nm. In one embodiment, the emission spectrum is collected with a 15 nm bandwidth (e.g., 525 nm + / - 7.5 nm). In one embodiment, the excitation and emission spectra of RiboGreen® are 485 nm and 525 nm, respectively. In another embodiment, the second fluorophore is SYBR® Gold. SYBR® 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 SYBR® Gold is in the range of 491 nm to 499 nm. In one embodiment, the excitation spectrum of SYBR® Gold is 495 nm. In one embodiment, excitation is performed with a 9 nm bandwidth (e.g., 495 nm + / - 4.5 nm). In one embodiment, the emission spectrum of SYBR® Gold is 537 nm. In one embodiment, the emission spectrum is collected with a 15 nm bandwidth (e.g., 537 nm + / - 7.5 nm). In one embodiment, the emission spectrum of SYBR® Gold is in the range of 530-544 nm.In one embodiment, the excitation and emission spectra of SYBR® Gold are 495 and 537 nm, respectively.

[0069] As will be readily understood by those skilled in the art, to accurately determine encapsulation efficiency, the fluorophore must not generate nonspecific fluorescence, for example, due to interactions between the fluorophore and the lipid components of the LNP. In some embodiments, a control sample containing no nucleic acid can be used to determine baseline fluorescence. This baseline can be subtracted from the fluorescence detected in a corresponding sample containing LNPs encapsulating nucleic acid.

[0070] In some embodiments, one or both fluorophores exhibit conditional fluorescence upon binding to a nucleic acid.

[0071] To ensure that the fluorescent signals from the conjugated first and second fluorophores can be individually identified, the fluorescent signals of the conjugated first and second fluorophores should be detectably distinct. In one embodiment, the fluorophores used in the method do not have overlapping excitation and emission spectra. If the spectra may overlap, an appropriate cutoff can be used to distinguish the signals.

[0072] To ensure that the fluorescent signal from the conjugated first and second fluorophores is determined without interference, the first and second fluorophores must not compete for binding to the nucleic acid.

[0073] To accurately determine encapsulation efficiency, the sensitivity range of the first and second fluorophores should encompass the total concentration of nucleic acid (i.e., both unencapsulated and encapsulated nucleic acid) present in the sample.

[0074] Fluorophore concentrations may be expressed in units (e.g., μg / mL) or as a dilution factor relative to the initial concentration provided by the manufacturer. For example, diluting a 200x concentrated solution to a 1x concentration may be expressed as a 1:200 dilution. Alternatively, fluorophore concentrations may be expressed as the 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 in the range of 1:1 to 20:1. In one embodiment, the ratio of the first fluorophore to the second fluorophore is in the range of 1.5:1 to 10:1. In one embodiment, the ratio of the first fluorophore to the second fluorophore is 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.

[0075] In one embodiment, the first and second fluorophores are each Quant-iT TM HS and RiboGreen®. TM The ratio of HS to RiboGreen® is 2:1. TM HS is provided at a final concentration of 0.5x and RiboGreen® is provided at a final concentration of 0.25x.

[0076] Prior to measuring fluorescence emission, the sample is contacted with the fluorophore for a time sufficient for the fluorophore to bind to the nucleic acid and form a fluorophore-nucleic acid complex. In one embodiment, the fluorophore is incubated with the sample for more than 5 minutes. In one embodiment, the fluorophore is incubated with the sample for more than 10 minutes. In one embodiment, the fluorophore is incubated with the sample for 15-30 minutes. In one embodiment, the fluorophore is incubated with the sample for 20 minutes. In one embodiment, the fluorophore is incubated with the sample for 25 minutes. In one embodiment, the incubation is performed in the dark. In one embodiment, the incubation is performed at room temperature. In one embodiment, the fluorophore is incubated with the sample for 15-30 minutes in the dark at room temperature.

[0077] The above fluorophores bind to RNA. If the nucleic acid is not RNA, other fluorophores may be used. As an example, if the nucleic acid is dsDNA, PicoGreen® fluorophore may be considered.

[0078] 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, addition of a first fluorophore and measurement of a first fluorescent signal of the first fluorophore is followed by addition of a second fluorophore and measurement of a second fluorescent signal, or vice versa.

[0079] A control can be used to quantify the amount of encapsulated nucleic acid. In some embodiments, the control comprises a sample with a predetermined amount of nucleic acid. In some embodiments, the control comprises a sample with a predetermined amount of free nucleic acid. In some embodiments, the control comprises a sample with a predetermined amount of encapsulated nucleic acid. In some embodiments, the control comprises a predetermined amount of total nucleic acid. In some embodiments, the control comprises a predetermined amount of both 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 comprising a predetermined amount of encapsulated RNA.

[0080] The efficiency of nucleic acid (eg, RNA or mRNA) encapsulation in lipid nanoparticles is determined based on the detected fluorescent signals of the conjugated first and second fluorophores.

[0081] In some embodiments, the assay is performed quantitatively by establishing a calibration curve (also called a standard curve). In other words, encapsulation efficiency can be determined qualitatively or semi-quantitatively by comparing the fluorescent signal detected in an unknown sample with that of a known standard sample, or quantitatively by comparing it to a standard curve created using samples with many known nucleic acid concentrations. For example, quantification can be performed by creating a set of standard or calibrator solutions with known amounts of total nucleic acid and / or known amounts of free nucleic acid. These standard or calibrator solutions can be serially diluted, and the signal values obtained from each test concentration of the standard or calibrator are used to generate a standard curve (plotting the concentration of the standard against the resulting signal value). Once the standard quantification curve is established, the levels of total and free nucleic acid in a sample can be determined by plotting the resulting signal on the corresponding standard curve.

[0082] Thus, in one embodiment, the method further comprises generating a standard curve of the first and second fluorescent signals. In one embodiment, the method further comprises determining the absolute amount of encapsulated RNA by plotting each fluorescent signal detected in step b) with the corresponding standard curve. The amount (percentage) of unencapsulated nucleic acid can then be determined by dividing the amount of nucleic acid detected with the second fluorophore (i.e., a fluorophore that does not permeate the LNP) by the amount of nucleic acid detected with the first fluorophore (i.e., a fluorophore that permeates the LNP). The corresponding formula is shown below:

number

[0083] In some embodiments, the ratio of fluorescence between two fluorophores can be used to directly determine encapsulation efficiency (i.e., without the need to generate a standard curve). In some embodiments, the fluorescence ratio between two fluorophores is first established over a range of nucleic acid concentrations. The fluorescence coefficient (C) is calculated 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, C f is 0.01. In some embodiments, the fluorescent signal provided by the second fluorophore (i.e., the fluorophore that does not permeate the LNP and therefore binds to unencapsulated nucleic acid) is divided by the fluorescent signal provided by the first fluorophore (i.e., the fluorophore that permeates the LNP and therefore binds to total nucleic acid). This allows the relative amount (percent) of free nucleic acid to be determined. An exemplary formula is shown below:

number

[0084] This formula can be expressed as follows:

number

[0085] The percent encapsulation efficiency can then be determined by subtracting the amount of free nucleic acid from 100, as described above.

[0086] In some embodiments, a sample containing no nucleic acid (i.e., a blank) can be measured and the corresponding fluorescent signal subtracted from the signal measured in the corresponding sample containing nucleic acid. In some embodiments, the blank can include empty LNPs.

[0087] In some embodiments, the fluorescence ratio is determined for two RNA-LNP dilutions and then averaged to provide the amount (percent) of free nucleic acid. In some embodiments, the estimated final concentration of RNA (i.e., total RNA) in the dilutions is between 0.25 and 10 μg / mL. In some embodiments, the estimated final concentration of RNA in the dilutions is between 0.25 and 5 μg / mL. In some embodiments, the estimated final concentrations of RNA are about 1 μg / mL and about 1.25 μg / mL for the two RNA-LNP dilutions, respectively.

[0088] In some embodiments, the methods provided herein are used to characterize the encapsulation of a batch of RNA-LNPs. In some embodiments, the methods provided herein are performed prior to releasing a batch of LNPs encapsulating nucleic acids. In some embodiments, the encapsulation efficiency determined according to the methods provided herein is at least 80% upon batch release. Thus, in one embodiment, a method for batch release includes a) determining the efficiency of RNA encapsulation into LNPs according to the methods provided herein, and b) releasing the batch of RNA-LNPs when the encapsulation efficiency is at least 80%.

[0089] The present invention further relates to a method for producing LNPs encapsulating nucleic acids, comprising step a) encapsulating nucleic acids in LNPs and step b) determining the encapsulation efficiency of the nucleic acids in the LNPs according to the methods provided herein. In one embodiment, the nucleic acid is RNA, such as mRNA, as described herein. Accordingly, a method for producing LNPs encapsulating RNA is further disclosed, comprising step a) encapsulating RNA in LNPs and step b) determining the encapsulation efficiency of the nucleic acids in the LNPs according to the methods provided herein.

[0090] In one embodiment, the LNP comprises one or more ionizable lipids, one or more helper lipids, and one or more PEG-modified lipids.

[0091] In one embodiment, the nucleic acid molecules and / or LNPs correspond to those disclosed in U.S. Patent Application Publication No. 2022 / 0142923, the entire contents of which are incorporated herein by reference. In particular, the LNPs comprise four categories of lipids: (i) ionizable lipids; (ii) PEGylated lipids; (iii) cholesterol-based lipids; and (iv) helper lipids.

[0092] Ionized lipids. Ionizable lipids facilitate encapsulation of mRNA and can be cationic lipids, which provide a positively charged environment at low pH to facilitate efficient encapsulation of negatively charged mRNA drug substances.

[0093] 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 diketopiperazine core and a degradable ester linkage for connecting the doubly unsaturated tail, while OF-02 contains the same diketopiperazine core and a non-degradable 1,2-amino-alcohol linkage for connecting the doubly unsaturated tail (Fenton et al., Adv Mater. (2016) 28:2939; U.S. Patent No. 10,201,618).

[0094] In some embodiments, the cationic lipid is cKK-E10 (Dong et al., PNAS (2014) 111(11):3955-60; U.S. Patent No. 9,512,073).

[0095] 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)disulfanayl)ethyl)piperazin-1-yl)ethyl 4-(bis(2-hydroxydecyl)amino)butanoate), GL-HEPES-E3-E12-DS-4-E10(2-(4-(2-((3-(bis(2-hydroxydecyl)amino)butyl)disulfanayl)ethyl)piperazin-1-yl)ethyl 4-(bis(2-hydroxydecyl)amino)butanoate), and GL-HEPES-E3-E12-DS-3-E14 (2-(4-(2-((3-(bis(2-hydroxytetradecyl)amino)propyl)disulfanayl)ethyl)piperazin-1-yl)ethyl 4-(bis(2-hydroxydodecyl)amino)butanoate), which are HEPES-based disulfide cationic lipids with a piperazine core.

[0096] Other cationic lipids that can be used include those described in Dong (see above) and US Pat. No. 10,201,618.

[0097] PEGylated lipids PEGylated lipid components provide control over nanoparticle size and stability. The addition of such components can prevent complex aggregation, extend circulation life, and provide a means to enhance delivery of lipid-nucleic acid pharmaceutical compositions to target tissues (Klibanov et al., FEBS Letters (1990) 268(1):235:7). These components can be selected to be rapidly removed from the pharmaceutical composition in vivo (see, for example, U.S. Patent No. 5,885,613).

[0098] Contemplated PEGylated lipids include C6-C 20 (e.g., C8, C 10 , C 12 , C 14 , C 15 , or C 18 Examples of PEGylated lipids include, but are not limited to, polyethylene glycol (PEG) chains up to 5 kDa in length covalently attached to lipids having alkyl chains up to 5 kDa in length, such as derivatized ceramides (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).

[0099] In certain exemplary embodiments, the PEG has a high molecular weight, e.g., 2000-2400 g / mol. In some embodiments, the PEG is PEG2000 (or PEG-2K). In certain embodiments, the PEGylated lipid herein is DMG-PEG2000, DSPE-PEG2000, DLPE-PEG2000, DSG-PEG2000, or C8 PEG2000.

[0100] Cholesterol-based lipids The cholesterol component provides stability to the lipid bilayer structure of the nanoparticle. In some embodiments, the LNP comprises one or more cholesterol-based lipids. Suitable cholesterol-based lipids include, for example, DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine (Gao et al., Biochem Biophys Res Comm. (1991) 179:280; Wolf et al., BioTechniques (1997) 23:139; U.S. Pat. No. 5,744,335), imidazole cholesterol ester ("ICE"; WO 2011 / 068810), 3-sitosterol, fucosterol, stigmasterol, and other modified forms of cholesterol. In some embodiments, the cholesterol-based lipid used in the LNP is cholesterol.

[0101] Helper lipids Helper lipids improve the structural stability of LNPs and aid in endosomal escape, which improves uptake and release of mRNA drug payloads. In some embodiments, the helper lipid is a neutral lipid, i.e., a lipid that has no net charge at the conditions in which the composition is formulated and / or administered. In some embodiments, the helper lipid is an "anionic lipid," i.e., a lipid that has a net negative charge at a selected pH, such as physiological pH. In some embodiments, the helper lipid is a zwitterionic lipid with fusogenic properties to improve uptake and release of drug payloads. 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-dielideyl-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).

[0102] 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-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), or combinations thereof.

[0103] In certain embodiments, the helper lipid is DOPE. In further embodiments, the LNPs comprise (i) a cationic lipid selected from OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, and GL-HEPES-E3-E12-DS-3-E14; (ii) DMG-PEG2000; (iii) cholesterol; and (iv) DOPE.

[0104] Molar ratio of lipid components When cationic lipid, PEGylated lipid, cholesterol-based lipid, and helper lipid are present, the molar ratios are provided as A:B:C:D (where A+B+C+D=100%). In some embodiments, the molar ratio of cationic lipid to total lipid in the LNP (i.e., A) is 35-45% (e.g., 38-42%, e.g., 40%). In some embodiments, the molar ratio of PEGylated lipid component to total lipid (i.e., B) is 0.25-2.75% (e.g., 1-2%, e.g., 1.5%). In some embodiments, the molar ratio of cholesterol-based lipid to total lipid (i.e., C) is 20-35% (e.g., 27-30%, e.g., 28.5%). In some embodiments, the molar ratio of helper lipid to total lipid (i.e., D) is 25-35% (e.g., 28-32%, e.g., 30%). In some embodiments, the (PEGylated lipid + cholesterol) components have the same molar amount as the helper lipid. In some embodiments, the LNPs contain a molar ratio of cationic lipid to helper lipid that is greater than one.

[0105] In certain embodiments, the LNPs contain a cationic lipid, a PEGylated lipid, a cholesterol-based lipid, and a helper lipid in a molar ratio of 40:1.5:28.5:30. In further specific embodiments, the LNPs contain (i) OF-02, cKK-E10, GL-HEPES-E3-E10-DS-3-E18-1, GL-HEPES-E3-E12-DS-4-E10, or GL-HEPES-E3-E12-DS-3-E14, (ii) DMG-PEG2000; (iii) cholesterol; and (iv) DOPE in a molar ratio of 40:1.5:28.5:30.

[0106] To calculate the actual amount of each lipid to be included in the LNP formulation, first determine the molar amount of the cationic lipid 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, calculate the molar amount of each of the other lipids 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.

[0107] In one embodiment, the nucleic acid is encapsulated in an LNP comprising 35%-45% molar cationic lipid, 0.25%-2.75% molar polyethylene glycol (PEG)-conjugated (PEGylated) lipid, 20%-35% molar cholesterol-based lipid, and 25%-35% molar helper lipid (all molar ratios are relative to the total lipid content of the LNP). 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. In some embodiments, the LNP comprises a 40% molar ratio of cationic lipid, a 1.5% molar ratio of PEGylated lipid, a 28.5% molar ratio of cholesterol-based lipid, and a 30% molar ratio of helper lipid.

[0108] 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 certain embodiments, the LNPs comprise a 40% molar ratio of 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, a 1.5% molar ratio of DMG-PEG2000, a 28.5% molar ratio of cholesterol, and a 30% molar ratio of DOPE.

[0109] In some embodiments, LNPs can have an average diameter of about 30 nm to about 200 nm, about 80 nm to about 150 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm, or 200 nm. In some embodiments, LNPs are substantially non-toxic. One or more nucleic acid molecules, when present in one or more LNPs, are typically resistant to degradation by nucleases in aqueous solution. In some embodiments, the RNA-LNP has an N / P ratio of 1 to 20, 1 to 15, 1 to 10, 2 to 8, 2 to 6, or 2 to 4. The term "N / P ratio" refers to the molar ratio of positively charged molecular units in the cationic lipids in the LNP to negatively charged molecular units in the RNA encapsulated within the LNP. Therefore, the N / P ratio is typically calculated as the ratio of moles of amine groups in the cationic lipids in the LNP to moles of phosphate groups in the RNA encapsulated within the LNP. In some 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 some 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 some 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 greater 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.

[0110] In some embodiments, the LNP comprises one or more mRNA molecules that encode an antigen (eg, a viral antigen, such as an influenza virus antigen, or a bacterial antigen).

[0111] LNPs can be prepared by various techniques currently known in the art. For example, multilamellar vesicles (MLVs) can be prepared according to conventional techniques, for example, by dissolving the lipids in an appropriate solvent, depositing selected lipids on the inner wall of a suitable container or vessel, and then evaporating the solvent to leave a thin film on the inside of the vessel, or by spray drying. MLVs can then be formed by adding an aqueous phase to the vessel with a vortex motion. 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.

[0112] Various methods are described in U.S. Patent Application Publication Nos. 2011 / 0244026, 2016 / 0038432, 2018 / 0153822, 2018 / 0125989, and 2021 / 0046192 and can be used to practice the present disclosure. One exemplary process encapsulates mRNA by mixing the mRNA with a mixture of lipids without first preforming the lipids into lipid nanoparticles, as described in U.S. Patent Application Publication No. 2016 / 0038432. Another exemplary process encapsulates mRNA by mixing preformed LNPs with the mRNA, as described in U.S. Patent Application Publication No. 2018 / 0153822.

[0113] In one embodiment, the nucleic acid is prepared in an aqueous buffer and mixed with an amphipathic solution containing the lipid components of the LNP. The amphipathic solution for dissolving the four lipid components of the LNP can be an alcohol solution. In some embodiments, the alcohol is ethanol. The aqueous buffer can be, for example, a citrate, phosphate, acetate, or succinate buffer and can have a pH of about 3.0 to 7.0, e.g., about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, or about 6.5. The buffer can contain other components, such as salts (e.g., sodium, potassium, and / or calcium salts). In a specific embodiment, the aqueous buffer has 1 mM citrate, 150 mM NaCl, and a pH of 4.5.

[0114] In another embodiment, in step a), preformed LNPs are mixed with nucleic acid under conditions that allow the formation of nucleic acid-LNPs.

[0115] In one embodiment, the method comprises synthesizing mRNA in vitro prior to step a), hi one embodiment, the in vitro synthesized mRNA is purified prior to encapsulation into LNPs.

[0116] In one embodiment, the encapsulation efficiency is at least 80%.

[0117] In some embodiments, the methods provided herein may further include removing nucleic acid molecules that are not properly encapsulated in the LNPs and are instead bound to the outer surface of the LNPs. In particular, RNA molecules bound to the outer surface of the LNPs may be removed by contacting the RNA-LNPs with high salt. Thus, the methods provided herein may further include dissociating the RNA bound to the outer surface of the LNPs. Specifically, the dissociating RNA step may include contacting the RNA-LNPs with high salt before determining the encapsulation efficiency. As used herein, "high salt" may refer to salt provided at a final concentration ranging from 500 mM to 5 M. In some embodiments, salt is provided at a final concentration of 1 M to 5 M. In some embodiments, salt is provided at a final concentration of 0.75 M to 3 M. The salt may be NaCl. In some embodiments, NaCl is used at a final concentration ranging from about 500 mM to 5 M, optionally 1 to 2 M.

[0118] In some embodiments, the step of dissociating RNA bound to the outer surface of the LNP may further require heating. Suitable temperatures for the dissociation step may be between about 60°C and 95°C. In some embodiments, the dissociation step is carried out at a temperature between about 70°C and about 90°C. In some embodiments, the dissociation step is carried out at a temperature between about 80°C and 90°C. In some embodiments, the dissociation step is carried out at a temperature of about 85°C.

[0119] The present invention further provides kits containing various reagents and materials useful for practicing the inventive methods according to the present invention. The quantitative procedures described herein can be performed by diagnostic laboratories, research laboratories, or commercial laboratories. The present invention provides kits that can be used in these different settings.

[0120] For example, materials and reagents for quantifying RNA encapsulation efficiency in a sample according to the methods provided herein can be assembled into a kit. Each kit preferably includes reagents that make the procedure specific. In certain embodiments, the kit includes two fluorophores (wherein the first fluorophore is permeable to the LNP and the second fluorophore is impermeable to the LNP), such as those described herein. The kit optionally includes additional reagents, such as buffers, and instructions for using the kit according to the methods of the invention.

[0121] The present disclosure further provides a kit for determining the efficiency of RNA encapsulation into LNPs, comprising: - a first fluorophore that is permeable to the LNP; a second fluorophore that is impermeable to the LNP; and - optionally, instructions for use in accordance with the methods provided herein A kit is provided comprising:

[0122] A kit or other product according to the present invention may include one or more containers for holding various reagents. Suitable containers include, for example, bottles, vials, syringes (e.g., pre-filled syringes), and ampoules. The containers may be made of various materials, such as glass or plastic.

[0123] In some embodiments, the kits of the present invention may include a suitable control level or control sample for determining the control level described herein. For example, the kit may include LNPs encapsulating a known concentration of RNA and / or a known level of RNA. In some embodiments, the kits of the present invention may include instructions for using the kit according to the methods provided herein. In some embodiments, the kits of the present invention may further include instructions for encapsulating RNA into LNPs.

[0124] The present invention further provides a novel method for quantifying LNP-encapsulated RNA using Quant-iT. TMThe present invention further relates to the use of Quant-iT in the quantification of LNP-encapsulated RNA. TM 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 and 10 μg / mL.

[0125] The provided methods can be used for quality control of LNP-encapsulated RNA and batch release of RNA-LNP compositions. Indeed, the present invention is particularly useful for quality control during manufacturing of LNP-encapsulated mRNA and for characterization of LNP-encapsulated mRNA as an active pharmaceutical ingredient (API) in a final therapeutic product.

[0126] The present invention includes the following embodiments.

[0127] Embodiment 1. A method for determining RNA encapsulation efficiency in lipid nanoparticles (LNPs), comprising: a) contacting a sample containing RNA encapsulated in LNPs with a first fluorophore and a second fluorophore, thereby forming a fluorophore-RNA complex; and b) detecting the fluorescent signals of the conjugated first and second fluorophores; Including, The first fluorophore is permeable to the LNP, and the second fluorophore is impermeable to the LNP. method.

[0128] Embodiment 2. The first fluorophore is Quant-iT TM HS or SYBR® Green II.

[0129] Embodiment 3. The method of embodiment 1 or 2, wherein the second fluorophore is RiboGreen® or SYBR® Gold.

[0130] Embodiment 4. The method of any one of embodiments 1 to 3, wherein the RNA is 10 to 50,000 nucleotides in length.

[0131] Embodiment 5. The method of any one of embodiments 1 to 4, wherein the RNA is 300 to 10,000 nucleotides in length.

[0132] Embodiment 6. The method of any one of embodiments 1 to 5, wherein the RNA is 500 to 5000 nucleotides in length.

[0133] Embodiment 7. The method of any one of embodiments 1 to 6, wherein the RNA is double-stranded RNA or single-stranded RNA.

[0134] Embodiment 8. The method of any one of embodiments 1 to 7, wherein the RNA comprises mRNA, miRNA, shRNA, rRNA, tRNA, snRNA, snoRNA, asRNA, siRNA, aiRNA, gRNA, and / or piRNA.

[0135] Embodiment 9. The method of any one of embodiments 1 to 8, wherein the first fluorophore and the second fluorophore are added to the sample simultaneously.

[0136] Embodiment 10. The method of any one of embodiments 1 to 8, wherein the first fluorophore and the second fluorophore are added to the sample sequentially.

[0137] Embodiment 11. The method of any one of embodiments 1 to 10, 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.

[0138] Embodiment 12 The method of any one of embodiments 1 to 11, wherein the ratio of the first fluorophore to the second fluorophore is 2:1.

[0139] Embodiment 13 The method of any one of embodiments 1 to 12, wherein the first fluorophore is present at a final concentration of 0.5x.

[0140] Embodiment 14 The method of any one of embodiments 1 to 13, wherein the second fluorophore is present at a final concentration of 0.25x.

[0141] Embodiment 15. The method of any one of embodiments 1 to 14, further comprising generating a standard curve of the first fluorescent signal and the second fluorescent signal.

[0142] Embodiment 16. The method of embodiment 15, comprising determining the absolute amount of encapsulated RNA by matching each fluorescent signal detected in step b) with a corresponding standard curve.

[0143] Embodiment 17. The method of any one of embodiments 1 to 14, further comprising determining a ratio of the second fluorescent signal to the first fluorescent signal.

[0144] Embodiment 18. A method for producing RNA-encapsulated LNPs, comprising: a) encapsulating RNA into LNPs; and b) Determining the efficiency of RNA encapsulation into LNPs according to any one of the methods of embodiments 1 to 17. A method comprising:

[0145] Embodiment 19. The method of embodiment 18, wherein the RNA is mRNA.

[0146] Embodiment 20. The method of embodiment 19, further comprising the step of synthesizing mRNA in vitro prior to step a).

[0147] Embodiment 21 The method of embodiment 20, wherein the in vitro synthesized mRNA is purified prior to encapsulation into LNPs.

[0148] Embodiment 22. The method of any one of embodiments 18-21, wherein the lipid is mixed with the RNA under conditions that allow for the formation of LNPs that encapsulate the RNA.

[0149] Embodiment 23. The method of any one of embodiments 18-22, wherein the encapsulation efficiency is at least 80%.

[0150] Embodiment 24. The method of any one of embodiments 18 to 23, performed prior to releasing a batch of RNA-encapsulated LNPs.

[0151] Embodiment 25. The method of any one of embodiments 1-24, wherein the LNP comprises one or more ionizable lipids, one or more helper lipids, and one or more PEG-modified lipids.

[0152] Embodiment 26. A kit for determining RNA encapsulation efficiency in LNPs, comprising: - a first fluorophore that is permeable to the LNP; a second fluorophore that is impermeable to the LNP; and -Instructions for use in accordance with the methods provided herein Kit including:

[0153] Embodiment 27. Quant-iT for quantification of LNP-encapsulated RNA TM Use of HS.

[0154] In order that this invention may be better understood, the following examples are set forth, which are for illustrative purposes only and should not be construed as limiting the scope of the disclosure in any way. [Example]

[0155] Example 1: Materials and Methods mRNA generation mRNA was produced as previously described (Kalnin et al. (2021), NPJ Vaccines 6(1):61 and US Patent Publication No. 2022 / 0142923). Briefly, mRNA incorporating a coding sequence was synthesized by in vitro transcription using RNA polymerase from a plasmid DNA template encoding the desired gene using unmodified or modified nucleotides. An exemplary mRNA (mRNA1) is approximately 2000 nucleotides in length. The resulting purified precursor mRNA was further reacted and purified by enzymatic addition of a 5' cap structure (Cap1) and a 3' poly(A) tail of approximately 200 nucleotides in length as determined by gel electrophoresis. All mRNA preparations were analyzed for purity, integrity, and Cap1 percentage before storage at -80°C.

[0156] Encapsulation of mRNA into LNPs To encapsulate mRNA into LNPs, an ethanolic mixture of lipids (cationic / ionizable lipids, helper lipids, cholesterol, and polyethylene glycol-lipids) was mixed with an aqueous buffer solution of target mRNA at a fixed lipid-to-mRNA ratio under controlled conditions at acidic pH to obtain a homogeneous LNP suspension. After ultrafiltration and diafiltration into a suitable dilution series, the resulting nanoparticle suspension was diluted to the final concentration, filtered, and stored frozen at -80°C until use. The mRNA-LNP formulations were characterized by size using 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, LNPs contained OF-02 cationic lipid.

[0157] Fluorescence measurements Unless otherwise indicated, fluorescence emission (also referred to herein as fluorescence) was measured after incubating samples in the presence of fluorophores for 20 minutes at room temperature in the dark. Measurements were performed using a Spectramax I3 microplate reader (Molecular Devices) with the following settings: RiboGreen®: excitation 485 nm and emission 525 nm; Quant-iT TM HS: excitation 644 nm and emission 673 nm; SYBR® Gold: excitation 495 nm and emission 537 nm; SYBR® Green II: excitation 495 nm and emission 520 nm; SYTO® 17: excitation 621 nm and emission 646 nm.

[0158] In both cases, the excitation bandwidth was 9 nm and the emission bandwidth was 15 nm.

[0159] RiboGreen assay (comparison example) Encapsulation efficiency was determined using a standard RiboGreen assay (each sample was tested in duplicate).

[0160] First, free mRNA and mRNA encapsulated in LNPs (mRNA-LNPs) were diluted to 2 ng / μL and 100 ng / μL mRNA, respectively, in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5). In parallel, mRNA-LNPs were diluted to a final concentration of 2 ng / μL mRNA in TET buffer (TE buffer containing 0.5% Triton X-100). Free mRNA and mRNA-LNP dilutions were performed in a black 96-well microplate with a flat, clear bottom, using a final volume of 100 μL. Eight independent dilutions of a standard scale of free mRNA in TE or TET buffer (0–1 ng / μL) were performed, along with four independent dilutions of 20 ng / μL mRNA-LNP in TE (0.5–20 ng / μL) and four independent dilutions of 2 ng / μL mRNP-LNP in TET (0.05–0.4 ng / μL).

[0161] The RiboGreen® reagent was diluted extemporaneously with TE buffer to 1x, and 100 μL of the 1x solution was added to each well so that RiboGreen® was present at a final concentration of 0.5x. After 10 minutes of incubation at room temperature in the dark, RiboGreen® fluorescence was measured as described in Example 1. RNA quantification was performed based on a standard simple linear regression curve calculated from the RiboGreen® fluorescence emission of mRNA-LNPs in TE or TET buffer. The non-encapsulated mRNA concentration was determined by RiboGreen® fluorescence in mRNA-LNPs in TE buffer, and the total mRNA concentration was determined by RiboGreen® emission in LNPs in TET buffer. The encapsulation efficiency was then calculated using the following formula:

number

[0162] Assessment of LNP permeability to fluorophores To determine LNP permeability of fluorophores, mRNA-LNPs and the corresponding free mRNA were diluted in TE to different total concentrations in a final volume of 100 μL in a black 96-well microplate with a flat, clear bottom. 100 μL of Quant-iT TM HS (2x), or 100 μL of SYTO® 17 (2 μM) was added, and the samples were incubated in the dark for 20 minutes at room temperature. Fluorescence emission was measured as described above. To determine whether the fluorophore was able to permeate the LNPs and specifically bind to RNA, fluorescence standard curves were obtained and compared between free mRNA and mRNA-LNP ranges. SYTO® 17 was provided at a final concentration of 1 μM and analyzed by Quant-iT TM HS was provided at a final concentration of 1×.

[0163] Evaluation of a dual fluorescence assay to measure encapsulation The properties of the two selected fluorophores are shown in Table 1 below.

[0164] [Table 1]

[0165] Quant-iT TM HS was combined with RiboGreen®.

[0166] The assay was performed in duplicate for each point, starting with an initial concentration of 2.5 ng / μL for free mRNA and 100 ng / μL for mRNA-LNP in TE buffer. Eight independent dilutions of free mRNA (0–2.5 ng / μL) and eight independent dilutions of mRNA-LNP (0.5–20 ng / μL) were performed in a black 96-well microplate with a flat, clear bottom, using a final volume of 100 μL. RiboGreen® and Quant-iT TM The HS reagents were diluted in TE buffer to 0.5x and 1x, respectively, and the RiboGreen and Quant-iT TMTo each well was added 100 μL of the fluorophore mixture so that HS was present at final concentrations of 0.25× and 0.5×, respectively. Fluorescence was measured as described in Example 1. A standard simple linear regression curve was established for each fluorophore (FIG. 3).

[0167] Free mRNA in TE was used to detect RNA fragments using RiboGreen® and Quant-iT TM RNA quantification was performed based on two standard curves calculated from the fluorescence of HS (see Figure 3). The free mRNA concentration was then determined by measuring RiboGreen® fluorescence in samples containing mRNA-LNPs and Quant-iT® fluorescence in samples containing mRNA-LNPs. TM Total mRNA concentration was determined by measuring HS fluorescence.

[0168] A summary of fluorophore concentrations, final mRNA concentration ranges, and number of points measured in the RiboGreen assay or the assay of the present invention is shown in Table 2 below.

[0169] [Table 2]

[0170] Free mRNA dilutions of known concentrations were used to establish the standard curves needed to quantify free and total mRNA concentrations in samples. Conc.: concentration, TE: Tris-EDTA, TET: Tris-EDTA-Triton.

[0171] Use of the Dual RNA Encapsulation Assay in High-Throughput Screening The assay was performed in triplicate at each point using mRNA-LNP samples at an initial concentration of 20 ng / μL in TE buffer. Two independent dilutions of mRNA-LNP samples (to 2 and 2.5 ng / μL) were performed in TE buffer in a black 96-well microplate with a flat, clear bottom, using a final volume of 100 μL. A blank without mRNA-LNP was also included to eliminate background fluorescence. RiboGreen® and Quant-iT TM The HS reagents were diluted extemporaneously with TE buffer to 0.5x and 1x, respectively, and 100 μL of the fluorophore mixture was added to each well. TM HS luminescence was measured as described above.

[0172] The encapsulation efficiency was determined using the following formula:

number

[0173] Example 2: Fluorophore Selection The use of two fluorophores in one well to determine RNA encapsulation efficiency (also referred to herein as the "dual RNA encapsulation assay") was evaluated. A schematic diagram of the method, compared to the RiboGreen assay, is shown in Figure 1.

[0174] First, compatible fluorophores were selected for use in this assay. To be used in the same well, the two selected fluorophores should not exhibit spectral overlap (i.e., in excitation and emission wavelengths). The two selected fluorophores should also detect a similar range of mRNA concentrations (i.e., have a similar sensitivity scale). This was demonstrated for RiboGreen® and Quant-iT®. TM This is particularly evident in the case of HS (see Table 1 above).

[0175] In the RiboGreen assay, detergent must be added to dissolve the LNPs in order to measure the fluorescence of total RNA emission. RiboGreen® does not permeate the LNPs. Therefore, the second fluorophore must be able to permeate the LNPs.

[0176] Quant-iT in the presence of free mRNA or encapsulated mRNA (i.e., mRNA-LNP) TM The luminescence of HS was measured to determine its ability to label total mRNA in LNPs. TM The emission of HS was the same between free and encapsulated mRNA, demonstrating the permeability of this fluorophore to LNPs (see Figure 2). These results were confirmed by Quant-iT TM It further indicates that HS does not interact with lipids, including LNPs.

[0177] The two fluorophores must also not exhibit binding to lipids or competition with each other. Quantification of unencapsulated or total mRNA concentration is based on a standard curve of free mRNA, which must fit a standard linear regression. Therefore, RiboGreen® and Quant-iT TM HS was tested in the same well for its ability to simultaneously label free mRNA at different concentrations (see Figure 3). Results showed that the emissions from both fluorophores yielded a linear regression that could be used as a standard curve. Furthermore, RiboGreen® was also used in the Quant-iT TM Neither HS nor Quant-iT lost any sensitivity range in the presence of the other. TM HS were the two fluorophores chosen to further evaluate the dual-labeling assay.

[0178] Example 3: Evaluation of the dual RNA encapsulation assay To validate the assay according to the present invention, it was performed in parallel with a RiboGreen assay using the fluorophore concentrations and standard mRNA and LNP ranges shown in Table 2. As shown in Table 3 below, the encapsulation efficiency was very similar between the two methods. Therefore, the method of the present invention can be successfully used to determine encapsulation efficiency. Furthermore, the present invention is advantageous because variability is reduced compared to the RiboGreen assay, as shown in Table 3.

[0179] [Table 3]

[0180] RiboGreen assays and dual RNA encapsulation assays 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 (%) was calculated for both mRNA concentrations from four independent dilution points of triplicate mRNA-LNPs.

[0181] Example 4: Determining the range of encapsulation efficiencies that can be measured in 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, the assay of the present invention was able to accurately quantify both free and total mRNA concentrations in samples containing LNPs without the addition of detergent, as opposed to that required by the classical RiboGreen assay. The determination of encapsulation efficiency was accurate at various encapsulation rates (here, down to as low as 50%).

[0182] [Table 4]

[0183] Dual RNA encapsulation assays were 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 a 95% encapsulation efficiency (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%. Dual RNA encapsulation assays were performed using RiboGreen® and Quant-iT TM Using a standard linear regression line calculated by labeling free mRNA with both fluorophores of HS, the non-encapsulated (free) mRNA concentration and the total mRNA concentration were then calculated using RiboGreen® and Quant-iT®, respectively. TM The encapsulation efficiency was determined by the ratio between the free mRNA concentration and the total mRNA concentration, as determined by HS fluorescence. Four independent dilution points of mRNA-LNPs were measured in triplicate for each concentration.

[0184] Example 5: Establishment of a high-throughput dual RNA encapsulation assay We developed a protocol for high-throughput determination of encapsulation efficiency that does not require measurement of mRNA concentration. This method directly determines the encapsulation rate by comparing the ratio of fluorophore emissions in a sample. First, we used RiboGreen® and Quant-iT TM Fluorescence coefficient (C) between HS f ) was determined for the same concentration of labeled mRNA (Figure 4). Different concentrations of non-encapsulated mRNA and the corresponding identical concentrations of total mRNA were labeled with fluorophores, and the emission ratio was calculated (Table 5). Independent of the mRNA concentration, the ratio was always 0.01. f The consistency of this allowed us to incorporate it into a new encapsulation efficiency formula, where the percentage of non-encapsulated mRNA was given by the simple ratio of RiboGreen® luminescence to Quant-iT HS luminescence in the same well (Figure 5).

[0185] [Table 5]

[0186] LNPs encapsulating unmodified mRNA were diluted at different ratios, and the mRNA was analyzed by RiboGreen® and Quant-iT TM HS was labeled with both fluorophores. TM The fluorescence coefficient (C) between the fluorescence emission of HS and f ) and the free mRNA concentration (RiboGreen® fluorescence) was compared with the total mRNA concentration (Quant-iT TM HS fluorescence) were determined under the same conditions (N=3).

[0187] We then designed a protocol to use two dilution points of mRNA-LNPs (final mRNA concentrations in the diluted samples of 1000 and 1250 ng / mL, respectively; see Table 6) that correspond to a more precise sensitivity range for both fluorophores. The accuracy of this high-throughput assay was verified at different levels of encapsulation efficiency (see Table 7).

[0188] [Table 6]

[0189] This table summarizes the fluorophore concentrations used to quantify free and total mRNA concentrations in mRNA-LNP samples, as well as the free and encapsulated mRNA (LNP) ranges. High-throughput (HT) screening based on the dual RNA encapsulation protocol provided in Example 4 was performed without absolute quantification of mRNA concentrations.

[0190] [Table 7]

[0191] 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%. The LNPs were diluted to 2000 and 2500 ng / mL of total RNA, followed by detection with RiboGreen® fluorophore and Quant-iT, respectively. TM Free and total mRNA were labeled with fluorophores. The percentage of free mRNA was determined using RiboGreen® and Quant-iT TM The encapsulation efficiency was determined by the fluorescence ratio between HS and ES (N=3). The experiment was performed as two independent dilutions, and the standard deviation of the encapsulation efficiency was calculated from both dilutions. Em: fluorescence emission.

[0192] As shown herein, the determination of encapsulation efficiency is highly accurate even when only two dilution points are used. Therefore, the method of the present invention can be effectively used for high-throughput applications such as screening where a completely quantitative method is not required.

[0193] Example 6: Evaluation of encapsulation efficiency with various mRNA-LNPs LNPs containing various mRNAs (containing unmodified or modified nucleotides) and various 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, indicating that this method can be used regardless of LNP composition, mRNA sequence, or the presence of modified nucleosides in the mRNA (see Table 8).

[0194] [Table 8]

[0195] The encapsulation efficiency of mRNA in five different mRNA-LNPs (containing three different cationic lipids and four different mRNAs with or without chemically modified nucleosides) was determined in parallel. The dual RNA encapsulation assay was performed using RiboGreen® (unencapsulated mRNA) and Quant-iT in the same well. TM The ratio of HS (total mRNA) luminescence is used (N=3). The standard deviation of encapsulation efficiency was calculated from two different LNP dilutions described in Example 5 (see Table 6).

[0196] Example 7: Evaluation of alternative fluorophores SYTO® 17 was evaluated to determine whether it could be used as an alternative fluorophore in the methods of the present invention. While RiboGreen® alone yields a linear standard curve, this is no longer the case when RiboGreen® is combined with SYTO® 17 (compare Figure 6, panels A and B). This indicates that RiboGreen® fluorescence is altered by the presence of SYTO® 17. Furthermore, as shown in Figure 6C, the standard curve established with SYTO® 17 differed between free mRNA and mRNA-LNPs. Notably, fluorescence was higher for mRNA-LNPs than for free mRNA. Without wishing to be bound by theory, this suggests that SYTO® 17 may interact with the lipid components of LNPs.

[0197] SYBR® Gold and SYBR® Green II were also evaluated to determine whether they could be used as alternative fluorophores. As shown in Figure 7, the standard curves obtained for free and encapsulated mRNA were identical when SYBR® Green II was used, indicating that this fluorophore permeates the LNPs. In contrast, the standard curve obtained for SYBR® Gold varied depending on the sample tested (i.e., free mRNA only or mRNA-LNPs). Notably, when mRNA-LNPs were contacted with SYBR® Gold, almost no fluorescence emission was detected. This suggests that this fluorophore does not permeate the LNPs.

[0198] Overall, the simple linear regression lines measured when mRNA was labeled with SYBR® Green II or SYBR® Gold indicate that either of these fluorophores, in combination with compatible fluorophores with detectably distinct fluorescence emissions, can be utilized in dual RNA encapsulation assays.

[0199] Example 8: Automation of the Dual RNA Encapsulation Assay and Validation of the Method on Two Different Spectrophotometers The dual RNA encapsulation assay was implemented on a Starlet Hamilton robotic platform to automate the method. A source sample containing unmodified mRNA encapsulated in LNPs containing OF-02 cationic lipids, known to have a total mRNA concentration of 1000 μg / mL and a 95% encapsulation efficiency (determined by the RiboGreen assay described in Example 1 above), was used. Unmodified free mRNA was added to the source sample to artificially adjust the encapsulation efficiency to 70% and 50%. Various concentrations of these mRNA-LNPs (ranging from 20 to 200 ng / μL) were loaded into a PCR 96-well plate using a final volume of 40 μL. Samples were diluted to 10 ng / μL with TE buffer in a separate PCR 96-well plate, if necessary. Finally, samples were diluted in a black 384-well microplate with a flat, clear bottom to provide two data points per sample, loaded in duplicate (i.e., a total of four replicates per condition). RiboGreen® and Quant-iT TM The HS reagents were robotically diluted on-site to 0.5x and 1x in TE buffer, respectively, and the RiboGreen® and Quant-iT TM To each well, 40 μL of the fluorophore mixture was added so that HS was present at a final concentration of 0.25× and 0.5×, respectively. Fluorescence was measured as described in Example 1 using either a Cytation 7 (Agilent Biotek) or a Spectramax i3 spectrophotometer, and encapsulation efficiency was then determined using the corresponding equation shown in Example 1. All steps, from the initial dilution of the sample to fluorescence measurement with the appropriate spectrophotometer, were performed on a robotic platform.

[0200] The results shown in Figure 8 demonstrate that this method is automatable. Furthermore, the results are accurate regardless of the spectrophotometer used.

[0201] conclusion In light of the above, the method of the present invention represents an improved method for determining encapsulation efficiency. In particular, because dual measurements can be determined from a single well, the number of samples required is reduced by at least two-fold. This method can be used in both quantitative and high-throughput screening approaches and demonstrates a high level of accuracy in determining the encapsulation efficiency of nucleic acids in LNPs. In particular, by using the ratiometric approach provided in Example 5 for measuring LNP-RNA encapsulation efficiency, many different RNAs can be evaluated without the need to provide standard curves for each sample for absolute quantification of free and encapsulated mRNA. Furthermore, using the automated dual RNA encapsulation assay of Example 8, the protocol is fully automated, up to 95 samples are processed in less than two hours, and only limited amounts of raw materials (e.g., mRNA-LNP sample, fluorophore) are required.

[0202] Other embodiments of the present 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 as exemplary only, with the true scope and spirit of the present disclosure being indicated by the following claims.

[0203] All patents and publications cited herein are incorporated by reference in their entirety.

Claims

1. 1. A method for determining RNA encapsulation efficiency in lipid nanoparticles (LNPs), comprising: a) contacting a sample containing RNA encapsulated in LNPs with a first fluorophore and a second fluorophore, thereby forming a fluorophore-RNA complex; and b) detecting the fluorescent signals of the conjugated first and second fluorophores. Including, The first fluorophore is permeable to the LNP, and the second fluorophore is impermeable to the LNP. method.

2. The first fluorophore is Quant-iT ＴＭ HS or SYBR® Green II.

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

4. The method of any one of claims 1 to 3, wherein the RNA is 10 to 50,000 nucleotides in length.

5. The method according to any one of claims 1 to 4, wherein the RNA is double-stranded RNA or single-stranded RNA.

6. The method of any one of claims 1 to 5, 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 claims 1 to 6, wherein the first fluorophore and the second fluorophore are added to the sample simultaneously.

8. 8. The method of claim 1, wherein the ratio of the first fluorophore to the second fluorophore is 2:

1.

9. The method of any one of claims 1 to 8, further comprising determining a ratio of the second fluorescent signal to the first fluorescent signal.

10. The method of any one of claims 1 to 9, further comprising generating a standard curve of the first fluorescent signal and the second fluorescent signal.

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

12. 1. A method for producing RNA-encapsulated LNPs, comprising: a) encapsulating RNA into LNPs; and b) determining the efficiency of RNA encapsulation into LNPs according to the method of any one of claims 1 to 11; A method comprising:

13. 13. The method of claim 12, further comprising the step of synthesizing mRNA in vitro prior to step a).

14. 1. A kit for determining the efficiency of RNA encapsulation in lipid nanoparticles (LNPs), comprising: - a first fluorophore that is permeable to the LNP; a second fluorophore that is impermeable to the LNP; and - instructions for use according to the method of any one of claims 1 to 11 Kit including:

15. Quant-iT for quantification of LNP-encapsulated RNA ＴＭ Use of HS.

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