Method for analyzing nucleic acid carrier of lipid nucleic acid assembly

Abstract

The present disclosure provides, inter alia, methods and compositions for isolating and / or determining the concentration of nucleic acids in lipid nucleic acid assembly compositions. In certain embodiments, the present disclosure provides methods and compositions for isolating and / or determining the concentration of nucleic acids, the isolation and / or determination being performed using size exclusion chromatography (SEC) and / or ion pair chromatography. In certain embodiments, the present disclosure provides methods and compositions for isolating and / or determining nucleic acid concentrations, where nucleic acids are extracted from a lipid nucleic acid assembly composition by a method including ethanol precipitation. In various embodiments, nucleic acids are encapsulated in lipid nanoparticles (LNPs). In various embodiments, the nucleic acids include messenger RNA (mRNA) and guide RNA (gRNA).

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 434,812, filed on December 22, 2022, the contents of which are hereby incorporated by reference in their entirety. Background Art

[0003] Lipid nucleic acid assembly compositions, such as lipid nanoparticles (LNPs) with nucleic acid cargo, are useful in a variety of therapeutic and molecular biology applications. In some LNA compositions, particularly for applications such as gene editing, multiple nucleic acids are present. Separating nucleic acids of varying sizes and determining their concentrations can be used to characterize such compositions. Summary of the Invention

[0004] The present disclosure provides, in particular, methods and compositions for separating nucleic acids and / or determining their absolute concentration and / or relative concentration in lipid nucleic acid assembly compositions. In certain embodiments, the present disclosure provides methods and compositions for separating nucleic acids and / or determining their absolute concentration and / or relative concentration by using size exclusion chromatography (SEC). In certain embodiments, the present disclosure provides methods and compositions for separating nucleic acids and / or determining their absolute concentration and / or relative concentration by using ion pair chromatography. In certain embodiments, the present disclosure provides methods and compositions for separating nucleic acids and / or determining their absolute concentration and / or relative concentration, wherein nucleic acids are extracted from lipid nucleic acid assembly compositions using a method comprising ethanol precipitation. In various embodiments, nucleic acids include messenger RNA (mRNA) and guide RNA (gRNA). In various embodiments, nucleic acids include first messenger RNA (mRNA) and guide RNA (gRNA). In various embodiments, nucleic acids include first messenger RNA (mRNA), second messenger RNA and guide RNA (gRNA). In various embodiments, nucleic acids include first messenger RNA (mRNA), second messenger RNA and guide RNA (gRNA).

[0005] The present disclosure particularly includes recognition that, when preparing a bulk drug or a medicine comprising a lipid nucleic acid assembly composition, it is useful and / or necessary to determine the absolute concentration and / or relative concentration of each nucleic acid. The present disclosure particularly includes recognition that the methods and compositions disclosed herein are particularly suitable for determining the absolute concentration and / or relative concentration of nucleic acids in the lipid nucleic acid assembly composition. The present disclosure particularly includes recognition that the methods and compositions disclosed herein are particularly suitable for determining the absolute concentration and / or relative concentration of RNA molecules present in the lipid nucleic acid assembly composition, wherein the RNA molecules are, for example, one or more gRNA molecules and one or more mRNA molecules (e.g., one mRNA or two mRNAs). In some embodiments, the lipid nucleic acid assembly composition includes an RNA molecule encapsulated by lipid nanoparticles.

[0006] Various existing methods for isolating nucleic acids and / or determining nucleic acid concentrations include organic solvent solutions that can cause turbidity and low recovery, for example, when applied to lipid-nucleic acid assembly compositions. In various embodiments, the nucleic acid extracts (e.g., RNA extracts) of the present disclosure are advantageously free of or substantially free of organic solvents.

[0007] Various existing methods for separating and / or determining nucleic acid concentrations include detergents that are detrimental to chromatographic performance and robustness. In various embodiments, the nucleic acid extracts (e.g., RNA extracts) of the present disclosure are advantageously free of or substantially free of detergents, such as detergents that are incompatible with chromatographic techniques such as size exclusion chromatography (SEC). In various embodiments, the nucleic acid extracts (e.g., RNA extracts) of the present disclosure are advantageously free of or substantially free of detergents, such as detergents that are incompatible with chromatographic techniques such as ion pair chromatography.

[0008] Some existing methods for determining nucleic acid concentration in lipid nucleic acid assembly compositions can be used to determine total nucleic acid concentration, but can not be used to determine the presence or concentration of a single nucleic acid species in a composition.For example, Ribogreen fluorometry (such as Goswami et al., Conjugation of Mannans to Enhance the Potency of LiposomeNanoparticles for the Delivery of RNA Vaccines.Pharmaceutics 2021,13 (2): Described in 240) can provide the total RNA concentration of the lipid nucleic acid assembly compositions measured, but can not be used to determine the presence or concentration of a single RNA species in a composition. Other advantages of the method and composition for separating nucleic acids and / or determining the absolute concentration and / or relative concentration of nucleic acids provided herein include high resolution separation, high accuracy, high precision, high recovery and robustness of different nucleic acids, including small nucleic acids (such as sgRNA) and low concentration nucleic acids (such as total nucleic acid concentration is less than 100 μg / mL).

[0009] Another advantage of the method and composition for separating nucleic acid and / or determining the absolute concentration of nucleic acid and / or relative concentration provided herein is the simplicity and / or program efficiency of lipid nucleic acid assembly composition preparation.Compared with various existing methods, method and composition provided herein has especially reduced quantity and / or the complexity of nucleic acid extraction required step, and excellent result is provided.For example, method and composition provided herein can provide from complicated lipid nucleic acid assembly composition (for example nucleic acid is by the lipid nucleic acid assembly composition of LNP encapsulation) efficient preparation high-purity nucleic acid extract for chromatography.

[0010] In at least one aspect, the present disclosure provides a method for isolating nucleic acids present in a lipid nucleic acid assembly composition, the method comprising: a) preparing a nucleic acid extract from the lipid nucleic acid assembly composition, wherein the preparation of the nucleic acid extract comprises ethanol precipitation of nucleic acids in the lipid nucleic acid assembly composition; and b) performing SEC on the nucleic acid extract, wherein the SEC separates nucleic acids. In at least one aspect, the present disclosure provides a method comprising: a) preparing a nucleic acid extract from the lipid nucleic acid assembly composition, wherein the preparation of the nucleic acid extract comprises ethanol precipitation of nucleic acids in the lipid nucleic acid assembly composition; and b) performing SEC on the nucleic acid extract. In certain embodiments, the method further comprises determining the total concentration of nucleic acids present in the lipid nucleic acid assembly composition.

[0011] In at least one aspect, the present disclosure provides a method for determining the total concentration of nucleic acids present in a lipid-nucleic acid assembly composition, the method comprising: a) preparing a nucleic acid extract from the lipid-nucleic acid assembly composition, wherein the preparation of the nucleic acid extract comprises ethanol precipitation of the nucleic acids in the lipid-nucleic acid assembly composition; b) performing SEC on the nucleic acid extract, wherein the SEC separates the nucleic acids; and c) determining the total concentration of nucleic acids in the lipid-nucleic acid assembly composition.

[0012] In at least one aspect, the present disclosure provides a method for determining the total concentration of nucleic acids present in a lipid-nucleic acid assembly composition, the method comprising: a) preparing a nucleic acid extract from the lipid-nucleic acid assembly composition, wherein the preparation of the nucleic acid extract comprises ethanol precipitation of the nucleic acids in the lipid-nucleic acid assembly composition; b) performing SEC on the nucleic acid extract, wherein the SEC separates the nucleic acids; and c) determining the total concentration of nucleic acids in the lipid-nucleic acid assembly composition.

[0013] In certain embodiments of the method provided herein, the nucleic acid present in the lipid nucleic acid assembly composition comprises at least the first nucleic acid and the second nucleic acid, and wherein SEC separates the first nucleic acid from the second nucleic acid. In certain embodiments, the method further comprises determining the concentration of the first nucleic acid and / or the second nucleic acid in the lipid nucleic acid assembly composition. In certain embodiments, the nucleic acid present in the lipid nucleic acid assembly composition comprises at least the first nucleic acid, the second nucleic acid and the third nucleic acid. In certain embodiments, the nucleic acid present in the lipid nucleic acid assembly composition comprises at least the first nucleic acid, the second nucleic acid and the third nucleic acid, and SEC separates the first nucleic acid from the second nucleic acid and / or the third nucleic acid.

[0014] In certain embodiments of the methods provided herein, the first nucleic acid is a ribonucleic acid (RNA) molecule. In certain embodiments, the first nucleic acid is a messenger RNA (mRNA) molecule. In certain embodiments, the mRNA molecule encodes a polypeptide, optionally wherein the polypeptide includes an RNA-guided DNA binder, optionally wherein the RNA-guided DNA binder includes a nuclease, optionally wherein the nuclease is a Cas protein, optionally wherein the RNA-guided DNA binder is a fusion polypeptide. In certain embodiments, the first nucleic acid encodes a base editor. In certain embodiments, the base editor is a cytosine base editor. In certain embodiments, the base editor is an adenine base editor. In certain embodiments, the first nucleic acid includes 1000-7000 nucleotides. In certain embodiments, the second nucleic acid is an RNA molecule. In certain embodiments, the second nucleic acid is a guide RNA (gRNA) molecule, optionally wherein the gRNA molecule is an sgRNA molecule. In certain embodiments, the second nucleic acid includes 75-200 nucleotides, optionally wherein the second nucleic acid includes 80-120 nucleotides. In certain embodiments, the nucleic acid present in the lipid nucleic acid assembly composition consists of or is substantially composed of the first nucleic acid and the second nucleic acid.

[0015] In certain embodiments of the methods provided herein, the total concentration of nucleic acid in the lipid nucleic acid assembly composition is the sum of the concentration of the first nucleic acid and the concentration of the second nucleic acid. In certain embodiments of the methods provided herein, the concentration of the first nucleic acid and / or the second nucleic acid is determined according to a calibration curve, wherein the calibration curve represents the concentration of nucleic acid in a dilution series of at least one reference nucleic acid. In certain embodiments, the concentration of the first nucleic acid and the second nucleic acid is determined respectively, wherein the concentration of the first nucleic acid is determined according to the first calibration curve, and the concentration of the second nucleic acid is determined according to the second calibration curve. In certain embodiments, the first nucleic acid is an mRNA molecule, and the first calibration curve is a dilution series with reference to the mRNA molecule. In certain embodiments, the second nucleic acid is a gRNA molecule, and the second calibration curve is a dilution series with reference to the gRNA molecule. In certain embodiments, the first calibration curve represents the first dilution series including the concentration of the reference mRNA molecule, and the concentration covers the range including 12 μg / mL and 60 μg / mL, and / or the range including 12 μg / mL and 36 μg / mL. In certain embodiments, the second calibration curve represents a second dilution series comprising a concentration of the reference gRNA molecule that encompasses a range comprising 8 μg / mL and 40 μg / mL, and / or a range comprising 8 μg / mL and 24 μg / mL. In certain embodiments, the first and second dilution series comprise concentrations such that the concentrations in the first dilution series are increased pairwise with the concentrations in the second dilution series to yield a total RNA concentration encompassing a range comprising 20 μg / mL and 100 μg / mL, or a range comprising 20 μg / mL and 60 μg / mL.

[0016] In certain embodiments of the methods provided herein, the method includes determining the concentration of the first nucleic acid and / or the second nucleic acid in the lipid nucleic acid assembly composition, and / or the total nucleic acid concentration, and the concentration is equal to or less than 2.5 mg / mL, 2.0 mg / mL, 1.5 mg / mL, 1.0 mg / mL, 0.5 mg / mL, 0.4 mg / mL or 0.3 mg / mL. In certain embodiments of the methods provided herein, the method includes determining the concentration of the first nucleic acid and / or the second nucleic acid, and / or the total nucleic acid concentration, wherein the total nucleic acid concentration is between about 0.5 mg / mL and about 2 mg / mL, optionally wherein the total nucleic acid concentration is between about 0.75 mg / mL and about 1.8 mg / mL. In certain embodiments of the methods provided herein, SEC produces a chromatogram, wherein the USP resolution between the nucleic acid peak representing the first nucleic acid and the nucleic acid peak representing the second nucleic acid is equal to or greater than 1.5. In certain embodiments of the methods provided herein, the preparation of the nucleic acid extract includes diluting the concentration of the nucleic acid extract to the midpoint of the calibration curve.

[0017] In certain embodiments of the method provided herein, lipid nucleic acid assembly compositions include the lipid nanoparticle (LNP) containing nucleic acid, consisting of or consisting essentially of it.In certain embodiments, the lipid component of LNP includes (i) amine lipid, (ii) helper lipid and (iii) stealth lipid, optionally wherein LNP includes neutral lipid, optionally wherein stealth lipid is PEG lipid.In certain embodiments, the first nucleic acid is messenger RNA (mRNA) molecule of the DNA binding agent that encoding RNA guides, and the second nucleic acid is gRNA molecule, lipid nucleic acid assembly compositions include the LNP containing the molecule of the first nucleic acid and the second nucleic acid, consisting of or consisting essentially of it, and the lipid component of LNP includes (i) amine lipid, (ii) helper lipid, (iii) stealth lipid and (iv) neutral lipid, optionally wherein stealth lipid is PEG lipid.

[0018] In certain embodiments of the methods provided herein, the total concentration of nucleic acids in the lipid-nucleic acid assembly composition is the total concentration of RNA in the lipid-nucleic acid assembly composition. In certain embodiments of the methods provided herein, ethanol precipitation comprises: a) mixing the lipid-nucleic acid assembly composition with ethanol to produce an ethanol mixture; b) centrifuging the ethanol mixture, wherein the centrifugation produces a nucleic acid precipitate and an ethanol supernatant; and c) separating the nucleic acid precipitate from the ethanol, wherein the nucleic acid precipitate comprises precipitated RNA. In certain embodiments, separating the precipitate comprises decanting the supernatant and / or drying the nucleic acid precipitate. In certain embodiments, preparing a nucleic acid extract comprises resuspending the nucleic acid precipitate in water to produce an aqueous nucleic acid solution. In certain embodiments, preparing the nucleic acid extract comprises heating the aqueous nucleic acid solution to a temperature between about 65°C and about 90°C for at least about 2 minutes to about 15 minutes, optionally wherein preparing the nucleic acid extract comprises cooling the aqueous nucleic acid solution after heating, optionally wherein the cooling is to 4°C. In certain embodiments of the methods provided herein, the nucleic acid extract is free of or substantially free of lipids, free of or substantially free of buffers, and / or free of or substantially free of detergents.

[0019] In certain embodiments of the method provided herein, SEC is high performance liquid chromatography (HPLC). In certain embodiments of the method provided herein, nucleic acid extract is carried out SEC including contacting nucleic acid extract with SEC matrix, optionally wherein said SEC matrix is ​​SEC post or is present in SEC post. In certain embodiments, nucleic acid extract is carried out SEC including contacting nucleic acid extract with SEC post, the internal diameter of described SEC post is 3mm to 6mm, optionally its internal diameter is about 4mm to about 5mm, optionally its internal diameter is about 4.6mm. In certain embodiments, nucleic acid extract is carried out SEC including contacting nucleic acid extract with SEC post, the length of described SEC post is about 100mm to about 400mm, optionally its length is about 300mm long. In certain embodiments of the method provided herein, nucleic acid extract is carried out SEC including contacting nucleic acid extract with SEC matrix and / or SEC post with an aperture of about 5nm to about 50nm, optionally wherein said aperture is about 12.5nm to about 25nm. In certain embodiments of the methods provided herein, performing SEC on the nucleic acid extract comprises contacting the nucleic acid extract with a SEC matrix and / or a SEC column having a particle size of about 3 μm to about 5 μm, optionally with a particle size of about 4 μm. In certain embodiments of the methods provided herein, performing SEC on the nucleic acid extract comprises contacting the nucleic acid extract with a SEC matrix and / or a SEC column comprising silica, optionally wherein the SEC matrix and / or SEC column comprises silica particles.

[0020] In certain embodiments of the methods provided herein, performing SEC on the nucleic acid extract comprises contacting the nucleic acid extract with a SEC matrix and / or a SEC column that is compatible with a mobile phase having a pH of about 7 to about 8, optionally wherein the SEC matrix and / or SEC column is compatible with a mobile phase having a pH of about 7.3 to about 7.7, optionally wherein the SEC matrix and / or SEC column is compatible with a mobile phase having a pH of about 7.5. In certain embodiments of the methods provided herein, performing SEC on the nucleic acid extract comprises contacting the nucleic acid extract with a SEC matrix and / or a SEC column that is compatible with a mobile phase comprising Tris-HCl, optionally wherein the SEC matrix and / or SEC column is compatible with a mobile phase comprising about 5 mM to about 50 mM Tris-HCl, optionally wherein the SEC matrix and / or SEC column is compatible with a mobile phase comprising about 20 mM Tris-HCl. In certain embodiments of the methods provided herein, performing SEC on the nucleic acid extract comprises contacting the nucleic acid extract with a SEC matrix and / or a SEC column that is compatible with a mobile phase comprising about 25 mM to about 200 mM NaCl, optionally wherein the SEC matrix and / or SEC column is compatible with a mobile phase comprising about 150 mM NaCl.

[0021] In certain embodiments of the methods provided herein, performing SEC on the nucleic acid extract comprises contacting the nucleic acid extract with a SEC mobile phase having a pH between about 7 and about 8, optionally wherein the pH of the mobile phase is about 7.3 to about 7.7, optionally wherein the pH of the mobile phase is about 7.5. In certain embodiments of the methods provided herein, performing SEC on the nucleic acid extract comprises contacting the nucleic acid extract with a SEC mobile phase comprising Tris-HCl, optionally wherein the SEC mobile phase comprises about 5 mM to about 50 mM Tris-HCl, optionally wherein the SEC mobile phase comprises about 20 mM Tris-HCl. In certain embodiments of the methods provided herein, performing SEC on the nucleic acid extract comprises contacting the nucleic acid extract with a SEC mobile phase comprising NaCl, optionally wherein the SEC mobile phase comprises about 25 mM to about 200 mM NaCl, optionally wherein the SEC mobile phase comprises about 150 mM NaCl. In certain embodiments of the methods provided herein, the method comprises measuring the absorbance of the SEC eluate at 260 nm. In certain embodiments of the methods provided herein, SEC is performed at a temperature of 20-40° C., optionally wherein SEC is performed at a temperature of 30° C.

[0022] In at least one aspect, the present disclosure provides a kit for isolating nucleic acids present in a lipid nucleic acid assembly composition, the kit comprising a SEC matrix and at least one reagent for ethanol precipitation of nucleic acids in the lipid nucleic acid assembly composition, optionally wherein the kit is for isolating two or more nucleic acids, optionally wherein the two or more nucleic acid molecules are or include gRNA and mRNA. In certain embodiments, the SEC matrix is ​​a SEC column or is present in a SEC column. In certain embodiments, the inner diameter of the SEC column is 3 mm to 6 mm, optionally its inner diameter is about 4 mm to about 5 mm, optionally its inner diameter is about 4.6 mm inner diameter. In certain embodiments, the length of the SEC column is about 100 mm to about 400 mm, optionally its length is about 300 mm long. In certain embodiments, the SEC matrix and / or SEC column has a pore size of about 5 nm to about 50 nm, optionally wherein the pore size is about 12.5 nm to about 25 nm. In certain embodiments, the SEC matrix and / or SEC column has a particle size of about 3 μm to about 5 μm, optionally its particle size is about 4 μm. In certain embodiments, the SEC matrix and / or SEC column comprises silica, optionally wherein the SEC matrix comprises silica particles. In certain embodiments, the SEC matrix and / or SEC column is compatible with a mobile phase having a pH of about 7 to about 8, optionally a mobile phase having a pH of about 7.3 to about 7.7, optionally a mobile phase having a pH of about 7.5. In certain embodiments, the SEC matrix and / or SEC column is compatible with a mobile phase comprising about 25 mM to about 200 mM NaCl, optionally a mobile phase comprising about 150 mM NaCl.

[0023] In certain embodiments of the kits provided herein, the kits include a dilution series of one or more reference nucleic acids, optionally wherein the kit includes a dilution series of reference gRNA molecules and / or reference mRNA molecules. In certain embodiments of the kits provided herein, the kits include instructions for preparing a nucleic acid extract by a method including ethanol precipitation. In certain embodiments of the kits provided herein, the kits include instructions for separating two or more nucleic acids by SEC, optionally wherein the two or more nucleic acids include or consist of RNA molecules, optionally wherein the RNA molecules include or consist of gRNA molecules and mRNA molecules. In certain embodiments of the kits provided herein, the kits include instructions for determining the concentration and / or total nucleic acid concentration of two or more nucleic acids, optionally wherein the two or more nucleic acids include or consist of RNA molecules, optionally wherein the RNA molecules include or consist of gRNA molecules and mRNA molecules.

[0024] In at least one aspect, the present disclosure provides a method for isolating nucleic acids present in a lipid nucleic acid assembly composition, the method comprising: a) preparing a nucleic acid extract from the lipid nucleic acid assembly composition, wherein the preparation of the nucleic acid extract comprises ethanol precipitation of nucleic acids in the lipid nucleic acid assembly composition; and b) subjecting the nucleic acid extract to ion pair chromatography, wherein the ion pair chromatography separates nucleic acids. In at least one aspect, the present disclosure provides a method comprising: a) preparing a nucleic acid extract from the lipid nucleic acid assembly composition, wherein the preparation of the nucleic acid extract comprises ethanol precipitation of nucleic acids in the lipid nucleic acid assembly composition; and b) subjecting the nucleic acid extract to ion pair chromatography. In certain embodiments, the method further comprises determining the total concentration of nucleic acids present in the lipid nucleic acid assembly composition.

[0025] In at least one aspect, the present disclosure provides a method for determining the total concentration of nucleic acids present in a lipid-nucleic acid assembly composition, the method comprising: a) preparing a nucleic acid extract from the lipid-nucleic acid assembly composition, wherein the preparation of the nucleic acid extract comprises ethanol precipitation of the nucleic acids in the lipid-nucleic acid assembly composition; b) subjecting the nucleic acid extract to ion-pair chromatography, wherein the ion-pair chromatography separates the nucleic acids; and c) determining the total concentration of nucleic acids in the lipid-nucleic acid assembly composition.

[0026] In at least one aspect, the present disclosure provides a method for determining the total concentration of nucleic acids present in a lipid-nucleic acid assembly composition, the method comprising: a) preparing a nucleic acid extract from the lipid-nucleic acid assembly composition, wherein the preparation of the nucleic acid extract comprises ethanol precipitation of the nucleic acids in the lipid-nucleic acid assembly composition; b) performing ion-pair chromatography on the nucleic acid extract; and c) determining the total concentration of nucleic acids in the lipid-nucleic acid assembly composition.

[0027] In certain embodiments, the nucleic acid present in the lipid nucleic acid assembly composition includes at least a first nucleic acid and a second nucleic acid, and wherein ion pair chromatography separates the first nucleic acid from the second nucleic acid. In certain embodiments, the method further comprises determining the concentration of the first nucleic acid and / or the second nucleic acid in the lipid nucleic acid assembly composition. In certain embodiments, the nucleic acid present in the lipid nucleic acid assembly composition includes at least a first nucleic acid, a second nucleic acid, and a third nucleic acid. In certain embodiments, the nucleic acid present in the lipid nucleic acid assembly composition includes at least a first nucleic acid, a second nucleic acid, and a third nucleic acid, and SEC separates the first nucleic acid from the second nucleic acid and / or the third nucleic acid. In certain embodiments, the first nucleic acid is a ribonucleic acid (RNA) molecule. In certain embodiments, the first nucleic acid is a messenger RNA (mRNA) molecule. In certain embodiments, the mRNA molecule encodes a polypeptide, optionally wherein the polypeptide includes an RNA-guided DNA binder, optionally wherein the RNA-guided DNA binder includes a nuclease, optionally wherein the nuclease is a Cas protein, optionally wherein the RNA-guided DNA binder is a fusion polypeptide. In certain embodiments, the first nucleic acid encodes a base editor. In certain embodiments, the base editor is a cytosine base editor. In certain embodiments, the first nucleic acid comprises 1000-7000 nucleotides. In certain embodiments, the second nucleic acid is an RNA molecule. In certain embodiments, the second nucleic acid is a guide RNA (gRNA) molecule, optionally wherein the gRNA molecule is an sgRNA molecule. In certain embodiments, the second nucleic acid comprises 75-200 nucleotides, optionally wherein the second nucleic acid comprises 80-120 nucleotides. In certain embodiments, the third nucleic acid is a ribonucleic acid (RNA) molecule. In certain embodiments, the third nucleic acid is a messenger RNA (mRNA) molecule. In certain embodiments, the mRNA molecule encodes a polypeptide, optionally wherein the polypeptide is a DNA glycosylase inhibitor, optionally wherein the DNA glycosylase inhibitor is UGI. In certain embodiments, the third nucleic acid comprises 750-3000 nucleotides. In certain embodiments, the nucleic acids present in the lipid nucleic acid assembly composition include (i) the first nucleic acid and the second nucleic acid; or (ii) the first nucleic acid, the second nucleic acid and the third nucleic acid.

[0028] In certain embodiments where the lipid nucleic acid assembly composition comprises only the first nucleic acid and the second nucleic acid of the present disclosure, the total concentration of nucleic acid in the lipid nucleic acid assembly composition is the sum of the concentration of the first nucleic acid and the second nucleic acid. In certain embodiments, (i) the concentration of the first nucleic acid and / or the second nucleic acid is determined according to a calibration curve, wherein the calibration curve represents the concentration of nucleic acids in a dilution series of at least one reference nucleic acid. In certain embodiments, the concentrations of the first nucleic acid and the second nucleic acid are determined separately, wherein the concentration of the first nucleic acid is determined according to a first calibration curve, and the concentration of the second nucleic acid is determined according to a second calibration curve.

[0029] In certain embodiments where the lipid nucleic acid assembly composition only includes the first nucleic acid, the second nucleic acid, and the third nucleic acid of the present disclosure, the total concentration of nucleic acid in the lipid nucleic acid assembly composition is the sum of the concentration of the first nucleic acid, the concentration of the second nucleic acid, and the concentration of the third nucleic acid. In certain embodiments, the concentration of the first nucleic acid, the second nucleic acid, and / or the third nucleic acid is determined according to a calibration curve, wherein the calibration curve represents the concentration of nucleic acids in a dilution series of at least one reference nucleic acid. In certain embodiments, the concentration of the first nucleic acid, the second nucleic acid, and the third nucleic acid is determined separately, wherein the concentration of the first nucleic acid is determined according to the first calibration curve, the concentration of the second nucleic acid is determined according to the second calibration curve, and the concentration of the third nucleic acid is determined according to the third calibration curve.

[0030] In certain embodiments, the first nucleic acid is an mRNA molecule, and the first calibration curve is a dilution series of a first reference mRNA molecule, optionally wherein the length and / or sequence of the first reference mRNA molecule is identical to the first nucleic acid or differs from the first nucleic acid by no more than 10%, no more than 5%, or no more than 1%. In certain embodiments, the second nucleic acid is a gRNA molecule, and the second calibration curve is a dilution series of a reference gRNA molecule, optionally wherein the length and / or sequence of the reference gRNA molecule is identical to the second nucleic acid or differs from the second nucleic acid by no more than 10%, no more than 5%, or no more than 1%. In certain embodiments, the third nucleic acid, when present, is an mRNA molecule, and the third calibration curve is a dilution series of a second reference mRNA molecule, optionally wherein the length and / or sequence of the second reference mRNA molecule is identical to the third nucleic acid or differs from the third nucleic acid by no more than 10%, no more than 5%, or no more than 1%.

[0031] In certain embodiments, the first calibration curve represents a first dilution series comprising a concentration of the first reference mRNA molecule, and the concentration covers a range including 10 μg / mL and 100 μg / mL. In certain embodiments, the second calibration curve represents a second dilution series comprising a concentration of the reference gRNA molecule, and the concentration covers a range including 5 μg / mL and 100 μg / mL, and / or a range including 5 μg / mL and 50 μg / mL. In certain embodiments, the third calibration curve represents a third dilution series comprising a concentration of the second reference mRNA molecule, and the concentration covers a range including 10 μg / mL and 100 μg / mL. In certain embodiments, the first dilution series and the second dilution series include concentrations such that the total concentration obtained by the first dilution series, the second dilution series, and the third dilution series obtains a total RNA concentration covering a range including 20 μg / mL and 200 μg / mL or a range including 20 μg / mL and 100 μg / mL.

[0032] In certain embodiments, the method includes determining the concentration of the first nucleic acid, the second nucleic acid and / or the third nucleic acid in the lipid nucleic acid assembly composition, and / or the total nucleic acid concentration, and the concentration is equal to or less than 2.5mg / mL, 2.0mg / mL, 1.5mg / mL, 1.0mg / mL, 0.5mg / mL, 0.4mg / mL or 0.3mg / mL. In certain embodiments, the method includes determining the concentration of the first nucleic acid, the second nucleic acid and / or the third nucleic acid, and / or the total nucleic acid concentration, wherein the total nucleic acid concentration is between about 0.5mg / mL and about 2mg / mL, optionally wherein the total nucleic acid concentration is between about 0.75mg / mL and about 1.8mg / mL. In certain embodiments, ion pair chromatography produces a chromatogram, in which the USP resolution between the nucleic acid peak representing the first nucleic acid and the nucleic acid peak representing the second nucleic acid is equal to or greater than 1, 1.5, 2, 2.5 or 3. In certain embodiments, the ion-pair chromatography produces a chromatogram in which the USP resolution between the nucleic acid peak representing the first nucleic acid and the nucleic acid peak representing the third nucleic acid is equal to or greater than 1, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5. In certain embodiments, the ion-pair chromatography produces a chromatogram in which the USP resolution between the nucleic acid peak representing the second nucleic acid and the nucleic acid peak representing the third nucleic acid is equal to or greater than 1, 1.5, or 2. In certain embodiments, the preparation of the nucleic acid extract comprises diluting the concentration of the nucleic acid extract to the midpoint of the calibration curve.

[0033] In certain embodiments, the first nucleic acid is a messenger RNA (mRNA) molecule encoding a cytosine base editor, the second nucleic acid is a gRNA molecule, optionally wherein the gRNA molecule is an sgRNA molecule, and the third nucleic acid is an mRNA molecule encoding a DNA glycosylase inhibitor, optionally wherein the DNA glycosylase inhibitor is UGI. In certain embodiments, the first nucleic acid does not encode a DNA glycosylase inhibitor and / or does not encode UGI. In certain embodiments, wherein the cytosine base editor includes a DNA glycosylase inhibitor, optionally wherein the DNA glycosylase inhibitor is UGI.

[0034] In certain embodiments, lipid nucleic acid assembly compositions include lipid nanoparticles (LNPs) containing nucleic acid.In certain embodiments, the lipid components of LNP include (i) amine lipids, (ii) helper lipids and (iii) stealth lipids, optionally wherein LNP includes neutral lipids, optionally wherein stealth lipids are PEG lipids.In certain embodiments, the first nucleic acid is messenger RNA (mRNA) molecules encoding the DNA binding agents guided by RNA.In certain embodiments, the first nucleic acid is messenger RNA (mRNA) molecules encoding cytosine base editors, the second nucleic acid is gRNA molecules, and the third nucleic acid is mRNA molecules encoding DNA glycosylase inhibitors, optionally wherein the DNA glycosylase inhibitor is uracil glycosylase inhibitor (UGI), lipid nucleic acid assembly compositions include the LNPs containing the first and second nucleic acid molecules, and the lipid components of LNP include (i) amine lipids, (ii) helper lipids, (iii) stealth lipids and (iv) neutral lipids, optionally wherein stealth lipids are PEG lipids.In certain embodiments, the total concentration of nucleic acid in lipid nucleic acid assembly compositions is the total concentration of RNA in lipid nucleic acid assembly compositions.

[0035] In certain embodiments, ethanol precipitation comprises: a) mixing the lipid-nucleic acid assembly composition with ethanol to produce an ethanol mixture; b) centrifuging the ethanol mixture, wherein the centrifugation produces a nucleic acid precipitate and an ethanol supernatant; and c) separating the nucleic acid precipitate from the ethanol, wherein the nucleic acid precipitate comprises precipitated RNA. In certain embodiments, separating the precipitate comprises decanting the supernatant, removing the supernatant with a pipette, and / or drying the nucleic acid precipitate. In certain embodiments, preparing a nucleic acid extract comprises resuspending the nucleic acid precipitate in water to produce an aqueous nucleic acid solution. In certain embodiments, preparing the nucleic acid extract comprises heating the aqueous nucleic acid solution to a temperature between about 65°C and about 90°C for at least about 2 minutes to about 15 minutes, optionally wherein preparing the nucleic acid extract comprises cooling the aqueous nucleic acid solution after heating, optionally wherein the cooling is to 4°C. In certain embodiments, the nucleic acid extract is free of or substantially free of lipids, free of or substantially free of buffers, and / or free of or substantially free of detergents.

[0036] In certain embodiments, the ion pair chromatography is reverse phase chromatography. In certain embodiments, the ion pair chromatography is high performance liquid chromatography (HPLC). In certain embodiments, the ion pair chromatography is reverse phase high performance liquid chromatography (HPLC).

[0037] In certain embodiments, performing ion-pair chromatography on the nucleic acid extract comprises contacting the nucleic acid extract with an ion-pair chromatography matrix, optionally wherein the ion-pair chromatography matrix is ​​an ion-pair chromatography column or is present in an ion-pair chromatography column. In certain embodiments, performing ion-pair chromatography on the nucleic acid extract comprises contacting the nucleic acid extract with an ion-pair chromatography column having an inner diameter of 1 mm to 4 mm, optionally having an inner diameter of about 1.5 mm to about 2.5 mm, optionally having an inner diameter of about 2.1 mm inner diameter. In certain embodiments, performing ion-pair chromatography on the nucleic acid extract comprises contacting the nucleic acid extract with an ion-pair chromatography column having a length of about 50 mm to about 400 mm, optionally having a length of about 100 mm long. In certain embodiments, performing ion-pair chromatography on the nucleic acid extract comprises contacting the nucleic acid extract with an ion-pair chromatography column having a pore size of about to about contacting an ion-pair chromatography matrix and / or an ion-pair chromatography column, optionally wherein the pore size is about to about In certain embodiments, subjecting the nucleic acid extract to ion-pair chromatography comprises contacting the nucleic acid extract with an ion-pair chromatography matrix and / or an ion-pair chromatography column having a particle size of about 3 μm to about 5 μm, optionally with a particle size of about 4 μm. In certain embodiments, subjecting the nucleic acid extract to ion-pair chromatography comprises contacting the nucleic acid extract with an ion-pair chromatography matrix and / or an ion-pair chromatography column comprising silica, optionally wherein the ion-pair chromatography matrix and / or the ion-pair chromatography column comprises silica particles.

[0038] In certain embodiments, performing ion-pair chromatography on a nucleic acid extract comprises contacting the nucleic acid extract with an ion-pair chromatography matrix and / or an ion-pair chromatography column compatible with at least one mobile phase comprising dibutylammonium acetate and / or triethylammonium acetate. In certain embodiments, the at least one mobile phase comprises 25 mM to 300 mM dibutylammonium acetate, optionally wherein the mobile phase comprises 50 mM dibutylammonium acetate. In certain embodiments, the at least one mobile phase comprises 25 mM to 300 mM triethylammonium acetate, optionally wherein the mobile phase comprises 100 mM triethylammonium acetate. In certain embodiments, the at least one mobile phase comprises 25% to 100% acetonitrile in water, optionally wherein the mobile phase comprises about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% acetonitrile in water. In certain embodiments, at least one mobile phase comprises about 50% to about 100% acetonitrile in water, or about 75% to about 100% acetonitrile in water. In certain embodiments, the method comprises measuring the absorbance of the ion pair chromatography eluent at about 240 nm to about 280 nm or at about 250 nm to about 270 nm, optionally wherein the method comprises measuring the absorbance of the ion pair chromatography eluent at about 240 nm, about 245 nm, about 250 nm, about 255 nm, about 260 nm, about 265 nm, about 270 nm, about 275 nm, or about 280 nm. In certain embodiments, ion pair chromatography is performed at a temperature of 20-80 ° C, optionally wherein the ion pair chromatography is performed at a temperature of 65 ° C.

[0039] In at least one aspect, the present disclosure provides a kit for separating nucleic acids present in a lipid nucleic acid assembly composition, the kit comprising an ion pair chromatography matrix and at least one reagent for ethanol precipitation of nucleic acids in a lipid nucleic acid assembly composition, optionally wherein the kit is for separating two or more nucleic acids, optionally wherein the two or more nucleic acid molecules are or include gRNA and mRNA; and / or for separating three or more nucleic acids, optionally wherein the three or more nucleic acid molecules are or include gRNA, a first mRNA, and a second mRNA. In certain embodiments, the ion pair chromatography matrix is ​​an ion pair chromatography column or is present in an ion pair chromatography column. In certain embodiments, the inner diameter of the ion pair chromatography column is 1 mm to 4 mm, optionally its inner diameter is about 1.5 mm to about 2.5 mm, optionally its inner diameter is about 2.1 mm inner diameter. In certain embodiments, the length of the ion pair chromatography column is about 50 mm to about 400 mm, optionally its length is about 100 mm long. In certain embodiments, the pore size of the ion pair chromatography matrix and / or the ion pair chromatography column is about to about Optionally wherein the pore size is about to about In certain embodiments, the particle size of the ion pair chromatography matrix and / or the ion pair chromatography column is about 3 μm to about 5 μm, optionally its particle size is about 4 μm. In certain embodiments, the ion pair chromatography matrix and / or the ion pair chromatography column comprises silica, optionally wherein the ion pair chromatography matrix comprises silica particles. In certain embodiments, the ion pair chromatography matrix and / or the ion pair chromatography column is compatible with a mobile phase comprising dibutylammonium acetate and / or triethylammonium acetate. In certain embodiments, the kit comprises a dilution series of one or more reference nucleic acids, optionally wherein the kit comprises a dilution series of a reference gRNA molecule, a first reference mRNA molecule, and / or a second reference mRNA molecule. In certain embodiments, the kit comprises instructions for preparing a nucleic acid extract by a method comprising ethanol precipitation. In certain embodiments, the kit comprises instructions for separating two or more nucleic acids, or three or more nucleic acids, by ion pair chromatography, optionally wherein the nucleic acids comprise RNA molecules, optionally wherein the RNA molecules comprise gRNA molecules, first mRNA molecules, and / or second mRNA molecules. In certain embodiments, the kit includes instructions for determining the concentration of two or more nucleic acids, three or more nucleic acids, and / or the total nucleic acid concentration, optionally wherein the nucleic acids include RNA molecules, optionally wherein the RNA molecules include gRNA molecules, first mRNA molecules, and / or second mRNA molecules.

[0040] definition

[0041] A, an, and the: As used herein, "a," "an," and "the" refer to one or more than one (i.e., at least one) of the grammatical object of the article. For example, "an element" discloses embodiments with exactly one element as well as embodiments that include more than one element. The term "or" is used in an inclusive sense, i.e., equivalent to "and / or," unless the context clearly dictates otherwise.

[0042] About: As used herein, the term "about," when used to refer to a value, refers to a value that is similar in context to the reference value. Generally, one skilled in the art familiar with the context will understand the relative degree of variation encompassed by "about" in this context. For example, in some embodiments, the term "about" can encompass a range of values ​​that are within 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of a reference value.

[0043] APOBEC3A: As used herein, the term "APOBEC3A" refers to a cytidine deaminase, such as a protein expressed by the human A3A gene. APOBEC3A may have catalytic DNA editing activity. The amino acid sequence of APOBEC3A has been described (UniPROT accession number: p31941). In some embodiments, the APOBEC3A protein is a human APOBEC3A protein and / or a wild-type protein. Variants include proteins having a sequence that differs from that of the wild-type APOBEC3A protein due to one or more mutations (i.e., substitutions, deletions, insertions, such as one or more single-point substitutions). For example, a shortened APOBEC3A sequence can be used, such as by deleting the N-terminus, C-terminus, or internal amino acids, preferably deleting one to four amino acids at the C-terminus of the sequence. As used herein, the term "variant" refers to allelic variants, splice variants, and natural or artificial mutants that are homologous to an APOBEC3A reference sequence. The variant is "functional" because it exhibits catalytic activity for DNA editing. In some embodiments, APOBEC3A (e.g., human APOBEC3A) has a wild-type amino acid position 57 (by numbering in the wild-type sequence). In some embodiments, APOBEC3A (e.g., human APOBEC3A) has an asparagine at wild-type amino acid position 57 (by numbering in the wild-type sequence).

[0044] Base editor: As used herein, the term "base editor" refers to an agent comprising a polypeptide capable of deaminating bases in a DNA molecule and an RNA-guided nickase (e.g., Cas9 nickase). The base editor is capable of deaminating cytidine (C) in DNA, or deaminating adenine (A) in DNA. For example, a base editor may include an RNA-guided nickase (e.g., Cas9 nickase, e.g., Nme2Cas9 (D16A) or Spy2Cas9 (D10A)) fused to a cytidine deaminase (e.g., APOBEC3A deaminase (A3A)) via an optional linker. In some embodiments, Cas9 is a nickase and comprises an inactivating mutation within the RuvC subdomain. In some embodiments, Cas9 is a nickase and comprises an inactivating mutation within the HNH subdomain. In some embodiments, Cas9 has no nuclease activity and comprises an inactivating mutation within the RuvC and HNH subdomains. In some cases, the base editor comprising a cytidine deaminase further comprises a DNA glycosylase inhibitor (e.g., UGI; that is, comprising one or more DNA glycosylase inhibitor domains, such as one or more UGI domains). In some cases, the base editor comprising a cytidine deaminase can be used in combination with a trans-delivered DNA glycosylase inhibitor (e.g., UGI). In some embodiments, the base editor does not include a DNA glycosylase inhibitor (e.g., produced by an mRNA encoding one or more DNA glycosylase inhibitor domains, such as one or more UGI domains). In various embodiments, the mRNA molecules of the present disclosure encode base editors.

[0045] Between or from: As used herein, the term "between" refers to what is between a stated upper and lower limit, or between a first boundary and a second boundary (or "limits"), inclusive. Similarly, the term "from," when used in the context of a range of values, indicates that the range includes what is between a stated upper and lower limit, or between a first boundary and a second boundary (or "limits"), inclusive.

[0046] Comparable: As used herein, the term "comparable" refers to members within a collection of two or more conditions, situations, agents, entities, populations, etc. that may be different from one another but are similar enough to allow comparisons to be made between them, thereby enabling one skilled in the art to understand that conclusions can be reasonably drawn based on the observed differences or similarities. In some embodiments, a collection of comparable conditions, situations, agents, entities, populations, etc. is typically characterized by a plurality of substantially identical features and zero, one, or more different features. One of ordinary skill in the art will understand what degree of identity is required to make the members of a collection comparable in context. For example, one of ordinary skill in the art will understand that when the members of a collection of conditions, situations, agents, entities, populations, etc. are characterized by a sufficient number and type of substantially identical features, the members are comparable to one another to warrant a reasonable conclusion that the observed differences can be attributed, in whole or in part, to their non-identical features.

[0047] Cytidine deaminase: As used herein, the term "cytidine deaminase" refers to a polypeptide or polypeptide complex capable of having cytidine deaminase activity that catalyzes the hydrolytic deamination of cytidine or deoxycytidine, typically producing uridine or deoxyuridine. Cytidine deaminase encompasses enzymes in the cytidine deaminase superfamily, particularly enzymes of the APOBEC family (APOBEC1, APOBEC2, APOBEC4, and APOBEC3 enzyme subgroups), activation-induced cytidine deaminase (AID or AICDA), and CMP deaminase (see, e.g., Conticello et al., Mol. Biol. Evol. 22:367-77, 2005; Conticello, Genome Biol. 9:229, 2008; Muramatsu et al., J. Biol. Chem. 274:18470-6, (1999); Carrington et al., Cells 9:1690 (2020)). In some embodiments, variants of any known cytidine deaminase or APOBEC protein are contemplated. Variants include proteins having a sequence different from that of the wild-type protein due to one or more mutations (i.e., substitutions, deletions, insertions), such as one or more single-point substitutions. For example, a shortened sequence can be used, for example, by deleting the N-terminus, the C-terminus, or internal amino acids, preferably one to four amino acids at the C-terminus of the sequence. As used herein, the term "variant" refers to allelic variants, splice variants, and natural or artificial mutants homologous to a reference sequence. The variant is "functional" in that it exhibits catalytic activity for DNA editing.

[0048] Drug Product: As used herein, the term "drug product" refers to a final pharmaceutically acceptable dosage form comprising a drug substance, typically but not necessarily in combination with one or more pharmaceutically acceptable carriers. A non-limiting example of a drug product is a lipid-nucleic acid assembly in a final pharmaceutically acceptable dosage form.

[0049] Drug Substance: As used herein, the term "drug substance" refers to an active agent (e.g., an active pharmaceutical ingredient (API) of a drug or an active pharmaceutical ingredient (API) for a drug) that is intended to provide pharmacological activity (e.g., for treating a disease or condition). A non-limiting example of a drug substance is a lipid nucleic acid assembly suitable for use in a final pharmaceutically acceptable dosage form.

[0050] Excipients: As used herein, "excipient" refers to non-therapeutic agents that may be included in a pharmaceutical composition, for example, to provide or contribute to a desired consistency or stabilizing effect. In some embodiments, suitable pharmaceutical excipients may include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, and the like.

[0051] Fusion polypeptide: As used herein, the term "fusion polypeptide" or "chimeric polypeptide" generally refers to a polypeptide that includes at least two parts or segments. Generally, a polypeptide containing at least two parts is considered to be a fusion polypeptide if the two parts (1) are not included in the same peptide in nature (e.g., the second part is heterologous to the first part), and / or (2) have been linked to each other by the hand of man. A fusion polypeptide can include amino acids in addition to the amino acids in the two parts of the fusion polypeptide, or in addition to the amino acids in at least two parts of the polypeptide. The parts of a fusion polypeptide can be referred to as "fusion partners," "fused," or "linked."

[0052] Guide RNA: "Guide RNA," "gRNA," and simply "guide" are used interchangeably herein to refer to a guide that directs an RNA-guided DNA binder to a target DNA, and can be a crRNA (also known as CRISPR RNA) or a combination of crRNA and trRNA (also known as tracrRNA). The crRNA and trRNA can be associated as a single RNA molecule (single guide RNA, sgRNA) or in two separate RNA molecules (dual guide RNA, dgRNA). "Guide RNA" or "gRNA" refers to each type. The trRNA can be a naturally occurring sequence, or a trRNA sequence that has modifications or variations compared to a naturally occurring sequence.

[0053] Guide sequence: As used herein, a "guide sequence" refers to a sequence within a guide RNA that is complementary to a target sequence and functions to direct the guide RNA to the target sequence for binding or modification (e.g., cleavage) by an RNA-guided DNA binder. A "guide sequence" may also be referred to as a "targeting sequence" or "spacer sequence." A guide sequence may be 20 base pairs long, such as in Streptococcus pyogenes (i.e., Spy Cas9) and related Cas9 homologs / orthologs. A guide sequence may be 24 base pairs long, such as in Neisseria meningitidis (i.e., Nme Cas9) and related Cas9 homologs / orthologs (e.g., Nme2Cas9 (D16A)). Shorter or longer sequences may also be used as guides, such as 15, 16, 17, 18, 19, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the target sequence is, for example, in a gene or on a chromosome, and is complementary to the guide sequence. In some embodiments, the degree of complementarity or identity between a guide sequence and its corresponding target sequence can be about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the guide sequence and target region can be 100% complementary or identical. In other embodiments, the guide sequence and target region can contain at least one mismatch. For example, the guide sequence and target sequence can contain 1, 2, 3, or 4 mismatches, wherein the total length of the target sequence is at least 17, 18, 19, 20, or more base pairs. In some embodiments, the guide sequence and target region can contain 1-4 mismatches, wherein the guide sequence comprises at least 17, 18, 19, 20, or more nucleotides. In some embodiments, the guide sequence and target region can contain 1, 2, 3, or 4 mismatches, wherein the guide sequence comprises 20 nucleotides.

[0054] The target sequence of an RNA-guided DNA binder includes both the positive and negative strands of genomic DNA (i.e., a given sequence and the reverse complement of the sequence) because the nucleic acid substrate of the RNA-guided DNA binder is a double-stranded nucleic acid. Therefore, when a guide sequence is referred to as "complementary to a target sequence," it is understood that the guide sequence can guide the guide RNA to bind to the reverse complement of the target sequence. Thus, in some embodiments, where the guide sequence binds to the reverse complement of the target sequence, the guide sequence is identical to certain nucleotides of the target sequence (e.g., a target sequence that does not include a PAM) except that U replaces T in the guide sequence.

[0055] Heterologous: As used herein, the term "heterologous" refers to a nucleotide or polypeptide sequence that is not present in a naturally occurring nucleic acid or protein, respectively. For example, in a chimeric Cas9 / Csnl protein, the RNA binding domain of a naturally occurring bacterial Cas9 / Csnl polypeptide (or a variant thereof) may be fused to a heterologous polypeptide sequence (i.e., a polypeptide sequence from a protein other than Cas9 / Csnl or a polypeptide sequence from another organism). The heterologous polypeptide sequence may exhibit an activity (e.g., an enzymatic activity) (e.g., methyltransferase activity, acetyltransferase activity, kinase activity, ubiquitination activity, etc.) that is also exhibited by the chimeric Cas9 / Csnl protein. A heterologous nucleic acid sequence may be linked to a naturally occurring nucleic acid sequence (or a variant thereof) (e.g., by genetic engineering) to produce a chimeric nucleotide sequence encoding a chimeric polypeptide. As another example, in a fusion variant Cas9 site-directed polypeptide, the variant Cas9 site-directed polypeptide may be fused to a heterologous polypeptide (i.e., a polypeptide other than Cas9) that exhibits an activity that is also exhibited by the fusion variant Cas9 site-directed polypeptide. A heterologous nucleic acid sequence can be linked to a variant Cas9 site-directed polypeptide (e.g., by genetic engineering) to generate a nucleotide sequence encoding a fusion variant Cas9 site-directed polypeptide.

[0056] Improve, increase, inhibit, decrease, or reduce: As used herein, the terms "improve," "increase," "inhibit," "decrease," and "reduce," and their grammatical equivalents, refer to a qualitative or quantitative difference from a reference value.

[0057] Isolated or purified: As used herein, the terms "isolated," "purified," or "separated" refer to a substance and / or entity that is (a) separated from at least some components with which it was originally associated when it was produced (whether in nature or in an experimental setting), and / or (b) is designed, produced, prepared, and / or manufactured by the hand of man. An isolated, purified, or separated substance and / or entity can be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of other components with which it was originally associated. In some embodiments, an isolated, purified, or separated substance and / or entity is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% pure. As used herein, a substance and / or entity is "pure" if it is substantially free of other components. In some embodiments, it will be understood by those skilled in the art that a substance and / or entity may still be considered "isolated," "separated," or "pure" after being combined with certain other components (e.g., one or more carriers or excipients, such as buffers, solvents, water, etc.); in such embodiments, the percent separation, separation, or purity of a substance and / or entity is calculated without including these carriers or excipients. By way of example only, in some embodiments, a polynucleotide of a lipid nucleic acid assembly composition can be referred to as "isolated," "separated," or "purified" when the polynucleotide is free, substantially free, or at least about 80% free (e.g., about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% free) of one or more or all lipid components of the lipid nucleic acid assembly composition. Alternatively or additionally, in some embodiments, a polypeptide or polynucleotide that has undergone one or more separation, separation, or purification techniques can be considered an "isolated," "separated," or "purified" polypeptide or polynucleotide if it has been separated from (a) other components with which it is associated in nature; and / or (b) components with which it was previously associated (e.g., when initially produced).

[0058] Messenger RNA: "Messenger RNA" or "mRNA" is used herein to refer to a polynucleotide comprising an open reading frame that can be translated into a polypeptide (i.e., can serve as a substrate for translation by ribosomes and aminoacylated tRNAs). The mRNA can include a phosphate-sugar backbone comprising a ribose residue, such as a 2'-methoxyribose residue, or an analog thereof. In some embodiments, the sugars in the mRNA phosphate-sugar backbone consist essentially of ribose residues, 2'-methoxyribose residues, or a combination thereof.

[0059] Polynucleotide or Nucleic Acid: "Polynucleotide" and "nucleic acid" are used herein to refer to polymeric compounds comprising nucleosides or nucleoside analogs, including conventional RNA, DNA, mixed RNA-DNA, and polymers as analogs thereof, wherein the nucleosides or nucleoside analogs have nitrogenous heterocyclic bases or base analogs linked together along the backbone. The nucleic acid "backbone" can be composed of a variety of linkages, including one or more sugar-phosphodiester linkages, peptide-nucleic acid linkages ("peptide nucleic acid" or PNA; PCT NO. WO 95 / 32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. The sugar portion of the nucleic acid can be ribose, deoxyribose, or similar compounds having substitutions such as 2' methoxy or 2' halo substitutions. The nitrogenous base can be a conventional base (A, G, C, T, U), an analog thereof (e.g., modified uridine, such as 5-methoxyuridine, pseudouridine, or N1-methylpseudouridine, etc.); inosine; a derivative of a purine or pyrimidine (e.g., N 4 -methyldeoxyguanosine, deaza or azapurine, deaza or azapyrimidine, a pyrimidine base having a substituent at the 5- or 6-position (e.g., 5-methylcytosine), a purine base having a substituent at the 2-, 6-, or 8-position, 2-amino-6-methylaminopurine, O 6 -methylguanine, 4-thiopyrimidine, 4-aminopyrimidine, 4-dimethylhydrazine and O 4-alkylpyrimidines; U.S. Pat. No. 5,378,825 and PCT No. WO 93 / 13121). For a general discussion, see The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11th ed., 1992). Nucleic acids can include one or more "absic" residues, in which the backbone does not include a nitrogenous base at each position in the polymer (U.S. Pat. No. 5,585,481). Nucleic acids can contain only conventional RNA or DNA sugars, bases, and linkages, or can include both conventional components and substitutions (e.g., conventional bases with 2' methoxy linkages, or polymers containing both conventional bases and one or more base analogs). Nucleic acids include "locked nucleic acids" (LNA), which are analogs containing one or more LNA nucleotide monomers whose bicyclic furanose units are locked in a sugar conformation that mimics RNA, which can enhance hybridization affinity for complementary RNA and DNA sequences (Vester and Wengel, 2004, Biochemistry 43(42):13233-41). RNA and DNA have different sugar moieties and may differ due to the presence of uracil or its analogs in RNA and thymine or its analogs in DNA. In some embodiments, the nucleic acid has a sequence that is or encodes a functional gene product. In some embodiments, the nucleic acid is or encodes a gRNA. In some embodiments, the nucleic acid is or encodes an mRNA. In some embodiments, the nucleic acid encodes a protein.

[0060] For the avoidance of doubt, unless the context otherwise defines, each reference to a nucleic acid disclosed herein (including, for example, an sgRNA and / or an mRNA) encompasses (i) a single nucleic acid molecule; (ii) a population of nucleic acid molecules having the same specific nucleic acid sequence; (iii) a population of nucleic acid molecules each having the same specific nucleic acid sequence or a sequence that is at least, for example, 95%, 96%, 97%, 98%, 99% or 100% identical to the specific nucleic acid sequence; and / or (iv) a population of nucleic acid molecules each having the same specific nucleic acid sequence or a sequence that differs from the specific nucleic acid sequence by no more than, for example, 1, 2, 3, 4 or 5 nucleotide positions (e.g., by nucleotide additions, deletions or substitutions). Generally, it will be understood that the lipid-nucleic acid assembly compositions and nucleic acid extracts disclosed herein include a population of nucleic acid molecules, the amount of which can be detected according to the techniques disclosed herein.

[0061] Nucleic acid extract: As used herein, the term "nucleic acid extract" refers to a composition comprising nucleic acids isolated from a lipid-nucleic acid assembly composition. The present disclosure contemplates that the nucleic acid extract can be the direct product of a nucleic acid extraction process (a "primary extract"), or can be a composition produced or obtained by treating a primary extract, for example, by adding or removing one or more agents, such as by diluting or concentrating the primary extract and / or by combining the primary extract with reagents that facilitate analysis.

[0062] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable" applies to one or more or all components of the formulation of the compositions disclosed herein, meaning that each component must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

[0063] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, which aids in the formulation of an agent (e.g., a pharmaceutical agent), alters the bioavailability of an agent, or aids in the transport of an agent from one organ or part of a subject to another organ or part. Some examples of materials that can be used as pharmaceutically acceptable carriers include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; pH buffered solutions; polyesters, polycarbonates, and / or polyanhydrides; and other nontoxic, compatible substances used in pharmaceutical formulations.

[0064] Pharmaceutical formulation: As used herein, the term "pharmaceutical composition" or "formulation" refers to a composition in which a therapeutic agent is formulated together with one or more pharmaceutically acceptable carriers.

[0065] Reference: As used herein, "reference" refers to a standard or control against which a comparison is made. For example, in some embodiments, an agent, sample, sequence, subject, animal, or individual, or a population thereof, or a measured value or representative characteristic thereof is compared to a reference, agent, sample, sequence, cell, subject, animal, or individual, or a population thereof, or a measured value or representative characteristic thereof. In some embodiments, a reference is a measured value. In some embodiments, a reference is a determined standard or expected value. In some embodiments, a reference is a historical reference. A reference can be quantitative or qualitative. Generally, it will be understood by those skilled in the art that a reference value and the value compared thereto represent an assessment made under comparable conditions. It will be understood by those skilled in the art that when there is sufficient similarity, judgments and / or comparisons can be reasonably made based on the similarity. In some embodiments, an appropriate reference can be an agent, sample, sequence, cell, subject, animal, or individual, or a population thereof, under conditions that are considered comparable by those skilled in the art, for example, for the purpose of assessing one or more specific variables (e.g., the presence, absence, or absolute and / or relative concentration of a certain agent or condition), or its measurement or representative characteristic.

[0066] RNA-guided DNA binding agent: As used herein, “RNA-guided DNA binding agent” means a polypeptide or polypeptide complex having RNA and DNA binding activity, or a DNA binding subunit of such a complex, wherein the DNA binding activity is sequence-specific and depends on the sequence of the RNA. Exemplary RNA-guided DNA binding agents include Cas lyases / nickases and inactivated forms thereof (“dCas DNA binders”). As used herein, “Cas nucleases”, also known as “Cas proteins”, encompass Cas lyases, Cas nickases, and dCas DNA binders. Cas lyases / nickases and dCas DNA binders include the Csm or Cmr complex of the type III CRISPR system; Cas10, Csml, or Cmr2 subunits thereof; the Cascade complex of the type I CRISPR system; the Cas3 subunits thereof; and class 2 Cas nucleases. As used herein, “class 2 Cas nucleases” are single-chain polypeptides with RNA-guided DNA binding activity. Class 2 Cas nucleases include: Class 2 Cas lyases / nickases (e.g., H840A, D10A, or N863A variants) that also have RNA-guided DNA lyase or nickase activity; and Class 2 dCas DNA binders in which the lyase / nickase activity is inactivated. Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2cl, C2c2, C2c3, HF Cas9 (e.g., N497A, R661A, Q695A, Q926A variants), HypaCas9 (e.g., N692A, M694A, Q695A, H698A variants), eSPCas9 (1.0) (e.g., K810A, K1003A, R1060A variants), and eSPCas9 (ll) (e.g., K848A, K1003A, R1060A variants) proteins, and modifications thereof. Class 2 Cas nucleases include, for example, Nme2Cas9 (D16A) nickases. Several Cas9 orthologs have been obtained from Neisseria meningitidis (Esvelt et al., NAT.METHODS, Vol. 10, 2013, 1116-1121; Hou et al., PNAS, Vol. 110, 2013, 15644-15649; Edraki et al., Mol.Cell 73:714-726, 2019) (NmelCas9, Nme2Cas9 and Nme3Cas9). Nme2Cas9 orthologs efficiently function in mammalian cells, recognize N4CC PAM, and can be used for in vivo editing (Ran et al., NATURE, p. 520, 2015, p. 186-191; Kim et al., NAT.COMMUN., Vol. 8, 2017, p. 14500).It has been shown that Nme2Cas9 has natural resistance to off-target editing (Lee et al., MOL.THER., Vol. 24, 2016, pp. 645-654; Kim et al., 2017). See also, for example, WO / 2020081568 (e.g., pages 28 and 42), which describes a Nme2Cas9 D16A nickase, the contents of which are incorporated herein by reference in their entirety. The Cpf1 protein (Zetsche et al., Cell, 163: 1-13 (2015)) is homologous to Cas9 and contains a RuvC-like nuclease domain. Zetsche's Cpf1 sequence is incorporated by reference in its entirety. See, for example, Tables S1 and S3 of Zetsche. See, e.g., Makarova et al., Nat Rev Microbiol, 13(11):722-36 (2015); Shmakov et al., Molecular Cell, 60:385-397 (2015).

[0067] Sample: As used herein, the term "sample" generally refers to an aliquot of material obtained or obtained from a source of interest. In some embodiments, the source of interest is a biological source or an environmental source. In some embodiments, the sample is a "raw sample" obtained directly from the source of interest. In some embodiments, as will be apparent from the context, the term "sample" refers to a preparation obtained by processing the raw sample (e.g., by removing one or more components of the raw sample and / or by adding one or more agents to the raw sample). Such "processed samples" can include, for example, cells, nucleic acids, or proteins extracted from the sample, or cells, nucleic acids, or proteins obtained by techniques such as nucleic acid amplification or reverse transcription of the raw sample, separation of certain components, separation, and / or purification.

[0068] Numerical ranges are inclusive of the values ​​delimiting the range. Measured and measurable values ​​should be understood to be approximate, taking into account significant digits and errors associated with measurements. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 is a schematic diagram of an exemplary method for nucleic acid extraction including ethanol precipitation, such as described in Example 1.

[0070] Figure 2 is an exemplary chromatogram showing that SEC performed according to Example 2 achieved robust separation of mRNA and sgRNA.

[0071] Figure 3is an exemplary chromatogram generated by performing SEC on a reference nucleic acid composition including mRNA and sgRNA (total nucleic acid concentration is 40 μg / mL) according to WS3 shown in Example 2, showing robust separation of mRNA and sgRNA.

[0072] Figure 4 Figure 2 is an exemplary chromatogram generated by performing SEC on a nucleic acid extract including mRNA and sgRNA, prepared from a lipid-nucleic acid assembly composition including LNPs and diluted to a total nucleic acid concentration of 40 μg / mL. The chromatogram shows robust separation of mRNA and sgRNA.

[0073] Figure 5 is a schematic diagram of an exemplary method for nucleic acid extraction including ethanol precipitation, such as described in Example 3.

[0074] Figure 6 is an exemplary chromatogram showing that ion-pair reversed-phase (IP RP) chromatography performed according to Example 4 achieves robust separation of three RNA species, including approximately 0.1 kb sgRNA, approximately 1.0 kb mRNA, and approximately 4.5 kb mRNA (the total nucleic acid concentration of the three RNA species is 100 μg / mL).

[0075] Figure 7 is an exemplary chromatogram showing that IP RP chromatography performed according to Example 4 achieves robust separation of three RNA species, including an approximately 0.1 kb sgRNA, an approximately 1.0 kb mRNA, and an approximately 4.5 kb mRNA (the total nucleic acid concentration of the three RNA species is 12.5 μg / mL).

[0076] Figure 8 is an exemplary chromatogram showing an overlay of the following three different samples separated according to Example 4: (1) a standard comprising three RNA species (approximately 0.1 kb sgRNA, approximately 1.0 kb mRNA, and approximately 4.5 kb mRNA) at a total nucleic acid concentration of 50 μg / mL; (2) a first replicate of the separation of the three mRNA species by IP RP chromatography (LNP extraction A); and (3) a second replicate of the separation of the three mRNA species by IP RP chromatography (LNP extraction B). The chromatograms show robust separation of the three RNA species present during the LNP extraction process, including the approximately 0.1 kb sgRNA, the approximately 1.0 kb mRNA, and the approximately 4.5 kb mRNA. DETAILED DESCRIPTION

[0077] The present disclosure provides a method for separating, quantifying and / or characterizing the nucleic acid present in a lipid nucleic acid assembly composition. In various embodiments, the lipid nucleic acid assembly composition includes LNPs that encapsulate nucleic acid (e.g., RNA). In various embodiments, the lipid nucleic acid assembly composition includes sgRNA, mRNA, or both. In various embodiments, the method for separating, quantifying and / or characterizing the nucleic acid present in a lipid nucleic acid assembly composition includes extracting nucleic acid from the lipid nucleic acid assembly composition. In specific embodiments, nucleic acid is extracted from a lipid nucleic acid assembly composition by ethanol precipitation, isopropanol precipitation, butanol precipitation, or acetonitrile precipitation. In various embodiments, the method provided herein can be used for, for example, separating, quantifying, and / or characterizing the nucleic acid present in a lipid nucleic acid assembly composition, wherein the nucleic acid is encapsulated by LNPs, because precipitation (e.g., ethanol precipitation) produces a nucleic acid extract that does not contain or is substantially free of other substances (e.g., lipids and / or detergents). In some embodiments, the extracted nucleic acid can be analyzed by chromatography, such as size exclusion chromatography (SEC) or ion pair chromatography. In various embodiments, the multiple nucleic acids in the nucleic acid extract can be separated by SEC. In various embodiments, the multiple nucleic acids in the nucleic acid extract can be separated by ion pair chromatography. In various embodiments, quantification can include determining the concentration (e.g., absolute concentration and / or relative concentration) of nucleic acids present in the lipid-nucleic acid assembly composition, the ratio of nucleic acids present in the lipid-nucleic acid assembly composition, and / or the total concentration (e.g., absolute total concentration and / or relative total concentration) of nucleic acids present in the lipid-nucleic acid assembly composition.

[0078] Exemplary lipid-nucleic acid assembly compositions

[0079] Provided herein are various embodiments utilizing lipid nucleic acid assemblies (e.g., lipid nanoparticles), the lipid nucleic acid assemblies comprising one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten or more) different nucleic acid molecules. The methods and kits provided herein can rapidly assess the nucleic acid molecules without interference from the lipid components in the lipid nucleic acid assemblies or the detergents traditionally used for nucleic acid extraction. In some embodiments, one or more of the nucleic acid molecules comprise RNA or consist of RNA. In some embodiments, the lipid nucleic acid assemblies may comprise genome editing tools, such as RNA, including CRISPR / Cas components and RNA encoding and / or expressing the components. The lipid nucleic acid assembly composition may comprise a nucleic acid (e.g., RNA) component, the nucleic acid component including one or more of the following: mRNA encoding an RNA-guided DNA binder, Cas nuclease mRNA, Class 2 Cas nuclease mRNA, Cas9 mRNA, mRNA encoding a base editor, mRNA encoding a fusion protein comprising an RNA-guided DNA binder, and / or gRNA. In some embodiments, the lipid nucleic acid assembly composition may include two RNA molecules. For example, in some embodiments, the lipid nucleic acid assembly composition may include (i) mRNA encoding a Class 2 Cas nuclease and (ii) gRNA. In some embodiments, the lipid nucleic acid assembly composition may include (i) mRNA encoding a base editor and (ii) gRNA. In some embodiments, the lipid nucleic acid assembly composition may include (i) mRNA encoding a cytosine base editor; (ii) mRNA encoding a DNA glycosylase inhibitor (e.g., UGI); and (ii) gRNA. In some embodiments, the lipid nucleic acid assembly includes RNA encoding and / or expressing a base editor, wherein the base editor includes a deaminase associated with a DNA binding domain, such as a catalytically impaired nuclease domain (e.g., a catalytically impaired CRISPR / Cas). In some embodiments, the lipid nucleic acid assembly includes RNA encoding and / or expressing a DNA glycosylase inhibitor (e.g., uracil DNA glycosylase inhibitor (UGI)).

[0080] As used herein, "lipid nucleic acid assembly composition" refers to a lipid-based delivery composition, including lipid nanoparticles (LNP) and lipid complexes (lipoplex). In some embodiments, "LNP composition" can be used interchangeably with "LNPs" or "LNP".

[0081] In some embodiments, LNP refers to lipid nanoparticles having a diameter of <100 nanometers (nm), or a population of LNPs having an average diameter of <100 nanometers (nm). In other embodiments, the diameter of the LNP can be about 1-250 nm, about 1-200 nm, about 1-175 nm, about 1-150 nm, about 1-125 nm, about 1-120 nm, about 1-100 nm, about 10-250 nm, about 10-200 nm, about 10-175 nm, about 10-150 nm, about 10-125 nm, about 10-120 nm, about 10-100 nm, about 20-250 nm, about 20-200 nm, about 20-175 nm, about 20-150 nm, about 20-125 nm, about 20-120 nm, about 20- In some embodiments, the present invention relates to a nanostructured carbonyl group comprising: a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, In certain embodiments, the mean diameter of the LNP population is about 1-250 nm, about 1-200 nm, about 1-175 nm, about 1-150 nm, about 1-125 nm, about 1-120 nm, about 1-100 nm, about 10-250 nm, about 10-200 nm, about 10-175 nm, about 10-150 nm, about 10-125 nm, about 10-120 nm, about 10-100 nm, about 20-250 nm, about 20-200 nm, about 20-175 nm, about 10-150 nm, about 10-125 nm, about 10-120 nm, about 10-100 nm, about 20-250 nm, about 20-200 nm, about 20-175 nm, about 20-150 nm, about 20-125 nm, about 20-120 nm, about 20 In some embodiments, the LNP composition has a diameter of 10-150 nm, or the average diameter of the LNP population is 10-150 nm.In some embodiments, the LNP composition has a diameter of 75-150 nm, or the average diameter of a population of LNPs is 75-150 nm.

[0082] LNP is formed by precisely mixing a lipid component (e.g., in ethanol) with an aqueous nucleic acid component, and the LNP size is uniform. Lipoplexes are particles formed by mixing lipid and nucleic acid components as a whole, and their size is between about 100 nanometers and 1 micron. In certain embodiments, a lipid nucleic acid assembly is or includes LNP. As used herein, a "lipid nucleic acid assembly" comprises a plurality (i.e., more than one) of lipid molecules physically associated with each other by intermolecular forces. The lipid nucleic acid assembly may comprise a bioavailable lipid with a pKa value <7.5 or <7. The lipid nucleic acid assembly is formed by mixing a nucleic acid-containing aqueous solution with an organic solvent-based lipid solution (e.g., 100% ethanol). Suitable solutions or solvents include or may contain: water, PBS, Tris buffer, NaCl, citrate buffer, ethanol, chloroform, ether, cyclohexane, tetrahydrofuran, methanol, isopropanol. In some embodiments, the nucleic acid aqueous solution comprises RNA, such as mRNA or gRNA. In some embodiments, the nucleic acid aqueous solution comprises mRNA encoding a polypeptide, such as an RNA-guided DNA binder, such as Cas9.

[0083] In some embodiments, the lipid nucleic acid assembly formulation includes an amine lipid (sometimes described herein or elsewhere as "ionizable lipid" or "biodegradable lipid"), and optional helper lipids, neutral lipids, and stealth lipids, such as PEG lipids. The terms amine lipids, helper lipids, neutral lipids, and stealth lipids have meanings known to those skilled in the art, including but not limited to those shown in International Application Publication Nos. WO2021 / 222287A1, WO2022 / 221695A1, and WO2022 / 221697A1, which are incorporated herein by reference in their entirety regarding the meaning of these terms. In some embodiments, the amine lipid or ionizable lipid presents a cationic state depending on the pH.

[0084] In some embodiments, the lipid nucleic acid assembly may comprise (i) an amine lipid (sometimes described herein or elsewhere as an "ionizable lipid" or "biodegradable lipid"); (ii) an optional neutral lipid; (iii) an optional helper lipid; and (iv) a stealth lipid, such as a PEG lipid. In some embodiments, the lipid nucleic acid assembly may comprise an amine lipid, and one or more of a neutral lipid, a helper lipid, and a stealth lipid (e.g., a PEG lipid). In some embodiments, the lipid nucleic acid assembly may comprise (i) an amine lipid, (ii) a neutral lipid, (iii) a helper lipid, and (iv) a stealth lipid, such as a PEG lipid.

[0085] In some embodiments, the lipid nucleic acid assembly composition comprises an "amine lipid," which is, for example, an ionizable lipid, such as lipid A or lipid D, or equivalents thereof, including acetal analogs of lipid A or lipid D.

[0086] In some embodiments, the amine lipid is lipid A, which is (9Z,12Z)-3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadec-9,12-dienoate, also known as (9Z,12Z)-3-((4,4-bis(octyloxy)butyryl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadec-9,12-dienoate. Lipid A can be depicted as:

[0087]

[0088] Lipid A can be synthesized according to WO2015 / 095340 (e.g., pages 84-86). In some embodiments, the amine lipid is lipid A, or an amine lipid provided in WO2020 / 219876, which is incorporated herein by reference.

[0089] In some embodiments, the amine lipid is an analog of lipid A. In some embodiments, the lipid A analog is an acetal analog of lipid A. In certain lipid nucleic acid assembly compositions, the acetal analog is a C4-C12 acetal analog. In some embodiments, the acetal analog is a C5-C12 acetal analog. In other embodiments, the acetal analog is a C5-C10 acetal analog. In other embodiments, the acetal analog is selected from C4, C5, C6, C7, C9, C10, C11, and C12 acetal analogs.

[0090] In some embodiments, the amine lipid is a compound having the structure of Formula IA:

[0091]

[0092] in

[0093] X1A is O, NH or a direct bond;

[0094] X2A is C2-3 alkylene;

[0095] R3A is C1-3 alkyl;

[0096] R2A is C1-3 alkyl, or

[0097] R2A together with the nitrogen atom to which it is attached and 2-3 carbon atoms of X2A form a 5-membered or 6-membered ring, or

[0098] R2A and R3A together with the nitrogen atom to which they are attached form a 5-membered ring;

[0099] Y1A is C6-10 alkylene;

[0100] Y2A is selected from

[0101] R4A is C4-11 alkyl;

[0102] Z1A is C2-5 alkylene;

[0103] Z2A is or does not exist;

[0104] R5A is C6-8 alkyl or C6-8 alkoxy; and

[0105] R6A is C6-8 alkyl or C6-8 alkoxy;

[0106] or a salt thereof.

[0107] In some embodiments, the amine lipid is a compound of formula (IIA)

[0108]

[0109] in

[0110] X1A is O, NH or a direct bond;

[0111] X2A is C2-3 alkylene;

[0112] Z1A is C3 alkylene, and R5A and R6A are each C6 alkyl, or Z1A is a direct bond, and R5A and R6A are each C8 alkoxy; and

[0113] R8A is

[0114] or a salt thereof.

[0115] In certain embodiments, X1A is O. In other embodiments, X1A is NH. In still other embodiments, X1A is a direct bond.

[0116] In certain embodiments, X2A is C3 alkylene. In specific embodiments, X2A is C2 alkylene.

[0117] In certain embodiments, Z1A is a direct bond and R5A and R6A are each C8 alkoxy. In other embodiments, Z1A is C3 alkylene and R5A and R6A are each C6 alkyl.

[0118] In certain embodiments, R8A is In other embodiments, R8A is

[0119] In certain embodiments, the amine lipid is a salt.

[0120] Representative compounds of formula (IA) include:

[0121]

[0122]

[0123]

[0124]

[0125] or a salt thereof, such as a pharmaceutically acceptable salt thereof.

[0126] In some embodiments, the amine lipid is lipid D, which is nonyl 8-((7,7-bis(octyloxy)heptyl)(2-hydroxyethyl)amino)octanoate:

[0127] or a salt thereof.

[0128] Lipid D can be synthesized according to WO2020072605 and Mol.Ther.2018,26(6),1509-1519 ("Sabnis"), which are incorporated by reference in their entirety. In some embodiments, the amine lipid is lipid D, or an amine lipid provided in WO2020072605, which is incorporated by reference herein.

[0129] In some embodiments, the amine lipid is a compound having the structure of Formula IB:

[0130]

[0131] in

[0132] X 1B It is C 6-7 alkylene;

[0133] X 2B yes Or not exist, if X 2B yes Then R 2B Not an alkoxy group;

[0134] Z 1B It is C 2-3 alkylene;

[0135] Z 2BSelected from -OH, -NHC(=O)OCH3 and -NHS(=O)2CH3;

[0136] R 1B It is C 7-9 an unbranched alkyl group; and

[0137] Each R 2B are independently C8 alkyl or C8 alkoxy;

[0138] or a salt thereof.

[0139] In some embodiments, the amine lipid is a compound of formula (IIB)

[0140]

[0141] in

[0142] X 1B It is C 6-7 alkylene;

[0143] Z 1B It is C 2-3 alkylene;

[0144] R 1B It is C 7-9 an unbranched alkyl group; and

[0145] Each R 2B is a C8 alkyl group;

[0146] or a salt thereof.

[0147] In certain embodiments, X 1B In other embodiments, X 1B It is a C7 alkylene group.

[0148] In certain embodiments, Z 1B It is a direct key, and R 5B and R 6B Each is a C8 alkoxy group. In other embodiments, Z 1B is a C3 alkylene group, and R 5B and R 6B Each is a C6 alkyl group.

[0149] In certain embodiments, X 2B yes And R 2B In other embodiments, X 2B Does not exist.

[0150] In certain embodiments, Z 1B is C2 alkylene; in other embodiments, Z 1BIt is a C3 alkylene group.

[0151] In certain embodiments, Z 2B In other embodiments, Z 2B is -NHC(=O)OCH3. In other embodiments, Z 2B It is -NHS(=O)2CH3.

[0152] In certain embodiments, R 1B is C7 alkylene. In other embodiments, R 1B is C8 alkylene. In other embodiments, R 1B It is a C9 branched or unbranched alkylene group.

[0153] In certain embodiments, the amine lipid is a salt.

[0154] Representative compounds of formula (IB) include:

[0155]

[0156]

[0157] or a salt thereof, such as a pharmaceutically acceptable salt thereof.

[0158] Amine lipids and other "biodegradable lipids" suitable for lipid nucleic acid assemblies described herein can be biodegraded in vivo or in vitro. Amine lipids have low toxicity (e.g., tolerable in animal models when the dose is greater than or equal to 10 mg / kg, and have no adverse effects). In some embodiments, the lipid nucleic acid assemblies comprising amine lipids include at least 75% of the amine lipids removed from plasma or engineered cells within 8 hours, 10 hours, 12 hours, 24 hours, or 48 hours, or 3 days, 4 days, 5 days, 6 days, 7 days, or 10 days. In some embodiments, the lipid nucleic acid assemblies comprising amine lipids include at least 50% of the nucleic acids (e.g., mRNA or gRNA) removed from plasma within 8 hours, 10 hours, 12 hours, 24 hours, or 48 hours, or 3 days, 4 days, 5 days, 6 days, 7 days, or 10 days. In some embodiments, the lipid nucleic acid assemblies comprising amine lipids include at least 50% of the lipid nucleic acid assemblies that are cleared from the plasma within 8 hours, 10 hours, 12 hours, 24 hours, or 48 hours, or within 3 days, 4 days, 5 days, 6 days, 7 days, or 10 days, as determined, for example, by measuring lipids (e.g., amine lipids), nucleic acids (e.g., RNA / mRNA), or other components. In some embodiments, the encapsulated lipids and free lipid, RNA, or nucleic acid components in the lipid nucleic acid assemblies are measured.

[0159] Biodegradable lipids include, for example, those of WO / 2020 / 219876 (e.g., pages 13-33, 66-87), WO / 2020 / 118041, WO / 2020 / 072605 (e.g., pages 5-12, 21-29, 61-68), WO / 2019 / 067992, WO / 2017 / 173054, WO 2015 / 095340, and WO2014 / 136086, and LNPs include the LNP compositions described therein, which are hereby incorporated by reference.

[0160] Lipid clearance can be measured as described in the literature. See Maier, MA et al. Biodegradable Lipids Enabling Rapidly Eliminated Lipid Nanoparticles for Systemic Delivery of RNAi Therapeutics. Mol. Ther. 2013, 21 (8), 1570-78 (" Maier "). For example, in Maier's study, 0.3 mg / kg of LNP-siRNA system containing siRNA targeting luciferase was administered to the lateral tail vein of six to eight week old male C57Bl / 6 mice by intravenous push injection. Blood, liver and spleen samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, 24, 48, 96 and 168 hours after administration. Mice were perfused with saline, tissues were subsequently collected, and blood samples were processed to obtain plasma. All samples were processed and analyzed by LC-MS. In addition, Maier describes a procedure for evaluating the toxicity after administering LNP-siRNA formulations. For example, siRNA targeting luciferase was administered to male Sprague-Dawley rats at a dose volume of 5 mL / kg by a single intravenous bolus of 0, 1, 3, 5, and 10 mg / kg (5 animals / group). After 24 hours, approximately 1 mL of blood was obtained from the jugular vein of the awake animals, and serum was separated. 72 hours after administration, all animals were euthanized for autopsy. Clinical signs, body weight, serum chemistry, organ weight, and histopathology were assessed. Although Maier describes methods for assessing siRNA-LNP formulations, these methods can also be used to assess clearance, pharmacokinetics, and toxicity of lipid nucleic acid assembly compositions of the present disclosure.

[0161] Ionizable and bioavailable lipids known in the art for LNP delivery of nucleic acids are suitable. Lipids can be ionized depending on the pH of the medium in which they are located. For example, in a weakly acidic medium, lipids, such as amine lipids, can be protonated and thus have a positive charge. In contrast, in a weakly alkaline medium, such as blood at a pH of about 7.35, lipids, such as amine lipids, can be unprotonated and thus have no charge.

[0162] The ability of a lipid to be charged is related to its intrinsic pKa. In some embodiments, the amine lipids of the present disclosure can each independently have a pKa in the range of about 5.1 to about 7.4. In some embodiments, the bioavailable lipids of the present disclosure can each independently have a pKa in the range of about 5.1 to about 7.4, for example, about 5.5 to about 6.6, about 5.6 to about 6.4, about 5.8 to about 6.2, or about 5.8 to about 6.5. In some embodiments, the amine lipids of the present disclosure can each independently have a pKa in the range of about 5.8 to about 6.5. Lipids with a pKa in the range of about 5.1 to about 7.4 can effectively deliver cargo in vivo, for example, to the liver. In addition, it has been found that lipids with a pKa in the range of about 5.3 to about 6.4 can effectively deliver cargo in vivo, for example, to tumors. See, for example, WO2014 / 136086.

[0163] "Neutral lipids" suitable for use in the lipid compositions of the present disclosure include, for example, a variety of neutral lipids, uncharged lipids, or zwitterionic lipids. Examples of neutral phospholipids suitable for use in the present disclosure include, but are not limited to, 5-heptadecanethiol-1,3-diol (resorcinol), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), phosphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (PMP C), 1-palmitoyl-2-stearoylphosphatidylcholine (PSPC), 1,2-diacaproyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), 1,2-di-eicosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoylphosphatidylcholine (POPC), lysophosphatidylcholine, dioleoylphosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine, distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), palmitoyloleoylphosphatidylethanolamine (POPE), lysophosphatidylethanolamine, and combinations thereof. In one embodiment, the neutral phospholipid may be selected from the group consisting of distearoylphosphatidylcholine (DSPC) and dimyristoylphosphatidylethanolamine (DMPE). In another embodiment, the neutral phospholipid can be distearoylphosphatidylcholine (DSPC).

[0164] "Helper lipids" include steroids, sterols, and alkylresorcinols. Helper lipids suitable for use in the present disclosure include, but are not limited to, cholesterol, 5-heptadecanylresorcinol, and cholesterol hemisuccinate. In one embodiment, the helper lipid can be cholesterol. In one embodiment, the helper lipid can be cholesterol hemisuccinate.

[0165] " Stealth lipid " is the lipid that changes the time length that nanoparticle can exist in vivo (for example in blood). Stealth lipid can help formulation process by, for example, reducing particle aggregation and controlling particle size. Stealth lipid used herein can regulate the pharmacokinetic properties of lipid nucleic acid assembly or contribute to the in vitro stability of nanoparticle. Stealth lipid suitable for lipid compositions of the present disclosure include but are not limited to stealth lipid with hydrophilic head group connected to lipid part. Stealth lipid suitable for lipid compositions of the present disclosure and information on the biochemistry of such lipids can be found in Romberg et al., Pharmaceutical Research, Vol. 25, No. 1, 2008, pp. 55-71 and Hoekstra et al., Biochimica et Biophysica Acta 1660 (2004) 41-52. Other suitable PEG lipids are disclosed in, for example, WO2006 / 007712.

[0166] In one embodiment, the hydrophilic head group of the stealth lipid comprises a polymer moiety selected from a PEG-based polymer. The stealth lipid may comprise a lipid moiety. In some embodiments, the stealth lipid is a PEG lipid.

[0167] In one embodiment, the stealth lipid comprises a polymer moiety selected from the group consisting of PEG (sometimes referred to as poly(ethylene oxide))-based polymers, poly(oxazoline), poly(vinyl alcohol), poly(glycerol), poly(N-vinylpyrrolidone), polyamino acids, and poly[N-(2-hydroxypropyl)methacrylamide].

[0168] In one embodiment, the PEG lipid comprises a polymer portion based on PEG (sometimes referred to as poly(ethylene oxide)).

[0169] PEG lipids also include a lipid moiety. In some embodiments, the lipid moiety can be derived from diacylglycerol or diacyl imidazole bisamides, including those containing dialkyl glycerol or dialkyl imidazole bisamide groups, wherein the dialkyl glycerol or dialkyl imidazole bisamide groups have an alkyl chain length independently comprising about C4 to about C40 saturated or unsaturated carbon atoms, wherein the chain can include one or more functional groups, such as amides or esters. In some embodiments, the alkyl chain length includes about C10 to C20. The dialkyl glycerol or dialkyl imidazole bisamide groups can also include one or more substituted alkyl groups. The chain length can be symmetrical or asymmetrical.

[0170] Unless otherwise indicated, the term "PEG" as used herein refers to any polyethylene glycol or other polyalkylene ether polymer. In one embodiment, PEG is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In one embodiment, PEG is unsubstituted. In one embodiment, PEG is substituted, for example, by one or more alkyl, alkoxy, acyl, hydroxyl, or aryl groups. In one embodiment, the term includes PEG copolymers, such as PEG-polyurethane or PEG-polypropylene (see, for example, J. Milton Harris, Poly(ethylene glycol) chemistry: biotechnical and biomedical applications (1992)); in another embodiment, the term does not include PEG copolymers. In one embodiment, the molecular weight of PEG is from about 130 to about 50,000, in a subembodiment from about 150 to about 30,000, in a subembodiment from about 150 to about 20,000, in a subembodiment from about 150 to about 15,000, in a subembodiment from about 150 to about 10,000, in a subembodiment from about 150 to about 6,000, in a subembodiment from about 150 to about 5,000, in a subembodiment from about 150 to about 4,000, in a subembodiment from about 150 to about 3,000, in a subembodiment from about 300 to about 3,000, in a subembodiment from about 1,000 to about 3,000, and in a subembodiment from about 1,500 to about 2,500.

[0171] In some embodiments, the PEG (e.g., conjugated to a lipid moiety or lipid such as a stealth lipid) is "PEG-2K," also known as "PEG 2000," which has an average molecular weight of about 2,000 Daltons. PEG-2K is represented herein by the following formula (IV), wherein n is 45, meaning that the number average degree of polymerization comprises about 45 subunits. However, other PEG embodiments known in the art may be used, including, for example, those having a number average degree of polymerization comprising about 23 subunits (n=23) and / or 68 subunits (n=68). In some embodiments, n may be in the range of about 30 to about 60. In some embodiments, n may be in the range of about 35 to about 55. In some embodiments, n may be in the range of about 40 to about 50. In some embodiments, n may be in the range of about 42 to about 48. In some embodiments, n may be 45. In some embodiments, R may be selected from H, substituted alkyl, and unsubstituted alkyl. In some embodiments, R may be unsubstituted alkyl. In some embodiments, R may be methyl.

[0172] In any of the embodiments described herein, the PEG lipid can be selected from PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG) (Cat. No. GM-020, from NOF, Tokyo, Japan), PEG-dipalmitoylglycerol, PEG-distearoylglycerol (PEG-DSPE) (Cat. No. DSPE-020CN, from NOF, Tokyo, Japan), PEG-dilaurylimidazolebisamide, PEG-dimyristoylimidazolebisamide, PEG-dipalmitoylimidazolebisamide, and PE G-distearoyl imidazole bisamide, PEG-cholesterol (1-[8'-(cholest-5-en-3[β]-oxy)formamido-3',6'-dioxaoctyl]carbamoyl-[ω]-methyl-poly(ethylene glycol)), PEG-DMB (3,4-di-tetradecyloxybenzyl-[ω]-methyl-poly(ethylene glycol) ether), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DMG) (Cat. No. 880150P, from Avanti In one embodiment, the PEG lipid may be PEG2k-DMG. In some embodiments, the PEG lipid may be PEG2k-DSG. In one embodiment, the PEG lipid may be PEG2k-DSPE. In one embodiment, the PEG lipid may be PEG2k-DMA. In one embodiment, the PEG lipid may be PEG2k-C-DMA. In one embodiment, the PEG lipid may be compound S027 disclosed in WO2016 / 010840 (paragraphs

[00240] to

[00244] ). In one embodiment, the PEG lipid may be PEG2k-DSA. In one embodiment, the PEG lipid may be PEG2k-C11. In some embodiments, the PEG lipid may be PEG2k-C14. In some embodiments, the PEG lipid may be PEG2k-C16. In some embodiments, the PEG lipid may be PEG2k-C18.

[0173] In some embodiments, the LNP composition includes a polymeric lipid, such as a PEG lipid, which can affect the length of time the nanoparticle exists in vivo or in vitro (e.g., in blood or a medium). The PEG lipid can facilitate the formulation process by, for example, reducing particle aggregation and controlling particle size. The PEG lipid used herein can regulate the pharmacokinetic properties of the LNP. Typically, the PEG lipid comprises a lipid portion and a polymer portion (PEG portion) based on PEG (sometimes referred to as poly(ethylene oxide)). PEG lipids suitable for lipid compositions with compounds of formula (I) or (II) of the present disclosure and information on the biochemistry of such lipids can be found in Romberg et al., Pharmaceutical Research 25(1), 2008, pp. 55-71 and Hoekstra et al., Biochimica et Biophysica Acta 1660(2004)41-52. Other suitable PEG lipids are disclosed, for example, in WO 2015 / 095340 (page 31, line 14 to page 37, line 6), WO 2006 / 007712, and WO 2011 / 076807 ("Stealth Lipids"), each of which is incorporated by reference in its entirety.

[0174] In a preferred embodiment, the PEG lipid comprises a glycerol group. In a preferred embodiment, the PEG lipid comprises a dimyristoylglycerol (DMG) group. In a preferred embodiment, the PEG lipid comprises PEG-2k. In a preferred embodiment, the PEG lipid is PEG-DMG. In a preferred embodiment, the PEG lipid is PEG-2k-DMG. In a preferred embodiment, the PEG lipid is 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000. In a preferred embodiment, PEG-2k-DMG is 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000.

[0175] In some embodiments, the lipid nucleic acid assembly composition can include an RNA component, an amine lipid, a helper lipid, a neutral lipid, and a stealth lipid. In some lipid nucleic acid assembly compositions, the helper lipid is cholesterol. In other compositions, the neutral lipid is DSPC. In other embodiments, the stealth lipid is PEG2k-DMG.

[0176] In some embodiments, lipid composition is to describe according to the corresponding mol ratio of component lipid in the lipid part of preparation.In one embodiment, the mol % of amine lipid can be about 30 mol % to about 60 mol %.In one embodiment, the mol % of amine lipid can be about 40 mol % to about 60 mol %.In one embodiment, the mol % of amine lipid can be about 45 mol % to about 60 mol %.In one embodiment, the mol % of amine lipid can be about 50 mol % to about 60 mol %.In one embodiment, the mol % of amine lipid can be about 55 mol % to about 60 mol %.In one embodiment, the mol % of amine lipid can be about 50 mol % to about 55 mol %.In one embodiment, the mol % of amine lipid can be about 50 mol %.In one embodiment, the mol % of amine lipid can be about 50 mol %.In one embodiment, the mol % of amine lipid can be about 55 mol %.In some embodiments, the mol % of lipid nucleic acid assembly batch will be ± 30%, ± 25%, ± 20%, ± 15%, ± 10%, ± 5% or ± 2.5% of target mol %. In some embodiments, the amine lipid mol% of the lipid nucleic acid assembly batch will be ±4 mol%, ±3 mol%, ±2 mol%, ±1.5 mol%, ±1 mol%, ±0.5 mol%, or ±0.25 mol% of the target mol%. All mol% values ​​are given as the fraction of lipid components in the lipid nucleic acid assembly composition. In some embodiments, the batch-to-batch variability of the amine lipid mol% in the lipid nucleic acid assembly will be less than 15%, less than 10%, or less than 5%.

[0177] In one embodiment, the mole % of neutral lipids may be from about 5 mole % to about 15 mole %. In one embodiment, the mole % of neutral lipids may be from about 7 mole % to about 12 mole %. In one embodiment, the mole % of neutral lipids may be about 9 mole %. In some embodiments, the mole % of neutral lipids in a lipid nucleic acid assembly batch will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5% or ±2.5% of the target neutral lipid mole %. In some embodiments, the batch-to-batch variability of lipid nucleic acid assemblies will be less than 15%, less than 10% or less than 5%.

[0178] In one embodiment, the mol% of helper lipids may be about 20 mol% to about 60 mol%. In one embodiment, the mol% of helper lipids may be about 25 mol% to about 55 mol%. In one embodiment, the mol% of helper lipids may be about 25 mol% to about 50 mol%. In one embodiment, the mol% of helper lipids may be about 25 mol% to about 40 mol%. In one embodiment, the mol% of helper lipids may be about 30 mol% to about 50 mol%. In one embodiment, the mol% of helper lipids may be about 30 mol% to about 40 mol%. In one embodiment, the mol% of helper lipids is based on the concentration adjustment of amine lipids, neutral lipids and PEG lipids to make the lipid component reach 100 mol%. In some embodiments, the mol% of the helper lipids of the lipid nucleic acid assembly batch will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5% or ±2.5% of the target mol%. In some embodiments, the batch-to-batch variability of the lipid nucleic acid assembly will be less than 15%, less than 10% or less than 5%.

[0179] In one embodiment, the mol% of PEG lipids may be from about 1 mol% to about 10 mol%. In one embodiment, the mol% of PEG lipids may be from about 2 mol% to about 10 mol%. In one embodiment, the mol% of PEG lipids may be from about 1 mol% to about 3 mol%. In one embodiment, the mol% of PEG lipids may be from about 2 mol% to about 4 mol%. In one embodiment, the mol% of PEG lipids may be from about 1.5 mol% to about 2 mol%. In one embodiment, the mol% of PEG lipids may be from about 2.5 mol% to about 4 mol%. In one embodiment, the mol% of PEG lipids may be about 3 mol%. In one embodiment, the mol% of PEG lipids may be about 2.5 mol%. In one embodiment, the mol% of PEG lipids may be about 2 mol%. In one embodiment, the mol% of PEG lipids may be about 1.5 mol%. In some embodiments, the mol% of PEG lipids in the lipid nucleic acid assembly batch will be ±30%, ±25%, ±20%, ±15%, ±10%, ±5% or ±2.5% of the target mol% of PEG lipids. In some embodiments, the batch-to-batch variability of the lipid-nucleic acid assembly composition (eg, LNP composition) will be less than 15%, less than 10%, or less than 5%.

[0180] Embodiments of the present disclosure also provide lipid compositions described according to the molar ratio between the positively charged amine groups in the amine lipids (N) and the negatively charged phosphate groups (P) in the nucleic acid to be encapsulated. This can be mathematically represented by the formula N / P. In some embodiments, the lipid nucleic acid assembly composition may include: a lipid component comprising an amine lipid, a helper lipid, a neutral lipid, and a helper lipid; and a nucleic acid component, wherein the N / P ratio is about 3 to 10. In some embodiments, the LNP comprises an amine lipid to RNA / DNA phosphate molar ratio (N:P) of about 4.5, 5.0, 5.5, 6.0, or 6.5. In some embodiments, the lipid nucleic acid assembly composition may include: a lipid component comprising an amine lipid, a helper lipid, a neutral lipid, and a helper lipid; and an RNA component, wherein the N / P ratio is about 3 to 10. In one embodiment, the N / P ratio may be about 5-7. In one embodiment, the N / P ratio may be about 4.5-8. In one embodiment, the N / P ratio may be about 6. In one embodiment, the N / P ratio may be 6±1. In one embodiment, the N / P ratio can be about 6 ± 0.5. In some embodiments, the N / P ratio will be ± 30%, ± 25%, ± 20%, ± 15%, ± 10%, ± 5%, or ± 2.5% of the target N / P ratio. In some embodiments, the batch-to-batch variability of the lipid-nucleic acid assembly will be less than 15%, less than 10%, or less than 5%.

[0181] In some embodiments, the lipid nucleic acid assembly composition comprises lipid nanoparticles (LNPs). In some embodiments, two or more nucleic acids in the lipid nucleic acid assembly composition can be encapsulated in LNPs. LNPs can include cationic lipids and other components, such as neutral phospholipids, phosphatidylcholines, sterols (e.g., cholesterol) and / or PEGylated phospholipids. Solid lipid nanoparticles (SLNs) are made using lipids that are solid at room temperature and body temperature. They are colloidal nanoparticles with a solid lipophilic core. The solid lipid core of SLN can include triglycerides (e.g., tristearin), glyceride mixtures or partial glycerides (e.g., Imwitor), fatty acids (e.g., stearic acid or palmitic acid), steroids (e.g., cholesterol) and / or waxes (e.g., cetyl palmitate), which are solid at room temperature and body temperature. Lipid nanoemulsions (LNEs) are colloidal nanoparticles whose core is liquid at room temperature. Nanostructured lipid carriers (NLCs) include mixtures of solid and liquid lipids, such as tricaprylin, ethyl oleate, isopropyl myristate, and / or diolein.

[0182] In some embodiments, the lipid nucleic acid assembly composition includes: Cas nuclease mRNA, such as Class 2 Cas mRNA; and at least one gRNA. In some embodiments, the lipid nucleic acid assembly composition includes a gRNA to Cas nuclease mRNA (such as Class 2 Cas nuclease mRNA) ratio of about 25: 1 to about 1: 25 wt / wt. In some embodiments, the lipid nucleic acid assembly composition includes a gRNA to Cas nuclease mRNA (such as Class 2 Cas nuclease mRNA) ratio of about 10: 1 to about 1: 10. In some embodiments, the lipid nucleic acid assembly preparation includes a gRNA to Cas nuclease mRNA (such as Class 2 Cas nuclease mRNA) ratio of about 8: 1 to about 1: 8. As measured herein, the ratio is by weight. In some embodiments, the lipid nucleic acid assembly preparation includes a gRNA to Cas nuclease mRNA (such as Class 2 Cas mRNA) ratio of about 5: 1 to about 1: 5. In some embodiments, the ratio ranges from about 3:1 to 1:3, about 2:1 to 1:2, about 5:1 to 1:2, about 5:1 to 1:1, about 3:1 to 1:2, about 3:1 to 1:1, about 3:1, about 2:1 to 1:1. In some embodiments, the ratio of gRNA to mRNA is about 3:1 or about 2:1. In some embodiments, the ratio of gRNA to Cas nuclease mRNA (e.g., Class 2 Cas nuclease) is about 1:1. In some embodiments, the ratio of gRNA to Cas nuclease mRNA (e.g., Class 2 Cas nuclease) is about 1:2. In other embodiments, the ratio can be about 25:1, 10:1, 5:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:5, 1:10, or 1:25.

[0183] The lipid nucleic acid assembly composition disclosed herein may include a template nucleic acid. The template nucleic acid can be co-formulated with an mRNA encoding a polypeptide such as a Cas nuclease (e.g., a Class 2 Cas nuclease mRNA). In some embodiments, the template nucleic acid can be co-formulated with a guide RNA. In some embodiments, the template nucleic acid can be co-formulated with an mRNA encoding a polypeptide such as a Cas nuclease and a guide RNA. In some embodiments, the template nucleic acid can be separately formulated from an mRNA or guide RNA encoding a polypeptide such as a Cas nuclease. The template nucleic acid can be delivered together with the lipid nucleic acid assembly composition or separately. In some embodiments, the template nucleic acid can be single-stranded or double-stranded, depending on the desired repair mechanism. The template may have a region homologous to the target DNA or to a sequence adjacent to the target DNA.

[0184] In some embodiments, the lipid nucleic acid assembly composition includes a nucleic acid encoding a DNA base editor. In specific embodiments, the base editing enzyme includes a cytidine deaminase domain or an adenine deaminase domain. Base editors including deaminases that deaminate cytosine can be referred to as cytosine base editors. Base editors including deaminases that deaminate adenosine can be referred to as adenosine base editors.

[0185] In some embodiments, the lipid-nucleic acid assembly composition may further include an mRNA encoding a DNA glycosylase inhibitor. In some embodiments, the DNA glycosylase inhibitor is a uracil glycosylase inhibitor (UGI).

[0186] In certain exemplary embodiments, the lipid nucleic acid assembly composition can include a gRNA (e.g., a gRNA of about 80 to about 120 nucleotides or about 80 to about 130 nucleotides in length), a first mRNA encoding a DNA glycosylase inhibitor (e.g., an mRNA of about 1000 nucleotides in length), and a second mRNA encoding a cytosine base editor (e.g., an mRNA of about 4500 nucleotides in length).

[0187] In some embodiments, the lipid nucleic acid assembly is formed by mixing an RNA aqueous solution with a lipid solution based on an organic solvent (e.g., 100% ethanol). Suitable solutions or solvents include or may contain: water, PBS, Tris buffer, NaCl, citrate buffer, ethanol, chloroform, ether, cyclohexane, tetrahydrofuran, methanol, isopropanol. Pharmaceutically acceptable buffers can be used, for example, for in vivo administration of lipid nucleic acid assemblies. In some embodiments, the pH of the composition comprising the lipid nucleic acid assembly is maintained at pH 6.5 or above using a buffer. In some embodiments, the pH of the composition comprising the lipid nucleic acid assembly is maintained at pH 7.0 or above using a buffer. In some embodiments, the pH of the composition is in the range of about 7.2 to about 7.7. In other embodiments, the pH of the composition is in the range of about 7.3 to about 7.7 or in the range of about 7.4 to about 7.6. In other embodiments, the pH of the composition is about 7.2, 7.3, 7.4, 7.5, 7.6, or 7.7. The pH of the composition can be measured with a micro pH probe. In some embodiments, a cryoprotectant is included in the composition. Non-limiting examples of cryoprotectants include sucrose, trehalose, glycerol, DMSO, and ethylene glycol. Exemplary compositions may include up to 10% cryoprotectant, such as sucrose. In some embodiments, the lipid nucleic acid assembly composition may include about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% cryoprotectant. In some embodiments, the lipid nucleic acid assembly composition may include about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% sucrose. In some embodiments, the lipid nucleic acid assembly composition may include a buffer. In some embodiments, the buffer may include phosphate buffered saline (PBS), Tris buffer, citrate buffer, and mixtures thereof. In some exemplary embodiments, the buffer includes NaCl. In some embodiments, NaCl is omitted. The exemplary amount of NaCl may be in the range of about 20mM to about 45mM. The exemplary amount of NaCl may be in the range of about 40mM to about 50mM. In some embodiments, the amount of NaCl is about 45mM. In some embodiments, the buffer is a Tris buffer. An exemplary amount of Tris can be in the range of about 20mM to about 60mM. An exemplary amount of Tris can be in the range of about 40mM to about 60mM. In some embodiments, the amount of Tris is about 50mM. In some embodiments, the buffer comprises NaCl and Tris. Certain exemplary embodiments of the lipid nucleic acid assembly composition contain 5% sucrose and 45mM NaCl in a Tris buffer. In other exemplary embodiments, the composition contains sucrose in an amount of about 5% w / v, about 45mM NaCl, and about 50mM Tris (pH 7.5).The amounts of salt, buffer, and cryoprotectant can be varied to maintain the osmotic pressure of the entire formulation. For example, the final osmotic pressure can be maintained below 450 mOsm / L. In other embodiments, the osmotic pressure is between 350 and 250 mOsm / L. The final osmotic pressure of certain embodiments is 300 + / - 20 mOsm / L.

[0188] In some embodiments, microfluidic mixing, T-type mixing or cross mixing is used to prepare lipid nucleic acid assembly compositions. In some aspects, flow rate, connector size, connector geometry, connector shape, tube diameter, solution and / or RNA and lipid concentrations can vary. The lipid nucleic acid assembly or lipid nucleic acid assembly composition can be concentrated or purified, for example, by dialysis, tangential flow filtration or chromatography. The lipid nucleic acid assembly can be stored, for example, in the form of a suspension, an emulsion or a lyophilized powder. In some embodiments, the lipid nucleic acid assembly composition is stored at 2-8°C, and in some aspects, the lipid nucleic acid assembly composition is stored at room temperature. In other embodiments, the lipid nucleic acid assembly composition is stored frozen, for example, at -20°C or -80°C. In other embodiments, the lipid nucleic acid assembly composition is stored at a temperature in the range of about 0°C to about -80°C. The frozen lipid nucleic acid assembly composition can be thawed before use, for example, on ice, at 4°C, at room temperature or at 25°C. The frozen lipid-nucleic acid assembly composition can be maintained at various temperatures, for example, on ice, at 4°C, at room temperature, at 25°C, or at 37°C.

[0189] In some embodiments, the lipid nucleic acid assembly composition comprises: about 40-60 mol% amine lipids; about 5-15 mol% neutral lipids; and about 1.5-10 mol% PEG lipids, wherein the remainder of the lipid components are helper lipids, and wherein the N / P ratio of the LNP composition is about 3-10. In some embodiments, the lipid nucleic acid assembly composition comprises: about 50-60 mol% amine lipids; about 8-10 mol% neutral lipids; and about 2.5-4 mol% PEG lipids, wherein the remainder of the lipid components are helper lipids, and wherein the N / P ratio of the LNP composition is about 3-8. In some embodiments, the lipid nucleic acid assembly composition comprises: about 50-60 mol% amine lipids; about 5-15 mol% DSPC; and about 2.5-4 mol% PEG lipids, wherein the remainder of the lipid components are cholesterol, and wherein the N / P ratio of the LNP composition is 3-8±0.2.

[0190] In embodiments, the average diameter is the Z-average diameter. In certain embodiments, the Z-average diameter is measured by dynamic light scattering (DLS) using methods known in the art. For example, a Malvern Zetasizer DLS instrument can be used to measure the average particle size and polydispersity by dynamic light scattering (DLS). Before being measured by DLS, the LNP sample is diluted with PBS buffer. The Z-average diameter and the number-average diameter and the polydispersity index (PDI) can be determined. The Z-average value is the average hydrodynamic size of the intensity-weighted overall particle set. The number-average value is the average hydrodynamic size of the particle number-weighted overall particle set. The Malvern Zetasizer instrument can also be used to measure the zeta potential of the LNP using methods known in the art.

[0191] Lipid-nucleic acid assembly compositions comprising nucleic acids

[0192] The present disclosure provides, inter alia, compositions and methods for evaluating lipid-nucleic acid assembly compositions comprising nucleic acids (e.g., methods that may include isolating, quantifying, and / or characterizing one, more, or all of the nucleic acids present in the lipid-nucleic acid assembly composition). The various lipid-nucleic acid assembly composition evaluation methods provided herein can be used to isolate, quantify, and / or characterize nucleic acids present in a variety of lipid-nucleic acid assembly compositions, examples of which may include bulk drugs and pharmaceutical products.

[0193] In some embodiments, the lipid nucleic acid assembly composition may include a ribonucleic acid (RNA) molecule. In some embodiments, the RNA molecule may include a messenger RNA (mRNA) molecule or a guide RNA (gRNA). In various embodiments, the nucleic acid present in the lipid nucleic acid assembly composition may be composed of or substantially composed of mRNA and gRNA (e.g., a single mRNA and a single gRNA, or two mRNAs and a single gRNA).

[0194] In some embodiments, the mRNA molecule can encode a polypeptide, such as an RNA-guided DNA binder. In some embodiments, the RNA-guided DNA binder is a naturally occurring polypeptide. In some embodiments, the RNA-guided DNA binder is a variant of a naturally occurring polypeptide (e.g., a polypeptide having the sequence of a naturally occurring polypeptide modified by, for example, mutation, deletion, and / or insertion).

[0195] In some embodiments, the mRNA molecule can encode a nuclease (e.g., a nuclease of a gene editing system). In some embodiments, the mRNA molecule can encode a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease (e.g., a CRISPR-associated RNA-guided endonuclease).

[0196] Cas nucleases include three types of nucleases (referred to as type I, type II, and type III), and 10 subtypes, including 5 type I, 3 type II, and 2 type III proteins (see, e.g., Hochstrasser and Doudna, Trends Biochem Sci, 2015: 40(1): 58-66). Exemplary Cas nucleases include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 or Csx12; including, e.g., spCas9, dCas9, nCas9, and Cas9-SpRY), Cas10, Cas12 (e.g., Cas12a (e.g., LbCas12a, AsCas12a, FnCas12a, MB3Cas12a, Cas12a-M 11. Cas12a-M13 (e.g., Cas12a-M13-1), Cas12a-M26 (e.g., Cas12a-M26-1), Cas12a-M28 (e.g., Cas12a-M28-1), Cas12a-M29 (e.g., Cas12a-M29-1), Cas12a-M30 (e.g., Cas12a-M30-1), Cas12a-M31 (e.g., Cas12a-M31-1), Cas12a-M32 (e.g., Cas12a-M32-1), Cas12a-M57, Cas12a-M58, Cas12a-M59, Cas12a-M60 (e.g., Cas12a-M60-9), Cas12a-M61 or Cas12a-M62), Cas12b, Cas12c, Cas12g, Cas12h or Cas12i), Cas-Phi, CasX, CasY, Cpf1, C2c3, C2c2, C2c1, Csy1, Cs Csx1, Csx2, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and variants thereof. These nucleases are known to those skilled in the art.

[0197] In specific embodiments, Cas9 refers to an RNA-guided double-stranded DNA binding nuclease protein or nickase protein. The wild-type Cas9 nuclease has two functional domains, such as RuvC and HNH, which can cut different DNA strands. When both functional domains are active, Cas9 can induce double-strand breaks in genomic DNA (target DNA). In some embodiments, the Cas9 enzyme includes one or more catalytic domains of a Cas9 protein derived from bacteria, such as Corynebacter, Sutterella, Legionella, Treponema, Filif actor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseri, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, and Campylobacter. mpylobacter). In some embodiments, Cas9 is a fusion protein, for example, the two catalytic domains are derived from different bacterial species. In various embodiments, variants of the Cas9 nuclease include a single inactive catalytic domain, such as a RuvC" or HNH" enzyme or a nickase. The Cas9 nickase has only one active functional domain and, in some embodiments, only cuts one strand of the target DNA, thereby generating a single-strand break or nick. Exemplary Cas9 amino acid sequences include, for example, the wild-type Cas9 polypeptide of Streptococcus pyogenes shown in NCBI Accession No. NP_269215, and the wild-type Cas9 polypeptide of Streptococcus thermophilus shown in NCBI Accession No. WP_011681470.

[0198] In some embodiments, an mRNA molecule can encode a fusion polypeptide. In some embodiments, an mRNA molecule can encode a fusion polypeptide that is an RNA-guided DNA binder and includes a heterologous polypeptide. In some embodiments, an mRNA molecule can encode a fusion polypeptide that includes an RNA-guided nuclease and a heterologous polypeptide. Various such fusion polypeptides, including site-directed polypeptides, are disclosed in WO 2013 / 176772, which is incorporated herein by reference in its entirety regarding fusion partners and fusion polypeptides.

[0199] In various embodiments, the mRNA molecule can encode a fusion polypeptide that is a site-directed polypeptide comprising two portions: an RNA-binding portion and an active portion. In some embodiments, the site-directed polypeptide comprises: (i) an RNA-binding portion that interacts with a DNA-targeting RNA, wherein the DNA-targeting RNA comprises a nucleotide sequence that is complementary to a sequence in the target DNA; and (ii) an active portion that exhibits a site-directed enzymatic activity (e.g., activity for DNA methylation, activity for DNA cleavage, activity for histone acetylation, activity for histone methylation, etc.), wherein the site of enzymatic activity is determined by the DNA-targeting RNA. In some embodiments, the RNA-binding portion (i) further has an activity, such as nickase activity.

[0200] In some embodiments, the site-directed polypeptide comprises: (i) an RNA-binding portion that interacts with a DNA-targeting RNA, wherein the DNA-targeting RNA comprises a nucleotide sequence that is complementary to a sequence in the target DNA; and (ii) an activity portion that modulates transcription (e.g., increases or decreases transcription) within the target DNA, wherein the site within the target DNA at which transcription is modulated is determined by the DNA-targeting RNA.

[0201] In some embodiments, the site-directed polypeptide has an enzymatic activity that modifies the target DNA (e.g., nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer-forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, or glycosylase activity).

[0202] In some embodiments, the site-directed protein can have an enzymatic activity that modifies a polypeptide (e.g., a histone) associated with the target DNA (e.g., methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristoylation activity, or demyristoylation activity).

[0203] In some embodiments, the heterologous sequence of the fusion polypeptide can provide subcellular localization of the site-directed modifying polypeptide (e.g., a nuclear localization signal (NLS) for targeting to the nucleus; a mitochondrial localization signal for targeting to mitochondria; a chloroplast localization signal for targeting to chloroplasts; an ER retention signal, etc.).

[0204] In some embodiments, the heterologous sequence of the fusion polypeptide can provide (e.g., directly provide) increased transcription of the target nucleic acid (e.g., a transcriptional activator or fragment thereof; a protein or fragment thereof that recruits a transcriptional activator; a small molecule / drug-responsive transcriptional regulator, etc.).

[0205] In some embodiments, the mRNA may encode a base editor, including a deaminase associated with a DNA binding domain, such as a catalytically impaired nuclease domain (e.g., a nickase, e.g., a catalytically impaired CRISPR / Cas). Exemplary Cas nucleases are provided herein. Specific embodiments utilize Cas9 (dCas9) without nuclease activity as a catalytically inactivated nuclease. For example, in various embodiments, a cytosine base editor may include an Nme2Cas9 (D16A) nickase fused to an APOBEC3A deaminase, and in various embodiments, does not include a DNA glycosylase inhibitor. However, any nuclease of the CRISPR system can be engineered to produce a catalytically impaired nuclease domain (e.g., a nickase) for use in base editors including engineered nucleases and deaminases.

[0206] Cytidine deaminases encompass enzymes in the cytidine deaminase superfamily, particularly enzymes in the APOBEC family (APOBEC1, APOBEC2, APOBEC4, and APOBEC3 enzyme subgroups), activation-induced cytidine deaminases (AID or AICDA), and CMP deaminases (see, e.g., Conticello et al., Mol. Biol. Evol. 22:367-77, 2005; Conticello, Genome Biol. 9:229, 2008; Muramatsu et al., J. Biol. Chem. 274:18470-6, (1999); and Carrington et al., Cells 9:1690 (2020)). For more information on deaminases and base editors, see PCT Publication No. WO2022 / 125968, which is incorporated herein by reference in its entirety.

[0207] In some embodiments, the cytidine deaminase disclosed herein is an enzyme of the APOBEC family. In some embodiments, the cytidine deaminase disclosed herein is an enzyme of the APOBEC 1, APOBEC2, APOBEC4, and APOBEC3 subgroups. In some embodiments, the cytidine deaminase disclosed herein is an enzyme of the APOBEC3 subgroup. In some embodiments, the cytidine deaminase disclosed herein is an APOBEC3A deaminase (A3A).

[0208] In some embodiments, the APOBEC3A deaminase (A3A) disclosed herein is human A3A. In some embodiments, A3A is wild-type A3A. In some embodiments, wild-type A3A is human A3A (UniPROT Accession No.: p319411). In some embodiments, A3A is an A3A variant. A3A variants share homology with wild-type A3A or a fragment thereof. In some embodiments, A3A variants have at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, or at least about 99.9% identity with wild-type A3A. In some embodiments, the A3A variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to wild-type A3A.

[0209] Exemplary adenosine deaminases can include mutant TadA adenosine deaminases (TadA*) that accept DNA as a substrate. E. coli TadA typically acts as a homodimer to deaminize adenosine in transfer RNA (tRNA). TadA* deaminase catalyzes the conversion of target "A" to "I" (inosine), which is processed by cellular polymerases to "G". Subsequently, the original genomic AT base pair can be converted to a GC pair. In some embodiments, the ABE can include a wild-type E. coli tRNA-specific adenosine deaminase (TadA) monomer and a TadA* mutant TadA monomer that catalyzes the deamination of deoxyadenosine. In certain embodiments, there is a linker between TadA and TadA*, and in certain embodiments, a linker is placed between TadA* and a DNA binding domain (e.g., a Cas nickase).

[0210] Other non-limiting examples of base editors include BE1 (APOBEC1-16 amino acid (aa) linker-Sp dCas9 (D10A, H840A) (see, e.g., Komor 2016 Nature 533:420-424)), BE2 (APOBEC1-16aa linker-SpdCas9 (D10A, H840A)-4aa linker-UGI (see, e.g., Komor 2016 Nature 533:420–424)), BE3 (APOBEC1-16aa linker-SpnCas9 (D10A)-4aa linker-UGI (see, e.g., Komor 2016 Nature 533:420–424)), HF-BE2 (rAPOBEC1-HF2 nCas9-UGI), HF-BE3 (APOBEC1-16aa linker-HF nCas9(D10A)-4aa linker-UGI (see, e.g., Rees 2017 Nat. Commun. 8:15790)), BE4 (rAPOBEC1-Sp nCas9-UGI-UGI), BE4max (APOBEC1-32aa linker-Sp nCas9(D10A)-9aa linker-UGI-9aa linker-UGI (see, e.g., Koblan 2018 Nat. Biotechnol 36(9):843-846 and / or Komor 2017 Sci. Adv. 3(8):eaao4774)), BE4-GAM (Gam-16aa linker-APOBEC1-32aa linker-Sp nCas9(D10A)-9aa linker-UGI-9aa linker-UGI (see, e.g., Komor 2017 Sci. Adv. 3(8):eaao4774)), YE1-BE3 (APOBEC1(W90Y, R126E)-16aa linker-Sp nCas9(D10A)-4aa linker-UGI (see, e.g., Kim 2017 Nat. Biotechnol. 35:475–480)), EE-BE3 (APOBEC1(R126E, R132E)-16aa linker-Sp nCas9(D10A)-4aa linker-UGI (see, e.g., Kim 2017 Nat. Biotechnol. 35:475–480)), YE2-BE3 (APOBEC1(W90Y, R132E)-16aa linker-Sp nCas9(D10A)-4aa linker-UGI (see, e.g., Kim 2017Nat.Biotechnol.35:475–480)), YEE-BE3 (APOBEC1 (W90Y, R126E, R132E)-16aa linker-Sp nCas9 (D10A)-4aa linker-UGI (see, e.g., Kim 2017 Nat. Biotechnol. 35:475–480)), VQR-BE3 (APOBEC1-16aa linker-Sp VQRnC as9 (D10A)-4aa linker-UGI (see, e.g., Kim 2017 Nat. Biotechnol. 35:475–480)), EQR-BE3 (rAPOBEC1-EQR SpnCas9-UGI), VRER-BE3 (APOBEC1-16aa linker-Sp VRER nCas9 (D10A)-4aa linker-UGI (see, e.g., Kim 2017 Nat. 2017 Nat. Biotechnol. 35: 475–480)), Sa-BE3 (APOBEC1-16aa linker-Sa nCas9(D10A)-4aa linker-UGI (see, e.g., Kim 2017 Nat. Biotechnol. 35: 475–480)), SA-BE4 (APOBEC1-32aa linker-Sa nCas9(D10A)-9aa linker-UGI-9aa linker-UGI (see, e.g., Komor 2017 Sci. Adv. 3(8): eaao4774)), SaBE4-Gam (Gam-16aa linker-APOBEC1-32aa linker-Sa nCas9(D10A)-9aa linker-UGI-9aa linker-UGI (see, e.g., Komor 2017 Sci. Adv. 3(8): eaao4774)), SaBE4-Gam (Gam-16aa linker-APOBEC1-32aa linker-Sa nCas9(D10A)-9aa linker-UGI-9aa linker-UGI (see, e.g., Komor 2017 Sci. Adv. 3(8): eaao4774)). 2017 Sci. Adv. 3(8):eaao4774)), SaKKH-BE3 (APOBEC1-16aa linker-Sa KKH nCas9(D10A)-4aa linker-UGI (see, e.g., Kim 2017 Nat. Biotechnol. 35:475–480)), FNLS-BE3 (rAPOBEC1-Sp nCas9-UGI), RA-BE3 (rAPOBEC1(RA)-Sp nCas9-UGI), Cas12a-BE (APOBEC1-16aa linker-dCas12a-14aa linker-UGI (see, e.g., Li 2018 Nat. Biotechno l.36:324–327)), Target-AID (Sp nCas9 (D10A) -100aa linker -CDA1-9aa linker -UGI (see, e.g., Nishida 2016 Science 353(6305):aaf8729)), Target-AID-NG (Sp nCas9 (D10A) -NG -100aa linker -CDA1-9aa linker -UGI (see, e.g., Nishimasu 2018 Science 361(6408):1259–1262)), xBE 3 (APOBEC1-16aa linker -xCas9 (D10A) -4aa linker -UGI (see, e.g., Hu 2018 Nature 556:57–63)), eA3A-BE3 (APOBEC3A(N37G)-16aa linker-SpnCas9(D10A)-4aa linker-UGI (see, e.g., Gehrke 2018 Nat. Biotechnol. 36(10):977-982)), A3A-BE3 (hAPOBEC3A-16aa linker-Sp nCas9(D10A)-4aa linker-UGI (see, e.g., Wang 2018 Nat. Biotechnol. 36:946–949)), eA3A-HF1-BE3-2xUGI (APOBEC3A-HF1 Sp nCas9-UGI-UGI), eA3A-HypaBE3-2xUGI (APOBEC3A-Hypa Sp nCas9-UGI-UGI), hA3A-BE3 (hAPOBEC3A-SpnCas9-UGI), hA3B-BE3 (hAPOB EC3B-Sp nCas9-UGI), hA3G-BE3 (hAPOBEC3G-Sp nCas9-UGI), hAID-BE3 (hAPOBEC3A-Sp nCas9-UGI), SaCas9-BE3 (rAPOBEC1-SanCas9-UGI), xCas9-BE3 (rAPOBEC1-xnCas9-UGI), ScCas9-BE3 (rAPOBEC1-ScnCas9-UGI), Sn iperCas9-BE3(rAPOBEC1-SnipernCas9-UGI), iSpyMac-BE3(rAPOBEC1-iSpyMacnCas9-UGI), CRISPR-X(SpdCas9-MS2-hAID), TAM(Sp dCas9-hAID(P182X)), AncBE4-Max(rAPOBEC1-Sp nCas9-UGI-UGI), ABE7.8 / 9 / 10(ecTadA-ecTa dA*-Sp nCas9), xCas9-ABE7.10(ecTadA-ecTadA*-nxCas9), VQR-ABE(ecTadA-ecTadA*-Sp VQR nCas9), Sa(KKH)-ABE(ecTadA-ecT adA*-SaKKH nCas9), ABEmax(ecTadA-ecTadA*-Sp nCas9), ABE 7.10max(ecTadA-ecTadA*-SpnCas9), ABE8e(ecTadA-ecTadA*-Spn Cas9), PE1(dSpCas9-MMLV-RT), PE2(dSpCas9-MMLV-RT), PE3(nSpCas9-MMLV-RT) and BE-PLUS(10X GCN4-Sp nCas9(D10A) / ScFv-rAPOBEC1-UGI ​​(see, e.g., Jiang 2018 Cell Res. 28(8):855-861). .

[0211] In some embodiments, the lipid nucleic acid assembly includes an mRNA encoding and / or expressing a uracil DNA glycosylase inhibitor (UGI), for example, wherein the lipid nucleic acid assembly further includes an mRNA encoding a cytosine base editor (e.g., wherein the base editor does not include a DNA glycosylase inhibitor). The DNA glycosylase inhibitor can go beyond the natural DNA repair mechanism, it may repair the predetermined base editing in other ways, reduce the generation of insertions / deletions (indels), and / or be able to deaminate the target nucleic acid more efficiently than the base editing in the absence of UGI. The DNA glycosylase inhibitor can be a uracil DNA glycosylase inhibitory protein (UGI). Wang (1991Gene 99:31-37) describes an exemplary UGI.

[0212] The mRNA may include one or more of the following: a 5' cap, a 5' untranslated region (UTR), a 3' UTR, and a polyadenine tail. The mRNA may include a modified open reading frame, for example to encode a nuclear localization sequence or to use alternative codons to encode a protein.

[0213] In addition, suitable modification includes the change of one or more nucleotides in the codon, so that the codon encodes the same amino acid, but more stable than the codon seen in wild-type mRNA. For example, it has been shown that there is an inverse relationship between the stability of RNA and a large number of cytidine (C) and / or uridine (U) residues, and it is found that RNA lacking C and U residues is stable to most RNases (Heidenreich et al., J Biol Chem 269, 2131-8 (1994)). In some embodiments, the number of C and / or U residues in the mRNA sequence is reduced. In another embodiment, the number of C and / or U residues is reduced by replacing another codon encoding the same or related amino acids with a codon encoding a specific amino acid. The modification of mRNA nucleic acid considered also includes incorporating modified uridine, such as 5-methoxyuridine, pseudouridine or N1-methyl pseudouridine, etc. Pseudouridine is incorporated into mRNA nucleic acid to enhance stability and translation ability, and reduce immunogenicity in vivo. See, for example, Karikó, K. et al., Molecular Therapy 16(11): 1833-1840 (2008). Substitution and modification of mRNA can be performed by methods readily apparent to those skilled in the art.

[0214] The constraints on reducing the number of C and U residues in the sequence may be greater within the coding region of an mRNA than in the untranslated regions (i.e., it may not be possible to eliminate all C and U residues present in the message while still maintaining the ability of the message to encode the desired amino acid sequence). However, the degeneracy of the genetic code provides an opportunity to reduce the number of C and / or U residues present in the sequence while maintaining the same coding capacity (i.e., there may be several different possibilities for modifying the RNA sequence depending on the amino acid encoded by the codon).

[0215] The term modification also includes, for example, the incorporation of non-nucleotide linkages or modified nucleotides into the mRNA sequence (e.g., modifications to one or both of the 3' and 5' ends of an mRNA molecule encoding a functional secreted protein or enzyme). These modifications include the addition of bases to the mRNA sequence (e.g., the inclusion of a poly A tail or a longer poly A tail); alterations to the 3'UTR or 5'UTR; complexing the mRNA with an agent (e.g., a protein or a complementary nucleic acid molecule); and the inclusion of elements that alter the structure of the mRNA molecule (e.g., the formation of a secondary structure).

[0216] The poly A tail is believed to stabilize natural messengers. Therefore, a longer poly A tail can be added to an mRNA molecule, thereby making the mRNA more stable. A variety of techniques recognized in the art can be used to add the Poly A tail. For example, a long poly A tail can be added to a synthetic or in vitro transcribed mRNA using poly A polymerase (Yokoe et al., Nature Biotechnology 1996; 14: 1252-1256). The transcription vector can also encode a long poly A tail. In addition, the poly A tail can be added by directly transcribing from a PCR product. In some embodiments, the length of the poly A tail is at least about 90, 200, 300, 400, or at least 500 nucleotides. In certain embodiments, the length of the poly A tail is adjusted to control the stability of the modified mRNA A molecule, and thus control the transcription of the protein. For example, because the length of the poly A tail can affect the half-life of the mRNA molecule, the length of the poly A tail can be adjusted to change the resistance level of the mRNA to nucleases, and thus control the time course of protein expression in the cell. In some embodiments, the stabilized mRNA molecules are sufficiently resistant to degradation in vivo (eg, by nucleases) such that they can be delivered to target cells without a transfer vehicle.

[0217] In certain embodiments, mRNA can be modified by incorporating 3' and / or 5' untranslated (UTR) sequences that are not naturally present in wild-type mRNA. In some embodiments, 3' and / or 5' flanking sequences of natural flanking mRNA and encoding a second unrelated protein can be incorporated into the nucleotide sequence of the mRNA molecule encoding therapeutic or functional protein to modify it. For example, 3' or 5' sequences of stable mRNA molecules (such as globulin, actin, GAPDH, tubulin, histone or citric acid cycle enzymes) can be incorporated into 3' and / or 5' regions of sense mRNA nucleic acid molecules to increase the stability of sense mRNA molecules. See, for example, US2003 / 0083272.

[0218] A more detailed description of mRNA modification can be found in US 2017 / 0210698 A1, pages 57-68, the contents of which are incorporated herein.

[0219] In some embodiments, the RNA molecule can be or include a guide RNA (gRNA). In some embodiments, the RNA molecule (e.g., gRNA) can be or include a CRISPR RNA (crRNA). In various embodiments, crRNA can target a site in the genome based on complementarity. In some embodiments, the RNA molecule (e.g., gRNA) can be or include a transactivated CRISPR RNA (tracrRNA), which can associate with an endonuclease. In some embodiments, the gRNA includes crRNA and tracrRNA. The gRNA comprising crRNA and tracrRNA at the same time can be referred to as a single gRNA (sgRNA). sgRNA can target a nuclease (e.g., Cas nuclease) to a desired sequence. Therefore, the CRISPR / Cas system can be engineered to produce double-strand breaks at the desired target site in the cell genome and utilize the cell's endogenous mechanisms to repair the breaks induced by HDR or NHEJ.

[0220] In certain embodiments, nucleic acid molecules of the present disclosure may include modified nucleosides or nucleotides. Modified nucleosides or nucleotides may be present in RNA, such as gRNA or mRNA. For example, a gRNA or mRNA comprising one or more modified nucleosides or nucleotides is referred to as a "modified" RNA to describe the presence of one or more non-natural and / or naturally occurring components or configurations to replace or supplement typical A, G, C, and U residues. In some embodiments, the modified RNA is synthesized with atypical nucleosides or nucleotides, referred to herein as "modified."

[0221] Modified nucleosides and nucleotides may include one or more of the following: (i) alteration, such as replacement of one or two non-linked phosphate oxygens and / or one or more linked phosphate oxygens in a phosphodiester backbone linkage (exemplary backbone modifications); (ii) alteration, such as replacement of a component of the ribose sugar, such as the 2' hydroxyl group on the ribose sugar (exemplary sugar modifications); (iii) replacement of the phosphate moiety with a "dephospho" linker as a whole (exemplary backbone modifications); (iv) modification or replacement of naturally occurring nucleobases, including modification or replacement with atypical nucleobases (exemplary base modifications); (v) replacement or modification of the ribose-phosphate backbone (exemplary backbone modifications); (vi) modification of the 3' or 5' end of a polynucleotide, such as removal, modification, or replacement of a terminal phosphate group or conjugation of a moiety, cap, or linker (e.g., a 3' or 5' cap modification may comprise a sugar and / or backbone modification); and (vii) modification or replacement of a sugar (exemplary sugar modifications). Certain embodiments include 5' end modifications to an mRNA, gRNA, or nucleic acid. Certain embodiments include modifications to an mRNA, gRNA, or nucleic acid. Certain embodiments include 3' end modifications to mRNA, gRNA or nucleic acid. The modified RNA may contain 5' and 3' end modifications. The modified RNA may contain one or more modified residues at non-terminal positions. In certain embodiments, the gRNA includes at least one modified residue. In certain embodiments, the mRNA includes at least one modified residue. In certain embodiments, the modified gRNA includes a modification at one or more of the first five nucleotides at the 5' end. In certain embodiments, the modified gRNA includes a modification at one or more of the first five nucleotides at the 3' end.

[0222] Unmodified nucleic acids may tend to be degraded by those enzymes such as those seen in intracellular nucleases or serum. For example, nucleases can hydrolyze nucleic acid phosphodiester bonds. Therefore, on the one hand, RNA described herein (e.g., mRNA, gRNA) can contain one or more modified nucleosides or nucleotides, for example to introduce stability for intracellular or serum-based nucleases. In some embodiments, the modified RNA molecules described herein can show a reduced innate immune response when introduced into a cell population (whether in vivo or in vitro). The term "innate immune response" includes cellular responses to exogenous nucleic acids (including single-stranded nucleic acids), which relate to the expression and release (particularly interferon) and cell death of induced cytokines.

[0223] Therefore, in some embodiments, RNA or nucleic acid comprises at least one modification, and it confers the stability that increases or strengthens nucleic acid, including, for example, improved resistance to in vivo nuclease digestion.As used herein, the term "modification" and "modified" as terms related to nucleic acid provided herein include at least one change, and the change preferably strengthens stability and makes RNA or nucleic acid more stable (for example, resistant to nuclease digestion) than wild-type or naturally occurring form of RNA or nucleic acid.As used herein, the term "stable" and "stability" as terms related to nucleic acid described herein and particularly with respect to RNA, refer to resistance to increase or strengthen degradation by, for example, nucleases (that is, endonucleases or exonucleases) that are usually capable of degrading such RNA. Increased stability can include, for example, lower sensitivity to hydrolysis or other destruction of endogenous enzymes (for example, endonucleases or exonucleases) or conditions in target cells or tissues, thereby increasing or strengthening the stay of such RNA or nucleic acid in target cells, tissues, subjects and / or cytoplasm. Stabilized RNA molecules or nucleic acids provided herein exhibit longer half-lives relative to their naturally occurring unmodified counterparts (for example, wild-type forms of mRNA). As terms related to the mRNA of the LNP compositions disclosed herein, the terms "modification" and "modified" also encompass changes that improve or enhance translation of the mRNA nucleic acid, including, for example, the inclusion of sequences that play a role in the initiation of protein translation (e.g., Kozak consensus sequence). (Kozak, M., Nucleic Acids Res 15(20):8125-48 (1987)).

[0224] In some embodiments, the RNA or nucleic acid has been chemically or biologically modified to make it more stable. Exemplary modifications to RNA or nucleic acids include deletions of bases (e.g., by deletion or by substituting one nucleotide for another) or modifications of bases, such as chemical modifications of bases. As used herein, the phrase "chemical modification" includes modifications that introduce chemical properties different from those found in naturally occurring RNA or nucleic acids, such as covalent modifications, such as the introduction of modified nucleotides (e.g., nucleotide analogs, or side groups that are included in such RNAs, e.g., deoxynucleosides or nucleic acid molecules).

[0225] In some embodiments of backbone modifications, the phosphate groups in the modified residues can be modified by replacing one or more oxygens with different substituents. In addition, modified residues, such as those present in modified nucleic acids, can include the wholesale replacement of unmodified phosphate moieties with modified phosphate groups as described herein. In some embodiments, backbone modifications of the phosphate backbone can include changes that produce uncharged linkers or charged linkers with asymmetric charge distributions.

[0226] The example of the phosphate group of modification comprises thiophosphate, selenophosphate, borane phosphate, hydrogen phosphonate, phosphoramidate, alkyl or aryl phosphonate and phosphotriester.The phosphorus atom in the unmodified phosphate group is achiral.However, replacing a non-bridging oxygen with one of the above-mentioned atoms or atomic groups can make the phosphorus atom have chirality.The stereoisomerization source phosphorus atom can have " R " configuration (referred to as Rp herein) or " S " configuration (referred to as Sp herein).The main chain can also be modified by replacing the oxygen (that is, the oxygen connecting phosphate and nucleoside) of bridging with nitrogen (phosphoramidate of bridging), sulfur (phosphorothioate of bridging) and carbon (methylene phosphonate of bridging).Replacement can occur on any connection oxygen or two connection oxygens.In some main chain modifications, phosphate group can be replaced by the linker without phosphorus.In some embodiments, charged phosphate group can be replaced by neutral part. Examples of moieties that can replace the phosphate group can include, but are not limited to, for example, methylphosphonate, hydroxyamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformal, methylal, oxime, methyleneimino, methylenemethylimino, methylenehydrazinyl, methylenedimethylhydrazinyl, and methyleneoxymethylimino.

[0227] In various embodiments, the mRNA nucleotide sequence encoding a polypeptide (e.g., an RNA-guided DNA binder, an endonuclease, a base editor, and / or a DNA glycosylase inhibitor (e.g., UGI)) comprises a number of about 1000 to about 7000 nucleotides. In various embodiments, the mRNA nucleotide sequence encoding the polypeptide comprises a number of nucleotides of or greater than about 1000, about 1050, about 1100, about 1150, about 1200, about 1250, about 1300, about 1350, about 1400, about 1450, about 1500, about 1550, about 1600, about 1650, about 1700, about 1750, about 1800, about 1850, about 1900, about 1950, about 2000, about 2050, about 2100, about 2150, about 2200, about 2250 , about 2300, about 2350, about 2400, about 2450, about 2500, about 2550, about 2600, about 2650, about 2700, about 2750, about 2800, about 2850, about 2900, about 2950, ​​about 3000, about 3050, about 3100, about 3150, about 3200, about 3250, about 3300, about 3350, about 3400, about 3450, about 3500, about 3550, about 3600, about 3650, about 3700, about 3750, about 3800, about 38 50, about 3900, about 3950, about 4000, about 4050, about 4100, about 4150, about 4200, about 4250, about 4300, about 4350, about 4400, about 4450, about 4500, about 4550, about 4600, about 4650, about 4700, about 4750, about 4800, about 4850, about 4900, about 4950, about 5000, about 5050, about 5100, about 5150, about 5200, about 5250, about 5300, about 5350, about 5400, about about 6500, about 6600, about 6650, about 6700, about 6750, about 6800, about 6850, about 6900, about 6950, about 7000, about 7050, about 7100, about 7150, about 7200, about 7250, about 7300, about 7350, about 7400, about 7450, about 7500, about 7550, about 7600, about 7650, about 7700, about 7750, about 7800, about 7850, about 7900, about 7950, or about 7000.

[0228] In various embodiments, the mRNA nucleotide sequence encoding the polypeptide includes a number of nucleotides between about 500 and about 7000, between about 1000 and about 6000, between about 1000 and about 5000, between about 1000 and about 4000, between about 1000 and about 3000, or between about 1000 and about 2000. In various embodiments, the mRNA nucleotide sequence encoding the polypeptide includes a number of nucleotides between about 2000 and about 7000, between about 2000 and about 6000, between about 2000 and about 5000, between about 2000 and about 4000, or between about 2000 and about 3000. In various embodiments, the number of nucleotides included in the mRNA nucleotide sequence encoding the polypeptide is between about 3000 and about 7000, between about 3000 and about 6000, between about 3000 and about 5000, or between about 3000 and about 4000. In various embodiments, the number of nucleotides included in the mRNA nucleotide sequence encoding the polypeptide is between about 4000 and about 7000, between about 4000 and about 6000, or between about 4000 and about 5000. In various embodiments, the number of nucleotides included in the mRNA nucleotide sequence encoding the polypeptide is between about 5000 and about 7000, or between about 5000 and about 6000. In various embodiments, the number of nucleotides included in the mRNA nucleotide sequence encoding the polypeptide is between about 500 and about 1000, or between about 500 and about 2000. In some embodiments, the gRNA comprises a number of nucleotides between about 50 and about 200, between about 75 and about 200, between about 50 and about 150, between about 75 and about 150, between about 80 and about 150, between about 50 and about 140, between about 75 and about 140, between about 80 and about 140, between about 75 and about 120, between about 80 and about 130, or between about 80 and about 120. In some embodiments, the gRNA comprises a number of nucleotides between about 85 and about 115, between about 90 and about 110, or between about 95 and 105.In various embodiments, the gRNA is or includes 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 147, 148, 149, or 150 nucleotides.

[0229] In various embodiments, the mRNA nucleotide sequence encoding the base editor includes between about 3,000 and about 7,000 nucleotides. In various embodiments, the number of nucleotides included in the mRNA nucleotide sequence encoding the base editor is or is greater than about 3000, about 3050, about 3100, about 3150, about 3200, about 3250, about 3300, about 3350, about 3400, about 3450, about 3500, about 3550, about 3600, about 3650, about 3700, about 3750, about 3800, about 3850, about 3900, about 3950, about 4000, about 4050, about 4100, about 4150, about 4200, about 4250, about 4300, about 4350, about 4400, about 4450, about 4500, about 4550, about 4600, about 4650, about 4700, about 4750, about 4800, about 4850, about 4900, about 4950, about 5000, about 5050, about 5100, about 5150, about 5200, about 5250, about 5300, about 5350, about 5400, about 5450, about 5500, about 5550, about 5600, about 5650, about 5700, about 5750, about 5800, about 5850, about 5900, about 5950, about 6000, about 6050, about 6100, about 6150, about 6200, about 6250, about 6300, about 6350, about 6400, about 6450, about 6500, about 6550, about 6600, about 6650, about 6700, about 6750, about 6800, about 6850, about 6900, about 6950 or about 7000.

[0230] In various embodiments, the mRNA nucleotide sequence encoding the base editor includes a number of nucleotides between about 3000 and about 7000, between about 3000 and about 6500, between about 3000 and about 6000, between about 3000 and about 5500, between about 3000 and about 5000, between about 3000 and about 4500, or between about 3000 and about 4000. In various embodiments, the mRNA nucleotide sequence encoding the base editor includes a number of nucleotides between about 4000 and about 7000, between 4000 and about 6500, between about 4000 and about 6000, between 4000 and about 5500, or between about 4000 and about 5000.

[0231] In various embodiments, the number of nucleotides included in the mRNA nucleotide sequence encoding the DNA glycosylation inhibitor is between about 500 and about 3000. In various embodiments, the number of nucleotides included in the mRNA nucleotide sequence encoding the DNA glycosylase inhibitor is or is greater than about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, about 1000, about 1050, about 1100, about 1150, about 1200, about 1250, about 1300, about 1350, about 1400, about 1450, about 1500, about 1550, about 1600, about 1700, about 1750, about 1800, about 1850, about 1900, about 1950, about 2000, about 2100, about 2200, about 2300, about 2400, about 2500, about 2600, about 2700, about 2800, about 2900, about 3000, about 3100, about 3200, about 3300, about 3400, about 3500, about 3600, about 3700, about 3800, about 3900, about 4000, about 4100, about 4200, about 4300, about 4400 about 2700, about 2750, about 2800, about 2850, about 2900, about 2950, ​​or about 3000.

[0232] In various embodiments, the mRNA nucleotide sequence encoding the DNA glycosylase inhibitor includes nucleotides comprising between about 500 and about 3000, between about 500 and about 2500, between about 500 and about 2000, between about 500 and about 1500, between about 500 and about 1000, between about 750 and about 3000, between about 750 and about 2500, between about 750 and about 2000, between about 750 and about 1500, between about 750 and about 1000, between about 1000 and about 3000, between about 1000 and about 2500, between about 1000 and about 2000, or between about 1000 and about 1500.

[0233] In some embodiments, the gRNA comprises a number of nucleotides between about 50 and about 200, between about 75 and about 200, between about 50 and about 150, between about 75 and about 150, between about 80 and about 150, between about 50 and about 140, between about 75 and about 140, between about 80 and about 140, between about 75 and about 120, between about 80 and about 130, or between about 80 and about 120. In some embodiments, the gRNA comprises a number of nucleotides between about 85 and about 115, between about 90 and about 110, or between about 95 and 105. In various embodiments, the gRNA is or includes 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 147, 148, 149, or 150 nucleotides.

[0234] In various embodiments, the number of nucleotides included in a nucleic acid (e.g., a first nucleic acid) is between about 1000 and about 7000. In various embodiments, the number of nucleotides included in a nucleic acid (e.g., a first nucleic acid) is or is greater than about 1000, about 1050, about 1100, about 1150, about 1200, about 1250, about 1300, about 1350, about 1400, about 1450, about 1500, about 1550, about 1600, about 1650, about 1700, about 1750, about 1800, about 1850, about 1900, about 1950, about 2000, about 2050, about 2100, about 2150, about 2200, about 2250, about 2300, about 2350, about 2400, about 2450, about 2500, about 2550, about 2600, about 2650, about 2700, about 2750, about 2800, about 2850, about 2900, about 2950, ​​about 3000, about 3050, about 3100, about 3150, about 3200, about 3250, about 3300, about 3350, about 3400, about 3450, about 3500, about 3550, about 3600, about 3650, about 3700, about 3750, about 3800, about 3850 , about 3900, about 3950, about 4000, about 4050, about 4100, about 4150, about 4200, about 4250, about 4300, about 4350, about 4400, about 4450, about 4500, about 4550, about 4600, about 4650, about 4700, about 4750, about 4800, about 4850, about 4900, about 4950, about 5000, about 5050, about 5100, about 5150, about 5200, about 5250, about 5300, about 5350, about 5400, about 55 about 6500, about 6550, about 6600, about 6650, about 6700, about 6750, about 6800, about 6850, about 6900, about 6950, about 7000, about 7050, about 7100, about 7150, about 7200, about 7250, about 7300, about 7350, about 7400, about 7450, about 7500, about 7550, about 7600, about 7650, about 7700, about 7750, about 7800, about 7850, about 7900, about 7950, or about 8000.

[0235] In various embodiments, the nucleic acid (e.g., first nucleic acid) comprises a number of nucleotides between about 500 and about 7000, between about 1000 and about 6000, between about 1000 and about 5000, between about 1000 and about 4000, between about 1000 and about 3000, or between about 1000 and about 2000. In various embodiments, the nucleic acid (e.g., first nucleic acid) comprises a number of nucleotides between about 2000 and about 7000, between about 2000 and about 6000, between about 2000 and about 5000, between about 2000 and about 4000, or between about 2000 and about 3000. In various embodiments, the number of nucleotides included in a nucleic acid (e.g., a first nucleic acid) is between about 3000 and about 7000, between about 3000 and about 6000, between about 3000 and about 5000, or between about 3000 and about 4000. In various embodiments, the number of nucleotides included in a nucleic acid (e.g., a first nucleic acid) is between about 4000 and about 7000, between about 4000 and about 6000, or between about 4000 and about 5000. In various embodiments, the number of nucleotides included in a nucleic acid (e.g., a first nucleic acid) is between about 5000 and about 7000, or between about 5000 and about 6000. In various embodiments, the number of nucleotides included in a nucleic acid (e.g., a first nucleic acid) is between about 500 and about 1000, or between about 500 and about 2000. In some embodiments, the nucleic acid (e.g., second nucleic acid) comprises between about 50 and about 200, between about 75 and about 200, between about 50 and about 150, between about 75 and about 150, between about 80 and about 150, between about 50 and about 140, between about 75 and about 140, between about 80 and about 140, between about 75 and about 120, between about 80 and about 130, or between about 80 and about 120. In some embodiments, the nucleic acid (e.g., second nucleic acid) comprises between about 85 and about 115, between about 90 and about 110, or between about 95 and 105.In various embodiments, a nucleic acid (e.g., a second nucleic acid) is or includes 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 200

[0236] In various embodiments, the nucleic acid (eg, first nucleic acid) comprises between about 3,000 and about 7,000 nucleotides. In various embodiments, the number of nucleotides included in a nucleic acid (e.g., a first nucleic acid) is or is greater than about 3000, about 3050, about 3100, about 3150, about 3200, about 3250, about 3300, about 3350, about 3400, about 3450, about 3500, about 3550, about 3600, about 3650, about 3700, about 3750, about 3800, about 3850, about 3900, about 3950, about 4000, about 4050, about 4100, about 4150, about 4200, about 4250, about 4300, about 4350, about 4400, about 4450, about 4500, about 4550, about 4600, about 4650, about 4700, about 4750, about 4800, about 4850 , about 4900, about 4950, about 5000, about 5050, about 5100, about 5150, about 5200, about 5250, about 5300, about 5350, about 5400, about 5450, about 5500, about 5550, about 5600, about 5650, about 5700, about 5750, about 5800, about 5850, about 5900, about 5 about 6800, about 6850, about 6900, about 6950, or about 7000.

[0237] In various embodiments, the nucleic acid (e.g., first nucleic acid) comprises a number of nucleotides between about 3000 and about 7000, between about 3000 and about 6500, between about 3000 and about 6000, between about 3000 and about 5500, between about 3000 and about 5000, between about 3000 and about 4500, or between about 3000 and about 4000. In various embodiments, the nucleic acid (e.g., first nucleic acid) comprises a number of nucleotides between about 4000 and about 7000, between 4000 and about 6500, between about 4000 and about 6000, between 4000 and about 5500, or between about 4000 and about 5000.

[0238] In some embodiments, the nucleic acid (e.g., second nucleic acid) comprises between about 50 and about 200, between about 75 and about 200, between about 50 and about 150, between about 75 and about 150, between about 80 and about 150, between about 50 and about 140, between about 75 and about 140, between about 80 and about 140, between about 75 and about 120, between about 80 and about 130, or between about 80 and about 120. In some embodiments, the nucleic acid (e.g., second nucleic acid) comprises between about 85 and about 115, between about 90 and about 110, or between about 95 and 105. In various embodiments, a nucleic acid (e.g., a second nucleic acid) is or includes 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 200

[0239] In various embodiments, the number of nucleotides included in a nucleic acid (e.g., a third nucleic acid) is between about 500 and about 3000. In various embodiments, the number of nucleotides included in a nucleic acid (e.g., a third nucleic acid) is or is greater than about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, about 1000, about 1050, about 1100, about 1150, about 1200, about 1250, about 1300, about 1350, about 1400, about 1450, about 1500, about 1550, about 1600, about 1650 about 2700, about 2750, about 2800, about 2850, about 2900, about 2950, ​​or about 3000.

[0240] In various embodiments, the number of nucleotides included in a nucleic acid (e.g., a third nucleic acid) is between about 500 and about 3000, between about 500 and about 2500, between about 500 and about 2000, between about 500 and about 1500, between about 500 and about 1000, between about 750 and about 3000, between about 750 and about 2500, between about 750 and about 2000, between about 750 and about 1500, between about 750 and about 1000, between about 1000 and about 3000, between about 1000 and about 2500, between about 1000 and about 2000, or between about 1000 and about 1500.

[0241] In various embodiments, the lipid nucleic acid assembly composition includes a gRNA and an mRNA (e.g., one mRNA or two mRNAs), wherein the length difference between the gRNA and the mRNA (e.g., independently, between the gRNA and the first mRNA, between the gRNA and the second mRNA, or between the gRNA and each of the first mRNA and the second mRNA) is at least or about 500 nucleotides to about 7000 nucleotides. In various embodiments, the lipid nucleic acid assembly composition includes a gRNA and an mRNA, wherein the length difference between the gRNA and the mRNA (e.g., independently, between the gRNA and the first mRNA, between the gRNA and the second mRNA, or between the gRNA and each of the first mRNA and the second mRNA) is at least or about 500, at least or about 550, at least or about 600, at least or about 650, at least or about 700, at least or about 750, at least or about 800, at least or about 850, to at least or about 1500, at least or about 1600, at least or about 1650, at least or about 1700, at least or about 1750, at least or about 1800, at least or about 1850, at least or about 1900, at least or about 1950, at least or about 2000, at least or about 2100, at least or about 2200, at least or about 2300, at least or about 2400, at least or about 2500, at least or about 2600, at least or about 2700, at least or about 2800, at least or about 2900, at least or about 3000, at least or about 3100, at least or about 3200, at least or about 3300, at least or about 3400, at least or about 3500, at least or about 3600, at least or about 3700, at least or about 3800, at least or about 3900 , at least or about 1900, at least or about 1950, at least or about 2000, at least or about 2050, at least or about 2100, at least or about 2150, at least or about 2200, at least or about 2250, at least or about 2300, at least or about 2350, at least or about 2400, at least or about 2450, at least or about 2500, at least or about 2550, at least or about 2600, at least or about 2650, at least or about 2700, at least or about 2750, at least or about 2800, at least or about 2800, at least or about 2900, at least or about 3000, at least or about 3100, at least or about 3100, at least or about 32 ...400, at least or about 3500, at least or about 3600, at least or about 3700, at least or about 3800, at least or about 3900 2850, at least or about 2900, at least or about 2950, ​​at least or about 3000, at least or about 3050, at least or about 3100, at least or about 3150, at least or about 3200, at least or about 3250, at least or about 3300, at least or about 3350, at least or about 3400, at least or about 3450, at least or about 3500, at least or about 3550, at least or about 3600, at least or about 3650, at least or about 3700, at least or about 3750, at least or about 3800,at least or about 4400, at least or about 4450, at least or about 4500, at least or about 4550, at least or about 4600, at least or about 4650, at least or about 4700, at least or about 4750, at least or about 4800, at least or about 4850, at least or about 4900, at least or about 5010, at least or about 5100, at least or about 5150, at least or about 5200, at least or about 5250, at least or about 5300, at least or about 5350, at least or about 5400, at least or about 5450, at least or about 5500, at least or about 5600, at least or about 4650, at least or about 4700, at least or about 4750, at least or about 4800, at least or about 4850, at least or about 4900, at least or about 4950, at least or about 5000, at least or about 5050, at least or about 5100, at least or about 5150, at least or about 5200, at least or about 5250, at least or about 5300, at least or about 5350, at least or about 5400, at least or about 5450, at least or about 5500, at least or about 5550, at least or about 5600, at least or about 5650, at least or about 5700, at least or about 5750, at least or about 5800, at least or about 5850, at least or about 5900, at least or about 5910, at least or about 5920, at least or about 5930, at least or about 5940, at least or about 5950 at least or about 5450, at least or about 5450, at least or about 5500, at least or about 5550, at least or about 5600, at least or about 5650, at least or about 5700, at least or about 5750, at least or about 5800, at least or about 5850, at least or about 5900, at least or about 5950, at least or about 6000, at least or about 6050, at least or about 6100, at least or about 6150, at least or about 6200, at least or about 6250, at least or about 6300, at least or about 6350, at least or about 6400, at least or about 6450, at least or about 6500, at least or about 6600, at least or about 6700, at least or about 6750, at least or about 6800, at least or about 6850, at least or about 6900, at least or about 7010, at least or about 7150, at least or about 7200, at least or about 7300, at least or about 7350 at least or about 6250, at least or about 6300, at least or about 6350, at least or about 6400, at least or about 6450, at least or about 6500, at least or about 6550, at least or about 6600, at least or about 6650, at least or about 6700, at least or about 6750, at least or about 6800, at least or about 6850, at least or about 6900, at least or about 6950, or at least or about 7000.

[0242] In various embodiments, the lipid-nucleic acid assembly composition includes a gRNA and an mRNA, wherein the difference in length between the gRNA and the mRNA (e.g., independently, between the gRNA and a first mRNA, between the gRNA and a second mRNA, or between the gRNA and each of the first mRNA and the second mRNA) is a certain number of nucleotides, and the number of nucleotides is or is at least between about 500 and about 7000, between about 500 and about 6000, between about 500 and about 5000, between about 500 and about 4000, between about 500 and about 3000, between about 500 and about 2000, or between about 500 and about 1000. In various embodiments, the lipid-nucleic acid assembly composition includes a gRNA and an mRNA, wherein the difference in length between the gRNA and the mRNA (e.g., independently, between the gRNA and a first mRNA, between the gRNA and a second mRNA, or between the gRNA and each of the first mRNA and the second mRNA) is a certain number of nucleotides, and the number of nucleotides is or is at least between about 500 and about 7000, between about 1000 and about 7000, between about 1000 and about 6000, between about 1000 and about 5000, between about 1000 and about 4000, between about 1000 and about 3000, between about 1000 and about 2000, or between about 500 and about 1000. In various embodiments, the lipid-nucleic acid assembly composition includes a gRNA and an mRNA, wherein the difference in length between the gRNA and the mRNA (e.g., independently, between the gRNA and a first mRNA, between the gRNA and a second mRNA, or between the gRNA and each of the first mRNA and the second mRNA) is a certain number of nucleotides, and the number of nucleotides is or is at least between about 2000 and about 7000, between about 2000 and about 6000, between about 2000 and about 5000, between about 2000 and about 4000, or between about 2000 and about 3000. In various embodiments, the lipid-nucleic acid assembly composition includes a gRNA and an mRNA, wherein the difference in length between the gRNA and the mRNA (e.g., independently, between the gRNA and a first mRNA, between the gRNA and a second mRNA, or between the gRNA and each of the first mRNA and the second mRNA) is a certain number of nucleotides, and the number of nucleotides is or is at least between about 3000 and about 7000, between about 3000 and about 6000, between about 3000 and about 5000, or between about 3000 and about 4000.In various embodiments, the lipid-nucleic acid assembly composition includes a gRNA and an mRNA, wherein the difference in length between the gRNA and the mRNA (e.g., independently, between the gRNA and the first mRNA, between the gRNA and the second mRNA, or between the gRNA and each of the first and second mRNAs) is a number of nucleotides that is, or at least is, between about 4000 and about 7000, between about 4000 and about 6000, or between about 4000 and about 5000. In various embodiments, the lipid-nucleic acid assembly composition includes a gRNA and an mRNA, wherein the difference in length between the gRNA and the mRNA (e.g., independently, between the gRNA and the first mRNA, between the gRNA and the second mRNA, or between the gRNA and each of the first and second mRNAs) is a number of nucleotides that is, or at least is, between about 5000 and about 7000, or between about 5000 and about 6000.

[0243] In various embodiments, the lipid nucleic acid assembly composition includes a first mRNA and a second mRNA (e.g., a first mRNA encoding a cytosine base editor and a second mRNA encoding a DNA glycosylase inhibitor), wherein the length difference between the first mRNA and the second mRNA is at least or about 500 nucleotides to about 7000 nucleotides. In various embodiments, the lipid nucleic acid assembly composition includes a first mRNA and a second mRNA, wherein the length difference between the first mRNA and the second mRNA is at least or about 500, at least or about 550, at least or about 600, at least or about 650, at least or about 700, at least or about 750, at least or about 800, at least or about 850, at least or about 900, at least or about 950, at least or about 1000, at least or about 1050, at least or about 11 00, at least or about 1150, at least or about 1200, at least or about 1250, at least or about 1300, at least or about 1350, at least or about 1400, at least or about 1450, at least or about 1500, at least or about 1550, at least or about 1600, at least or about 1650, at least or about 1700, at least or about 1750, at least or about 1800, at least or about 1850, at least or about 1900, at least or about 1950, at least or about 2000 0, at least or about 2050, at least or about 2100, at least or about 2150, at least or about 2200, at least or about 2250, at least or about 2300, at least or about 2350, at least or about 2400, at least or about 2450, at least or about 2500, at least or about 2550, at least or about 2600, at least or about 2650, at least or about 2700, at least or about 2750, at least or about 2800, at least or about 2850, at least or about 2900 , at least or about 2950, ​​at least or about 3000, at least or about 3050, at least or about 3100, at least or about 3150, at least or about 3200, at least or about 3250, at least or about 3300, at least or about 3350, at least or about 3400, at least or about 3450, at least or about 3500, at least or about 3550, at least or about 3600, at least or about 3650, at least or about 3700, at least or about 3750, at least or about 3800,at least or about 4400, at least or about 4450, at least or about 4500, at least or about 4550, at least or about 4600, at least or about 4650, at least or about 4700, at least or about 4750, at least or about 4800, at least or about 4850, at least or about 4900, at least or about 5010, at least or about 5100, at least or about 5150, at least or about 5200, at least or about 5250, at least or about 5300, at least or about 5350, at least or about 5400, at least or about 5450, at least or about 5500, at least or about 5600, at least or about 4650, at least or about 4700, at least or about 4750, at least or about 4800, at least or about 4850, at least or about 4900, at least or about 4950, at least or about 5000, at least or about 5050, at least or about 5100, at least or about 5150, at least or about 5200, at least or about 5250, at least or about 5300, at least or about 5350, at least or about 5400, at least or about 5450, at least or about 5500, at least or about 5550, at least or about 5600, at least or about 5650, at least or about 5700, at least or about 5750, at least or about 5800, at least or about 5850, at least or about 5900, at least or about 5910, at least or about 5920, at least or about 5930, at least or about 5940, at least or about 5950 at least or about 5450, at least or about 5450, at least or about 5500, at least or about 5550, at least or about 5600, at least or about 5650, at least or about 5700, at least or about 5750, at least or about 5800, at least or about 5850, at least or about 5900, at least or about 5950, at least or about 6000, at least or about 6050, at least or about 6100, at least or about 6150, at least or about 6200, at least or about 6250, at least or about 6300, at least or about 6350, at least or about 6400, at least or about 6450, at least or about 6500, at least or about 6600, at least or about 6700, at least or about 6750, at least or about 6800, at least or about 6850, at least or about 6900, at least or about 7010, at least or about 7150, at least or about 7200, at least or about 7300, at least or about 7350 at least or about 6250, at least or about 6300, at least or about 6350, at least or about 6400, at least or about 6450, at least or about 6500, at least or about 6550, at least or about 6600, at least or about 6650, at least or about 6700, at least or about 6750, at least or about 6800, at least or about 6850, at least or about 6900, at least or about 6950, or at least or about 7000.

[0244] In various embodiments, the lipid-nucleic acid assembly composition includes a first mRNA and a second mRNA, wherein the difference in length between the first mRNA and the second mRNA is a certain number of nucleotides, and the number of nucleotides is or is at least between about 500 and about 7000, between about 500 and about 6000, between about 500 and about 5000, between about 500 and about 4000, between about 500 and about 3000, between about 500 and about 2000, or between about 500 and about 1000. In various embodiments, the lipid-nucleic acid assembly composition comprises a first mRNA and a second mRNA, wherein the difference in length between the first mRNA and the second mRNA is a number of nucleotides that is, or at least is, between about 1000 and about 7000, between about 1000 and about 6000, between about 1000 and about 5000, between about 1000 and about 4000, between about 1000 and about 3000, or between about 1000 and about 2000. In various embodiments, the lipid-nucleic acid assembly composition comprises a first mRNA and a second mRNA, wherein the difference in length between the first mRNA and the second mRNA is a number of nucleotides that is, or at least is, between about 2000 and about 7000, between about 2000 and about 6000, between about 2000 and about 5000, between about 2000 and about 4000, or between about 2000 and about 3000. In various embodiments, the lipid-nucleic acid assembly composition comprises a first mRNA and a second mRNA, wherein the difference in length between the first mRNA and the second mRNA is a number of nucleotides that is, or at least between about 3000 and about 7000, between about 3000 and about 6000, between about 3000 and about 5000, or between about 3000 and about 4000. In various embodiments, the lipid-nucleic acid assembly composition comprises a first mRNA and a second mRNA, wherein the difference in length between the first mRNA and the second mRNA is a number of nucleotides that is, or at least between about 4000 and about 7000, between about 4000 and about 6000, or between about 4000 and about 5000. In various embodiments, the lipid-nucleic acid assembly composition comprises a first mRNA and a second mRNA, wherein the difference in length between the first mRNA and the second mRNA is a number of nucleotides that is, or at least between about 5000 and about 7000, or between about 5000 and about 6000.

[0245] In various embodiments, the lipid nucleic acid assembly composition comprises a first nucleic acid, a second nucleic acid, and an optional third nucleic acid, wherein the difference in length between the first nucleic acid and the second nucleic acid, between the first nucleic acid and the third nucleic acid (if present), or between the second nucleic acid and the third nucleic acid (if present) is at least or about 500 nucleotides to about 7000 nucleotides. In various embodiments, the lipid nucleic acid assembly composition comprises a first nucleic acid, a second nucleic acid, and an optional third nucleic acid, wherein the difference in length between the first nucleic acid and the second nucleic acid, between the first nucleic acid and the third nucleic acid (if present), or between the second nucleic acid and the third nucleic acid (if present) is at least or about 500, at least or about 550, at least or about 600, at least or about 650, at least or about 700, at least or about 750, at least or about 800, at least or about 850, at least or about 90 ... 50, at least or about 1000, at least or about 1050, at least or about 1100, at least or about 1150, at least or about 1200, at least or about 1250, at least or about 1300, at least or about 1350, at least or about 1400, at least or about 1450, at least or about 1500, at least or about 1550, at least or about 1600, at least or about 1650, at least or about 1700, at least or about 1750, at least or about 1800, at least or about 1850, at least or about 1900 0, at least or about 1950, at least or about 2000, at least or about 2050, at least or about 2100, at least or about 2150, at least or about 2200, at least or about 2250, at least or about 2300, at least or about 2350, at least or about 2400, at least or about 2450, at least or about 2500, at least or about 2550, at least or about 2600, at least or about 2650, at least or about 2700, at least or about 2750, at least or about 2800, at least or about 2850 , at least or about 2900, at least or about 2950, ​​at least or about 3000, at least or about 3050, at least or about 3100, at least or about 3150, at least or about 3200, at least or about 3250, at least or about 3300, at least or about 3350, at least or about 3400, at least or about 3450, at least or about 3500, at least or about 3550, at least or about 3600, at least or about 3650, at least or about 3700, at least or about 3750, at least or about 3800,at least or about 4400, at least or about 4450, at least or about 4500, at least or about 4550, at least or about 4600, at least or about 4650, at least or about 4700, at least or about 4750, at least or about 4800, at least or about 4850, at least or about 4900, at least or about 5010, at least or about 5100, at least or about 5150, at least or about 5200, at least or about 5250, at least or about 5300, at least or about 5350, at least or about 5400, at least or about 5450, at least or about 5500, at least or about 5600, at least or about 4650, at least or about 4700, at least or about 4750, at least or about 4800, at least or about 4850, at least or about 4900, at least or about 4950, at least or about 5000, at least or about 5050, at least or about 5100, at least or about 5150, at least or about 5200, at least or about 5250, at least or about 5300, at least or about 5350, at least or about 5400, at least or about 5450, at least or about 5500, at least or about 5550, at least or about 5600, at least or about 5650, at least or about 5700, at least or about 5750, at least or about 5800, at least or about 5850, at least or about 5900, at least or about 5910, at least or about 5920, at least or about 5930, at least or about 5940, at least or about 5950 at least or about 5450, at least or about 5450, at least or about 5500, at least or about 5550, at least or about 5600, at least or about 5650, at least or about 5700, at least or about 5750, at least or about 5800, at least or about 5850, at least or about 5900, at least or about 5950, at least or about 6000, at least or about 6050, at least or about 6100, at least or about 6150, at least or about 6200, at least or about 6250, at least or about 6300, at least or about 6350, at least or about 6400, at least or about 6450, at least or about 6500, at least or about 6600, at least or about 6700, at least or about 6750, at least or about 6800, at least or about 6850, at least or about 6900, at least or about 7010, at least or about 7150, at least or about 7200, at least or about 7300, at least or about 7350 at least or about 6250, at least or about 6300, at least or about 6350, at least or about 6400, at least or about 6450, at least or about 6500, at least or about 6550, at least or about 6600, at least or about 6650, at least or about 6700, at least or about 6750, at least or about 6800, at least or about 6850, at least or about 6900, at least or about 6950, or at least or about 7000.

[0246] In various embodiments, the lipid-nucleic acid assembly composition includes a first nucleic acid, a second nucleic acid, and optionally a third nucleic acid, wherein the difference in length between the first nucleic acid and the second nucleic acid, between the first nucleic acid and the third nucleic acid (if present), or between the second nucleic acid and the third nucleic acid (if present) is a certain number of nucleotides, and the number of nucleotides is or is at least between about 500 and about 7000, between about 500 and about 6000, between about 500 and about 5000, between about 500 and about 4000, between about 500 and about 3000, between about 500 and about 2000, or between about 500 and about 1000. In various embodiments, the lipid nucleic acid assembly composition includes a first nucleic acid, a second nucleic acid, and optionally a third nucleic acid, wherein the difference in length between the first nucleic acid and the second nucleic acid, between the first nucleic acid and the third nucleic acid (if present), or between the second nucleic acid and the third nucleic acid (if present) is a certain number of nucleotides, and the number of nucleotides is or is at least between about 500 and about 7000, between about 1000 and about 7000, between about 1000 and about 6000, between about 1000 and about 5000, between about 1000 and about 4000, between about 1000 and about 3000, between about 1000 and about 2000, or between about 500 and about 1000. In various embodiments, the lipid-nucleic acid assembly composition includes a first nucleic acid, a second nucleic acid, and optionally a third nucleic acid, wherein the difference in length between the first nucleic acid and the second nucleic acid, between the first nucleic acid and the third nucleic acid (if present), or between the second nucleic acid and the third nucleic acid (if present) is a certain number of nucleotides, and the number of nucleotides is or is at least between about 2000 and about 7000, between about 2000 and about 6000, between about 2000 and about 5000, between about 2000 and about 4000, or between about 2000 and about 3000. In various embodiments, the lipid-nucleic acid assembly composition comprises a first nucleic acid, a second nucleic acid, and optionally a third nucleic acid, wherein the difference in length between the first nucleic acid and the second nucleic acid, between the first nucleic acid and the third nucleic acid (if present), or between the second nucleic acid and the third nucleic acid (if present) is a number of nucleotides that is, or at least is, between about 3000 and about 7000, between about 3000 and about 6000, between about 3000 and about 5000, or between about 3000 and about 4000. In various embodiments, the lipid-nucleic acid assembly composition comprises a first nucleic acid, a second nucleic acid, and optionally a third nucleic acid, wherein the difference in length between the first nucleic acid and the second nucleic acid, between the first nucleic acid and the third nucleic acid (if present), or between the second nucleic acid and the third nucleic acid (if present) is a number of nucleotides that is, or at least is, between about 4000 and about 7000, between about 4000 and about 6000, or between about 4000 and about 5000.In various embodiments, the lipid nucleic acid assembly composition includes a first nucleic acid, a second nucleic acid, and optionally a third nucleic acid, wherein the difference in length between the first nucleic acid and the second nucleic acid, between the first nucleic acid and the third nucleic acid (if present), or between the second nucleic acid and the third nucleic acid (if present) is a certain number of nucleotides, which is or is at least between about 5000 and about 7000 or between about 5000 and about 6000.

[0247] In various embodiments, the lipid nucleic acid assembly composition comprises a first nucleic acid, a second nucleic acid, and an optional third nucleic acid, wherein the difference in length between the first nucleic acid and the second nucleic acid, between the first nucleic acid and the third nucleic acid (if present), or between the second nucleic acid and the third nucleic acid (if present) is at least or about 500 nucleotides to about 7000 nucleotides. In various embodiments, the lipid nucleic acid assembly composition comprises a first nucleic acid, a second nucleic acid, and an optional third nucleic acid, wherein the difference in length between the first nucleic acid and the second nucleic acid, between the first nucleic acid and the third nucleic acid (if present), or between the second nucleic acid and the third nucleic acid (if present) is at least or about 500, at least or about 550, at least or about 600, at least or about 650, at least or about 700, at least or about 750, at least or about 800, at least or about 850, at least or about 90 ... 50, at least or about 1000, at least or about 1050, at least or about 1100, at least or about 1150, at least or about 1200, at least or about 1250, at least or about 1300, at least or about 1350, at least or about 1400, at least or about 1450, at least or about 1500, at least or about 1550, at least or about 1600, at least or about 1650, at least or about 1700, at least or about 1750, at least or about 1800, at least or about 1850, at least or about 1900 0, at least or about 1950, at least or about 2000, at least or about 2050, at least or about 2100, at least or about 2150, at least or about 2200, at least or about 2250, at least or about 2300, at least or about 2350, at least or about 2400, at least or about 2450, at least or about 2500, at least or about 2550, at least or about 2600, at least or about 2650, at least or about 2700, at least or about 2750, at least or about 2800, at least or about 2850 , at least or about 2900, at least or about 2950, ​​at least or about 3000, at least or about 3050, at least or about 3100, at least or about 3150, at least or about 3200, at least or about 3250, at least or about 3300, at least or about 3350, at least or about 3400, at least or about 3450, at least or about 3500, at least or about 3550, at least or about 3600, at least or about 3650, at least or about 3700, at least or about 3750, at least or about 3800,at least or about 4400, at least or about 4450, at least or about 4500, at least or about 4550, at least or about 4600, at least or about 4650, at least or about 4700, at least or about 4750, at least or about 4800, at least or about 4850, at least or about 4900, at least or about 5010, at least or about 5100, at least or about 5150, at least or about 5200, at least or about 5250, at least or about 5300, at least or about 5350, at least or about 5400, at least or about 5450, at least or about 5500, at least or about 5600, at least or about 4650, at least or about 4700, at least or about 4750, at least or about 4800, at least or about 4850, at least or about 4900, at least or about 4950, at least or about 5000, at least or about 5050, at least or about 5100, at least or about 5150, at least or about 5200, at least or about 5250, at least or about 5300, at least or about 5350, at least or about 5400, at least or about 5450, at least or about 5500, at least or about 5550, at least or about 5600, at least or about 5650, at least or about 5700, at least or about 5750, at least or about 5800, at least or about 5850, at least or about 5900, at least or about 5910, at least or about 5920, at least or about 5930, at least or about 5940, at least or about 5950 at least or about 5450, at least or about 5450, at least or about 5500, at least or about 5550, at least or about 5600, at least or about 5650, at least or about 5700, at least or about 5750, at least or about 5800, at least or about 5850, at least or about 5900, at least or about 5950, at least or about 6000, at least or about 6050, at least or about 6100, at least or about 6150, at least or about 6200, at least or about 6250, at least or about 6300, at least or about 6350, at least or about 6400, at least or about 6450, at least or about 6500, at least or about 6600, at least or about 6700, at least or about 6750, at least or about 6800, at least or about 6850, at least or about 6900, at least or about 7010, at least or about 7150, at least or about 7200, at least or about 7300, at least or about 7350 at least or about 6250, at least or about 6300, at least or about 6350, at least or about 6400, at least or about 6450, at least or about 6500, at least or about 6550, at least or about 6600, at least or about 6650, at least or about 6700, at least or about 6750, at least or about 6800, at least or about 6850, at least or about 6900, at least or about 6950, or at least or about 7000.

[0248] In various embodiments, the lipid-nucleic acid assembly composition includes a first nucleic acid, a second nucleic acid, and optionally a third nucleic acid, wherein the difference in length between the first nucleic acid and the second nucleic acid, between the first nucleic acid and the third nucleic acid (if present), or between the second nucleic acid and the third nucleic acid (if present) is a certain number of nucleotides, and the number of nucleotides is or is at least between about 500 and about 7000, between about 500 and about 6000, between about 500 and about 5000, between about 500 and about 4000, between about 500 and about 3000, between about 500 and about 2000, or between about 500 and about 1000. In various embodiments, the lipid-nucleic acid assembly composition includes a first nucleic acid, a second nucleic acid, and optionally a third nucleic acid, wherein the difference in length between the first nucleic acid and the second nucleic acid, between the first nucleic acid and the third nucleic acid (if present), or between the second nucleic acid and the third nucleic acid (if present) is a certain number of nucleotides, and the number of nucleotides is or is at least between about 1000 and about 7000, between about 1000 and about 6000, between about 1000 and about 5000, between about 1000 and about 4000, between about 1000 and about 3000, or between about 1000 and about 2000. In various embodiments, the lipid-nucleic acid assembly composition includes a first nucleic acid, a second nucleic acid, and optionally a third nucleic acid, wherein the difference in length between the first nucleic acid and the second nucleic acid, between the first nucleic acid and the third nucleic acid (if present), or between the second nucleic acid and the third nucleic acid (if present) is a certain number of nucleotides, and the number of nucleotides is or is at least between about 2000 and about 7000, between about 2000 and about 6000, between about 2000 and about 5000, between about 2000 and about 4000, or between about 2000 and about 3000. In various embodiments, the lipid-nucleic acid assembly composition comprises a first nucleic acid, a second nucleic acid, and optionally a third nucleic acid, wherein the difference in length between the first nucleic acid and the second nucleic acid, between the first nucleic acid and the third nucleic acid (if present), or between the second nucleic acid and the third nucleic acid (if present) is a number of nucleotides that is, or at least is, between about 3000 and about 7000, between about 3000 and about 6000, between about 3000 and about 5000, or between about 3000 and about 4000. In various embodiments, the lipid-nucleic acid assembly composition comprises a first nucleic acid, a second nucleic acid, and optionally a third nucleic acid, wherein the difference in length between the first nucleic acid and the second nucleic acid, between the first nucleic acid and the third nucleic acid (if present), or between the second nucleic acid and the third nucleic acid (if present) is a number of nucleotides that is, or at least is, between about 4000 and about 7000, between about 4000 and about 6000, or between about 4000 and about 5000.In various embodiments, the lipid nucleic acid assembly composition includes a first nucleic acid, a second nucleic acid, and optionally a third nucleic acid, wherein the difference in length between the first nucleic acid and the second nucleic acid, between the first nucleic acid and the third nucleic acid (if present), or between the second nucleic acid and the third nucleic acid (if present) is a certain number of nucleotides, which is or is at least between about 5000 and about 7000 or between about 5000 and about 6000.

[0249] In certain exemplary embodiments, the lipid-nucleic acid assembly composition can include a gRNA (e.g., a gRNA of about 80 to about 120 nucleotides in length), a nucleic acid encoding a DNA glycosylase inhibitor (e.g., a UGI) (e.g., an mRNA of about 1000 nucleotides in length), and a nucleic acid encoding a cytosine base editor (e.g., an mRNA of about 4500 nucleotides in length).

[0250] In various embodiments, the lipid nucleic acid assembly composition can include at least, equal to or less than total nucleic acid concentration and / or the total RNA concentration of approximately 3mg / mL, 2.5mg / mL, 2.0mg / mL, 1.5mg / mL, 1.0mg / mL, 0.5mg / mL, 0.4mg / mL or 0.3mg / mL. In various embodiments, the lipid nucleic acid assembly composition can include total nucleic acid concentration and / or the total RNA concentration between 0.3mg / mL and 2.5mg / mL. In various embodiments, the lipid nucleic acid assembly composition can include total nucleic acid concentration and / or the total RNA concentration between 0.75mg / mL and 1.8mg / mL or between approximately 0.5mg / mL and approximately 2mg / mL. In various embodiments, the lipid nucleic acid assembly composition can include total nucleic acid concentration and / or the total RNA concentration of approximately 1.5mg / mL.

[0251] In various embodiments, the lipid nucleic acid assembly composition can include at least, be equal to or be less than the mRNA concentration of about 3mg / mL, 2.5mg / mL, 2.0mg / mL, 1.5mg / mL, 1.0mg / mL, 0.5mg / mL, 0.4mg / mL or 0.3mg / mL. In various embodiments, the lipid nucleic acid assembly composition can include the mRNA concentration between 0.3mg / mL and 2.5mg / mL or between about 0.5mg / mL and about 2mg / mL. In various embodiments, the lipid nucleic acid assembly composition can include the mRNA concentration between 0.75mg / mL and 1.8mg / mL. In various embodiments, the lipid nucleic acid assembly composition can include the mRNA concentration of about 1.5mg / mL.

[0252] In various embodiments, the lipid nucleic acid assembly composition can include at least, equal to or less than about 3 mg / mL, 2.5 mg / mL, 2.0 mg / mL, 1.5 mg / mL, 1.0 mg / mL, 0.5 mg / mL, 0.4 mg / mL or 0.3 mg / mL of sgRNA concentration. In various embodiments, the lipid nucleic acid assembly composition can include an sgRNA concentration between 0.3 mg / mL and 2.5 mg / mL or between about 0.5 mg / mL and about 2 mg / mL. In various embodiments, the lipid nucleic acid assembly composition can include an sgRNA concentration between 0.75 mg / mL and 1.8 mg / mL. In various embodiments, the lipid nucleic acid assembly composition can include an sgRNA concentration of about 1.5 mg / mL.

[0253] In various embodiments where the lipid-nucleic acid assembly composition includes mRNA and sgRNA, the ratio of mRNA:sgRNA can be between about 1:1 and about 5:1, for example, the ratio of mRNA:sgRNA can be about 3:2, 2:1, about 3:1, or about 4:1. In certain specific embodiments, the ratio of mRNA:sgRNA can be about 2:1. In certain specific embodiments, the ratio of mRNA:sgRNA can be about 3:2.

[0254] The present disclosure contemplates that any reagent or material disclosed herein or used in the methods provided herein that contacts or may contact the lipid-nucleic acid assembly composition and / or nucleic acid may be free or substantially free of RNases.

[0255] Extraction of nucleic acids from lipid-nucleic acid assembly compositions

[0256] In various embodiments, the present disclosure includes extracting nucleic acid from the lipid nucleic acid assembly composition.The extraction of nucleic acid has produced nucleic acid extract so that nucleic acid extract comprises a part or subset of the material existing in the lipid nucleic acid assembly composition.Extracting nucleic acid from the lipid nucleic acid assembly composition comprises the technology of separating or purifying one or more or all nucleic acids existing in the lipid nucleic acid assembly composition.The present disclosure includes especially unexpected discovery, and carrying out nucleic acid extraction according to the technology comprising ethanol precipitation, isopropanol precipitation, butanol precipitation or acetonitrile precipitation is particularly advantageous in some cases, for example, for purifying nucleic acid (for example RNA) from the lipid nucleic acid assembly composition comprising LNP, for example, nucleic acid extract is carried out chromatography subsequently.Although the present disclosure includes ethanol precipitation as the example of a preferred embodiment for nucleic acid extraction, it will be appreciated by those skilled in the art that the present disclosure includes using isopropanol precipitation, butanol precipitation or acetonitrile precipitation to realize similar results and advantages.

[0257] Various nucleic acid extraction techniques are known in the art. For example, the technology of extracting nucleic acid from the lipid nucleic acid assembly composition present in the encapsulation structure (for example, cell) can include mechanical and / or chemical steps to release nucleic acid from the encapsulation structure, for example, by grinding, freeze-thaw, bead milling, enzyme treatment (for example, with hydrolase) or exposure to detergent. After the step of mechanical or chemical release of nucleic acid from the encapsulation structure, nucleic acid purification from the processed lipid nucleic acid assembly composition can include washing, filtering and / or precipitation with multiple solutions, buffer and / or detergent (for example, phenol, chloroform or cetyl trimethylammonium bromide, polyethylene glycol, magnetic beads, ion exchange resin or gel). Various such methods all need to invest a large amount of time and resources to prepare nucleic acid extracts. In addition, as mentioned above, various such methods may all be incompatible with downstream applications. For example, the various prior art techniques for extracting nucleic acid (e.g., RNA, for example from LNP) include organic solvents, buffers and / or detergents that make nucleic acid extracts incompatible with chromatography (e.g., SEC or ion-pair chromatography, for example, ion-pair reversed-phase HPLC), which can be caused by one or both of product turbidity, low nucleic acid recovery, inconsistent nucleic acid recovery and / or inconsistent chromatography applicability (e.g., irreproducible chromatography results, low precision of confirmation and / or low accuracy of confirmation). In addition, various extraction and / or chromatography techniques may cause and / or promote residue when applied to the chromatography purification and / or separation of nucleic acids present in lipid nucleic acid assembly compositions. Residual characteristics are detected in the process of subsequent samples using the same device to analyze analytes from the previous sample. Therefore, the present disclosure particularly includes recognizing that there is a problem, namely how to identify a scheme for extracting nucleic acid (e.g., RNA, for example from LNP) to produce a nucleic acid extract that is compatible with chromatography (e.g., SEC or ion-pair chromatography, for example, ion-pair reversed-phase HPLC), can be used for chromatography and / or improved chromatography.

[0258] The present disclosure includes methods for nucleic acid extraction characterized by unexpectedly advantageous properties for producing nucleic acid extracts, including but not limited to producing nucleic acid extracts that are compatible with chromatography (e.g., SEC or ion-pair chromatography, e.g., ion-pair reversed-phase HPLC). The present disclosure also includes methods characterized by unexpectedly reducing carryover. The present disclosure includes methods for nucleic acid extraction, wherein the nucleic acids are separated by ethanol precipitation, isopropanol precipitation, butanol precipitation, or acetonitrile precipitation. In an exemplary nucleic acid extraction of the present disclosure, a lipid-nucleic acid assembly composition comprising nucleic acids (e.g., a lipid-nucleic acid assembly composition in which nucleic acids are encapsulated in LNPs) is mixed with ethanol to produce an ethanol mixture, the ethanol mixture is centrifuged to produce a nucleic acid precipitate, and the nucleic acid precipitate is separated from the ethanol to produce an isolated nucleic acid precipitate. In an exemplary nucleic acid extraction process of the present disclosure, a lipid-nucleic acid assembly composition comprising nucleic acids (e.g., a lipid-nucleic acid assembly composition in which nucleic acids are encapsulated in LNPs) is mixed with ethanol to produce an ethanol mixture, the ethanol mixture is centrifuged to produce a nucleic acid precipitate, and the nucleic acid precipitate is separated from the ethanol to produce an isolated nucleic acid precipitate, and the isolated nucleic acid precipitate is resuspended in an aqueous solution, thereby producing a nucleic acid extract. As disclosed elsewhere herein, in various embodiments, the nucleic acid can be or include two or more RNA molecules, such as an mRNA and an sgRNA, one or both of which can be encapsulated in an LNP as a lipid-nucleic acid assembly composition. As disclosed elsewhere herein, in various embodiments, the nucleic acid can be or include three or more RNA molecules, such as a first mRNA, a second mRNA, and an sgRNA, one or more of which, or all of which can be encapsulated in an LNP as a lipid-nucleic acid assembly composition.

[0259] In various embodiments, the ethanol mixed with the lipid nucleic acid assembly composition in the nucleic acid extraction process of the present disclosure is 200 proof. In various embodiments, the ethanol mixed with the lipid nucleic acid assembly composition in the nucleic acid extraction process of the present disclosure may not contain or substantially contain RNase. In various embodiments, the ethanol mixed with the sample in the nucleic acid extraction process of the present disclosure may include RNase-free water and / or nuclease-free water. In various embodiments, the lipid nucleic acid assembly composition can optionally undergo mechanical treatment to improve mixing with ethanol, such as by shaking, inverting, flicking or vortexing to mix, such as for example, continuing 1-3 seconds.

[0260] In various embodiments, the centrifugation used in the nucleic acid extraction process of the present disclosure can be performed using at least or about 10,000 RCF (e.g., at least or about 10,000, at least or about 15,000, at least or about 20,000, at least or about 25,000, or at least or about 30,000 RCF). In various embodiments, the centrifugation used in the nucleic acid extraction process of the present disclosure can be performed for at least or about 10 minutes (e.g., at least or about 10 minutes, at least or about 15 minutes, at least or about 20 minutes, at least or about 25 minutes, or at least or about 30 minutes).

[0261] In various embodiments, nucleic acid can be separated from ethanol by decantation. In various embodiments, nucleic acid can be separated from ethanol by evaporation and drying. In various embodiments, nucleic acid can be separated from ethanol by vacuum centrifugal concentrator (vacufuge). In various embodiments, nucleic acid can be separated from ethanol by pipetting alcohol. In various embodiments, nucleic acid can be separated from ethanol by decantation, evaporation and drying, vacuum centrifugal concentrator and / or pipetting any one combination to carry out. In various embodiments, nucleic acid can be separated from ethanol by those skilled in the art, so that in the container that holds nucleic acid precipitate, residual ethanol is not seen. In various embodiments, the extraction of nucleic acid and / or nucleic acid can be separated from ethanol by those skilled in the art, so that there is no or substantially no ethanol residual associated with nucleic acid precipitate.

[0262] In various embodiments, the following steps can be repeated: mixing ethanol with the lipid nucleic acid assembly composition to produce an ethanol mixture; centrifuging the ethanol mixture to produce a nucleic acid precipitate; and separating the nucleic acid precipitate from the ethanol to produce an isolated nucleic acid precipitate. In various embodiments, the nucleic acid extraction method disclosed herein can include two, three, four, or five repetitions of the following steps: mixing ethanol with the lipid nucleic acid assembly composition to produce an ethanol mixture, centrifuging the ethanol mixture to produce a nucleic acid precipitate, and separating the nucleic acid precipitate from the ethanol to produce an isolated nucleic acid precipitate. In various embodiments, this repetition can be continuous and / or substantially continuous in terms of the time and / or steps of the protocol.

[0263] In various embodiments, the nucleic acid precipitate of separation can be resuspended in water. In various embodiments, the nucleic acid precipitate of separation can be resuspended in RNase-free water. In various embodiments, the nucleic acid precipitate of separation can be resuspended in nuclease-free water. In various embodiments, compared with the lipid nucleic acid assembly composition, the concentration of nucleic acid in the resuspension of the present disclosure is diluted (for example, compared with the lipid nucleic acid assembly composition, the nucleic acid concentration is diluted at least or about 2 times, at least or about 3 times, at least or about 4 times, at least or about 5 times, or at least or about 10 times). It will be understood by those skilled in the art that the nucleic acid extract can be diluted to a concentration suitable for the intended use, for example, to a concentration suitable for a predetermined determination or analysis. It will also be understood by those skilled in the art that the nucleic acid extract can be diluted to a concentration within the measurement or measurable range of the predetermined determination or analysis, and in various embodiments, this can be determined by a reference or control (for example, a calibration curve selected) selected by the user. In various embodiments, the nucleic acid extract can be diluted to a concentration (for example, total nucleic acid concentration) of the calibration curve at the midpoint or close to the midpoint.

[0264] In various embodiments, nucleic acid extract can be heated before chromatography purification and / or separation, wherein heating causes denaturation. In various embodiments, heating nucleic acid extract before separation can include being heated to a temperature between about 65 DEG C and about 90 DEG C (e.g., being heated to a temperature of about 65 DEG C, about 70 DEG C, about 75 DEG C, about 80 DEG C, about 85 DEG C, or about 90 DEG C). In various embodiments, heating nucleic acid extract before separation can include being heated to a temperature between about 65 DEG C and 85 DEG C, between about 65 DEG C and 80 DEG C, between about 65 DEG C and 75 DEG C, between about 65 DEG C and 70 DEG C, between about 70 DEG C and 90 DEG C, between about 70 DEG C and 85 DEG C, between about 70 DEG C and 80 DEG C, or between about 70 DEG C and 75 DEG C. In various embodiments, heating nucleic acid extract before separation can include being heated to a specified temperature, keeping at least 2 minutes, for example, keeping at a time between about 2 minutes and about 15 minutes (e.g., about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 10 minutes, or about 15 minutes). In various embodiments, heating the nucleic acid extract prior to separation can include heating to an indicated temperature for a time between about 5 minutes and 15 minutes.

[0265] In various embodiments, the heating step may be followed by a cooling step in which the sample is cooled, for example, to a temperature between 1°C and 10°C (e.g., to about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, or about 10°C). In various embodiments, cooling the nucleic acid extract prior to separation may comprise cooling to a temperature between about 1°C and 5°C, between about 3°C ​​and 10°C, or between about 3°C ​​and 5°C. In various embodiments, cooling the nucleic acid extract prior to separation may comprise cooling to a specified temperature for at least 2 minutes, for example, for a time between about 2 minutes and about 15 minutes (e.g., about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 10 minutes, or about 15 minutes). In various embodiments, cooling the nucleic acid extract prior to separation may comprise cooling to a specified temperature for a time between about 5 minutes and 15 minutes. In various embodiments, the nucleic acid extract may be heated and / or cooled prior to transfer to an HPLC vial for separation. In various embodiments, nucleic acid extraction according to the present disclosure can include extracting RNA molecules (e.g., mRNA and sgRNA molecules) from a lipid nucleic acid assembly composition, wherein the RNA molecules are encapsulated in LNPs, wherein the nucleic acids are separated by ethanol precipitation, isopropanol precipitation, butanol precipitation, or acetonitrile precipitation as disclosed herein. In various embodiments, nucleic acid extraction according to the present disclosure is characterized by unexpectedly advantageous properties for producing nucleic acid extracts, including but not limited to producing nucleic acid extracts that are compatible with chromatography (e.g., SEC or ion pair chromatography, such as ion pair reversed-phase HPLC). Without wishing to be bound by any particular scientific theory, at least one reason that the nucleic acid extracts produced according to the present disclosure are compatible with chromatography (e.g., SEC or ion pair chromatography, such as ion pair reversed-phase HPLC) may be that the nucleic acid extracts are unexpectedly free of or substantially free of lipids, organic solvents, buffers, and / or detergents. Without wishing to be bound by any particular scientific theory, properties of nucleic acid extracts produced according to the present disclosure that contribute to compatibility with chromatography methods (e.g., SEC or ion-pair chromatography, such as ion-pair reversed-phase HPLC) may include low turbidity, high nucleic acid recovery, consistent nucleic acid recovery, and downstream properties observed in chromatography methods (e.g., SEC or ion-pair chromatography, such as ion-pair reversed-phase HPLC), which may include, but are not limited to, reproducibility of lipid-nucleic acid assembly composition chromatography results, accuracy of lipid-nucleic acid assembly composition chromatography results, and / or precision of lipid-nucleic acid assembly composition chromatography results.

[0266] Isolation of nucleic acid extracts

[0267] The present disclosure includes that molecules (e.g., nucleic acid molecules) present in a lipid nucleic acid assembly composition (e.g., present in a nucleic acid extracted from a lipid nucleic acid assembly composition) can be purified and / or isolated by a variety of methods and / or based on a variety of molecular characteristics, examples of which include chromatography, capillary zone electrophoresis, or capillary isoelectric focusing. Examples of chromatography include gas chromatography, liquid chromatography, high performance liquid chromatography (HPLC), size exclusion chromatography (SEC), affinity chromatography, anion exchange chromatography, cation exchange chromatography, gel filtration chromatography, hydrophobic interaction chromatography, ion exchange chromatography, reverse phase chromatography, paper chromatography, and thin layer chromatography.

[0268] In certain specific embodiments, the method of the present disclosure includes purification and / or separation of nucleic acids by size exclusion chromatography. In various embodiments of size exclusion chromatography, an aqueous solution or an organic solvent is applied to a matrix (e.g., a matrix present in a chromatographic column). The solution can be referred to as a mobile phase, and the matrix can be referred to as a stationary phase. Size exclusion chromatography includes chromatographic techniques for purifying and / or separating molecules based on molecular size and / or hydrodynamic volume, wherein differences cause molecules to elute (filter) through a stationary phase (e.g., a chromatographic column) at different rates. The present disclosure includes unexpected observations, i.e., compared with other forms of chromatography, such as affinity chromatography or using certain chromatographic columns other than the chromatographic column of the method and composition disclosed herein, using the size exclusion chromatography provided herein is relevant to residual reduction.

[0269] In certain specific embodiments, the methods of the present disclosure include purification and / or separation of nucleic acids by HPLC. HPLC includes a form of column chromatography in which a pump moves the mobile phase through a chromatographic column. HPLC can be used in conjunction with a variety of chromatographic matrices, and thus various modes of chromatographic analysis can be performed using HPLC. For example, SEC can be performed using HPLC.

[0270] In various embodiments, the method of the present disclosure includes the SEC of a matrix and / or a column characterized by one or more specific features provided herein. In various embodiments, the SEC of the present disclosure includes contacting a nucleic acid extract with a SEC matrix and / or a column characterized by a pore size of about 5 nm to about 50 nm, optionally wherein the pore size is about 12.5 nm to about 25 nm. In various embodiments, the SEC of the present disclosure includes contacting a nucleic acid extract with a SEC matrix and / or a column, wherein the SEC matrix and / or the column are characterized by an internal diameter of 3 mm to 6 mm, optionally with an internal diameter of about 4 mm to about 5 mm, optionally with an internal diameter of about 4.6 mm. In various embodiments, the SEC of the present disclosure includes contacting a nucleic acid extract with a SEC matrix and / or a column, wherein the SEC matrix and / or the column are characterized by a length of about 100 mm to about 400 mm (e.g., about 100 mm, about 150 mm, about 200 mm, about 250 mm, about 300 mm, about 350 mm, or about 400 mm), optionally with a length of about 300 mm. In various embodiments, the SEC of the present disclosure comprises contacting the nucleic acid extract with a SEC matrix and / or column characterized by a length of about 200 mm to about 400 mm, about 250 mm to about 350 mm, or about 275 mm to about 325 mm. In various embodiments, the SEC of the present disclosure comprises contacting the nucleic acid extract with a SEC matrix and / or column characterized by a particle size of about 3 μm to about 5 μm, optionally about 4 μm. In various embodiments, the SEC of the present disclosure comprises contacting the nucleic acid extract with a SEC matrix and / or column characterized by compatibility with an aqueous mobile phase. In various embodiments, the SEC of the present disclosure comprises contacting the nucleic acid extract with a SEC matrix and / or column characterized by compatibility with a salt-based mobile phase. In various embodiments, the SEC of the present disclosure comprises contacting the nucleic acid extract with a SEC matrix and / or column characterized by being compatible with a mobile phase having a pH of about 7 to about 8, optionally a mobile phase having a pH of about 7.3 to about 7.7, optionally a mobile phase having a pH of about 7.5. In various embodiments, the SEC of the present disclosure comprises contacting the nucleic acid extract with a SEC matrix and / or column characterized by being compatible with a mobile phase comprising about 25 mM to about 200 mM NaCl, optionally wherein the SEC is compatible with a mobile phase comprising about 150 mM NaCl. In various embodiments, the SEC of the present disclosure comprises contacting the nucleic acid extract with a SEC matrix and / or column characterized by being compatible with a SEC mobile phase having a pH between about 7 and about 8, optionally wherein the pH of the mobile phase is about 7.3 to about 7.7, optionally wherein the pH of the mobile phase is about 7.5.

[0271] In some embodiments, the exemplary SEC column can have a material composed of silica particles. In some embodiments, the exemplary silica material is compatible with a pH range of 7.5+ / -0.2 and a salt concentration of 150mM NaCl. In some embodiments, the exemplary column can have a pore size in the range of 12.5 to 25nm. In some embodiments, the internal dimensions of the exemplary column can be 4.6mm inner diameter (ID), 300mm length and 4μm particle size to efficiently separate two or more types of nucleic acids. In some embodiments, a useful column can be a Waters Biosuite UHR 125SEC column, 4.6mm×30cm, 4μm. Part number 186002161; or Tosoh TSKgel SuperSW30, 4.6mm ID×30cm L, part number 0018675. In some aspects, the above-mentioned column is used together with an Agilent, Shimazhu or Waters HPLC system.

[0272] In various embodiments, SEC of the present disclosure includes a mobile phase characterized in that it is an aqueous mobile phase. In various embodiments, SEC of the present disclosure includes a mobile phase characterized in that it is an aqueous mobile phase based on salt. In various embodiments, SEC of the present disclosure includes a mobile phase characterized in that it includes Tris-HCl. In various embodiments, SEC of the present disclosure includes a mobile phase characterized in that it includes about 5mM to about 50mM Tris-HCl (e.g., about 20mM Tris-HCl). In various embodiments, SEC of the present disclosure includes a mobile phase characterized in that it includes NaCl. In various embodiments, SEC of the present disclosure includes a mobile phase characterized in that it includes about 25mM to about 200mM NaCl (e.g., about 150mM NaCl). In various embodiments, SEC of the present disclosure includes a mobile phase characterized in that SEC separation is carried out at a temperature of about 20°C to about 40°C (e.g., about 30°C). In various embodiments, SEC of the present disclosure includes a mobile phase characterized by a pH between about 7 and about 8. In various embodiments, the SEC of the present disclosure comprises a mobile phase characterized by a pH between about 7.3 and about 7.7, such as about 7.5.

[0273] In various embodiments, the SEC of the present disclosure comprises a mobile phase characterized in that it comprises Tris-HCl and NaCl. In various embodiments, the SEC of the present disclosure comprises a mobile phase characterized in that it comprises about 5 mM to about 50 mM Tris-HCl (e.g., about 20 mM Tris-HCl) and about 25 mM to about 200 mM NaCl (e.g., about 150 mM NaCl).

[0274] In various embodiments, the SEC of the present disclosure comprises a mobile phase characterized in that it comprises Tris-HCl and NaCl and has a pH between about 7 and about 8. In various embodiments, the SEC of the present disclosure comprises a mobile phase characterized in that it comprises about 5 mM to about 50 mM Tris-HCl (e.g., about 20 mM Tris-HCl) and about 25 mM to about 200 mM NaCl (e.g., about 150 mM NaCl) and has a pH between about 7.3 and about 7.7 (e.g., about 7.5).

[0275] In various embodiments, the SEC of the present disclosure comprises a mobile phase characterized in that it comprises Tris-HCl and NaCl, and is performed at a temperature of about 20° C. to about 40° C. In various embodiments, the SEC of the present disclosure comprises a mobile phase characterized in that it comprises about 5 mM to about 50 mM Tris-HCl (e.g., about 20 mM Tris-HCl) and about 25 mM to about 200 mM NaCl (e.g., about 150 mM NaCl), and is performed at a temperature of about 20° C. to about 40° C. (e.g., about 30° C.).

[0276] In various embodiments, the SEC of the present disclosure comprises a mobile phase characterized in that it comprises Tris-HCl and NaCl, has a pH between about 7 and about 8, and is performed at a temperature of about 20° C. to about 40° C. In various embodiments, the SEC of the present disclosure comprises a mobile phase characterized in that it comprises about 5 mM to about 50 mM Tris-HCl (e.g., about 20 mM Tris-HCl) and about 25 mM to about 200 mM NaCl (e.g., about 150 mM NaCl), has a pH between about 7.3 and about 7.7 (e.g., about 7.5), and is performed at a temperature of about 20° C. to about 40° C. (e.g., about 30° C.).

[0277] In various embodiments, nucleic acids separated by a chromatography matrix and / or column (e.g., SEC column) are retained in the chromatography matrix and / or column (e.g., SEC column) at different retention times, leave the chromatography matrix and / or column (e.g., SEC column) at different times, and / or are detected at different times, any or all of which constitute separation as disclosed herein. In various embodiments, separation can also include physical separation of eluates containing different nucleic acid populations and / or detection of different nucleic acid populations.

[0278] In various embodiments, the SEC of the present disclosure separates the first nucleic acid and the second nucleic acid in the resulting chromatogram and differs by at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, for example, at a flow rate of 0.3 mL / min. In various embodiments, the SEC of the present disclosure separates the first nucleic acid and the second nucleic acid in the resulting chromatogram and differs by at least about 1 minute, for example, at a flow rate of 0.3 mL / min. In various embodiments, the SEC of the present disclosure separates the first nucleic acid and the second nucleic acid in the resulting chromatogram and differs by at least about 1.5, for example, at a flow rate of 0.3 mL / min. The separation of the first nucleic acid and the second nucleic acid can be calculated according to various methods known in the art, for example, based on peak or based on half-peak width.

[0279] In various embodiments, the methods of the present disclosure include ion pair chromatography (e.g., ion pair reversed-phase HPLC) using a matrix and / or column characterized by one or more specific features provided herein. In various embodiments, the ion pair chromatography of the present disclosure includes contacting a nucleic acid extract with an ion pair chromatography matrix and / or column that is considered to be useful for separating nucleic acids, such as DNA (e.g., single-stranded and / or double-stranded DNA) and / or RNA (e.g., single-stranded and / or double-stranded RNA). In various embodiments, the ion pair chromatography of the present disclosure includes contacting a nucleic acid extract with an ion pair chromatography matrix and / or column characterized by hydrophobic particles.

[0280] In various embodiments, the ion-pair chromatography methods (e.g., ion-pair reversed-phase HPLC) of the present disclosure comprise contacting a nucleic acid extract with an ion-pair chromatography matrix and / or column characterized by particles having a mean, median, and / or mode size between about 2 μm and about 6 μm, e.g., having a size of about 2 μm, about 3 μm, about 4 μm, about 5 μm, or about 6 μm. In various embodiments, the ion-pair chromatography methods of the present disclosure comprise contacting a nucleic acid extract with an ion-pair chromatography matrix and / or column characterized by particles having a mean, median, and / or mode size of between about 2 μm and about 6 μm, between about 2 μm and about 5 μm, between about 2 μm and about 4 μm, between about 2 μm and about 3 μm, between about 3 μm and about 6 μm, between about 3 μm and about 5 μm, between about 3 μm and about 4 μm, between about 4 μm and about 6 μm, or between about 4 μm and about 5 μm. In various embodiments, the ion-pair chromatography methods of the present disclosure comprise contacting a nucleic acid extract with an ion-pair chromatography matrix and / or column characterized by particles having a mean, median, and / or mode size of about 4 μm.

[0281] In various embodiments, the ion-pair chromatography methods of the present disclosure (e.g., ion-pair reversed-phase HPLC) comprise contacting a nucleic acid extract with an ion-pair chromatography matrix and / or column characterized by particles having about to about The average, median and / or mode pore size of about about about about about about about about about about about about about about about or about In various embodiments, the ion-pair chromatography methods of the present disclosure comprise contacting a nucleic acid extract with an ion-pair chromatography matrix and / or column characterized by particles having approximately to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about about to about or about to about In various embodiments, the ion-pair chromatography methods of the present disclosure (e.g., ion-pair reversed-phase HPLC) comprise contacting a nucleic acid extract with an ion-pair chromatography matrix and / or column characterized by particles having about to about The mean, median and / or mode pore size of

[0282] In various embodiments, the ion pair chromatography of the present disclosure (e.g., ion pair reversed phase HPLC) comprises contacting the nucleic acid extract with an ion pair chromatography column characterized by a diameter of about 1 mm to about 4 mm, e.g., about 1 mm, about 2 mm, about 3 mm, or about 4 mm. In various embodiments, the ion pair chromatography of the present disclosure comprises contacting the nucleic acid extract with an ion pair chromatography column characterized by a diameter of about 1 mm to about 4 mm, 1.5 mm to about 4 mm, 2 mm to about 4 mm, 2.5 mm to about 4 mm, 3 mm to about 4 mm, 3.5 mm to about 4 mm, 1 mm to about 4 mm, 1.5 mm to about 3 mm, 2 mm to about 3 mm, 2.5 mm to about 3 mm, about 1 mm to about 2.5 mm, about 1.5 mm to about 2.5 mm, about 2 mm to about 2.5 mm, about 1 mm to about 2 mm, or about 1.5 mm to about 2 mm. In various embodiments, the ion pair chromatography of the present disclosure comprises contacting the nucleic acid extract with an ion pair chromatography column characterized by a diameter of about 2.1 mm.

[0283] In various embodiments, the ion-pair chromatography methods (e.g., ion-pair reversed-phase HPLC) of the present disclosure comprise contacting the nucleic acid extract with an ion-pair chromatography column characterized by a length of about 10 mm to about 300 mm, e.g., about 25 mm, about 50 mm, about 75 mm, about 100 mm, about 125 mm, about 150 mm, about 175 mm, about 200 mm, about 225 mm, about 250 mm, about 275 mm, or about 300 mm. In various embodiments, the ion-pair chromatography methods of the present disclosure (e.g., ion-pair reversed-phase HPLC) comprise contacting the nucleic acid extract with an ion-pair chromatography column characterized by a length of about 25 mm to about 300 mm, about 50 mm to about 300 mm, about 75 mm to about 300 mm, about 100 mm to about 300 mm, about 125 mm to about 300 mm, about 150 mm to about 300 mm, about 175 mm to about 300 mm, about 200 mm to about 300 mm, about 225 mm to about 300 mm, about 250 mm to about 300 mm, or about 275 mm to about 300 mm. In various embodiments, the ion-pair chromatography methods of the present disclosure comprise contacting the nucleic acid extract with an ion-pair chromatography column characterized by a length of about 25 mm to about 150 mm, about 50 mm to about 150 mm, about 75 mm to about 150 mm, about 100 mm to about 150 mm, or about 125 mm to about 150 mm. In various embodiments, the ion-pair chromatography methods of the present disclosure comprise contacting the nucleic acid extract with an ion-pair chromatography column characterized by a length of about 100 mm.

[0284] In various embodiments, the ion pair chromatography of the present disclosure (e.g., ion pair reversed-phase HPLC) includes an ion pairing agent. The ion pairing agent can include an ionic functional group and a hydrophobic portion, such as a hydrocarbon chain and / or a lipophilic alkyl chain. The ion pairing agent can be a sulfonic acid derivative, such as hexanesulfonic acid, heptanesulfonic acid, octanesulfonic acid; a quaternary ammonium salt, such as tetramethylammonium hydroxide or tetrabutylammonium hydroxide; or a volatile reagent, such as trifluoroacetic acid and triethylamine. In certain exemplary embodiments, the ion pairing agent can be triethylamine, tripropylamine, hexylamine, N,N-dimethylbutylamine, dibutylamine, N,N-diisopropylethylamine, or hexafluoro-2-propanol. In certain exemplary embodiments, the ion pairing agent can be trimethylamine or hexylamine. In certain exemplary embodiments, the ion pairing agent can be dibutylammonium acetate. In certain exemplary embodiments, the ion pairing agent can be triethylammonium acetate. In certain exemplary embodiments, the ion pairing agent can be dibutylammonium acetate and triethylammonium acetate.

[0285] In various embodiments, the ion pair chromatography method of the present disclosure comprises contacting the nucleic acid extract with an ion pair chromatography matrix and / or column comprising silica, optionally wherein the matrix and / or column comprises silica particles. In various embodiments, the ion pair chromatography method of the present disclosure comprises contacting the nucleic acid extract with an ion pair chromatography matrix and / or column comprising particles that can be bound to an ion pairing agent. Those skilled in the art will be familiar with ion pair chromatography matrices and columns and will be able to select a column suitable for use with the methods and compositions provided herein based on the present disclosure.

[0286] In various embodiments, counter ion is also provided, for example, in mobile phase. In various embodiments, ion pair chromatography of the present disclosure (for example ion pair reversed phase HPLC) comprises mobile phase, it is characterized in that it comprises ion pairing agent, for example 10mM to 300mM ion pairing agent. In various embodiments, mobile phase can comprise for example about 25mM, about 50mM, about 75mM, about 100mM, about 125mM, about 150mM, about 175mM, about 200mM, about 225mM, about 250mM, about 275mM or about 300mM ion pairing agent. In various embodiments, the mobile phase may include, for example, about 25 mM to about 300 mM ion pairing agent, about 50 mM to about 300 mM ion pairing agent, about 75 mM to about 300 mM ion pairing agent, about 100 mM to about 300 mM ion pairing agent, about 150 mM to about 300 mM ion pairing agent, about 200 mM to about 300 mM ion pairing agent, about 25 mM to about 250 mM ion pairing agent, about 50 mM to about 250 mM ion pairing agent, about 75 mM to about 250 mM ion pairing agent, about 100 mM to about 250 mM ion pairing agent, about 150 mM to about 250 mM ion pairing agent, about 200 mM to about 250 mM ion pairing agent, about 25 mM to about 200 mM ion pairing agent, about 50 mM to about 200 mM ion pairing agent, about 75 mM to about 200 mM ion pairing agent, about 100 mM to about 200 mM ion pairing agent, about 150 mM to about 200 mM ion pairing agent, about 25 mM to about 150 mM ion pairing agent, about 50 mM to about 150 mM ion pairing agent, about 75 mM to about 150 mM ion pairing agent, or about 100 mM to about 150 mM ion pairing agent.

[0287] In various embodiments, counter ions are also provided, for example in the mobile phase. In various embodiments, the ion pair chromatography of the present disclosure (e.g., ion pair reversed phase HPLC) includes a mobile phase characterized in that it includes dibutylammonium acetate, for example, 10mM to 300mM dibutylammonium acetate. In various embodiments, the mobile phase can include, for example, about 25mM, about 50mM, about 75mM, about 100mM, about 125mM, about 150mM, about 175mM, about 200mM, about 225mM, about 250mM, about 275mM or about 300mM dibutylammonium acetate. In various embodiments, the mobile phase may include, for example, about 25 mM to about 300 mM dibutylammonium acetate, about 50 mM to about 300 mM dibutylammonium acetate, about 75 mM to about 300 mM dibutylammonium acetate, about 100 mM to about 300 mM dibutylammonium acetate, about 150 mM to about 300 mM dibutylammonium acetate, about 200 mM to about 300 mM dibutylammonium acetate, about 25 mM to about 250 mM dibutylammonium acetate, about 50 mM to about 250 mM dibutylammonium acetate, about 75 mM to about 250 mM dibutylammonium acetate, about 100 mM to about 250 mM dibutylammonium acetate, about 150 mM to about 250 mM dibutylammonium acetate, about 200 mM to about 250 mM dibutylammonium acetate, about 25 mM to about 200 mM dibutylammonium acetate, about 50 mM to about 200 mM dibutylammonium acetate, about 75 mM to about 200 mM dibutylammonium acetate, about 100 mM to about 200 mM dibutylammonium acetate, about 150 mM to about 200 mM dibutylammonium acetate, about 25 mM to about 150 mM dibutylammonium acetate, about 50 mM to about 150 mM dibutylammonium acetate, about 75 mM to about 150 mM dibutylammonium acetate, or about 100 mM to about 150 mM dibutylammonium acetate.

[0288] In various embodiments, counter ion is also provided, for example in mobile phase. In various embodiments, ion pair chromatography of the present disclosure (for example ion pair reversed phase HPLC) comprises mobile phase, it is characterized in that it comprises triethylammonium acetate, for example 10mM to 300mM triethylammonium acetate. In various embodiments, mobile phase can comprise for example about 25mM, about 50mM, about 75mM, about 100mM, about 125mM, about 150mM, about 175mM, about 200mM, about 225mM, about 250mM, about 275mM or about 300mM triethylammonium acetate. In various embodiments, the mobile phase may include, for example, about 25 mM to about 300 mM triethylammonium acetate, about 50 mM to about 300 mM triethylammonium acetate, about 75 mM to about 300 mM triethylammonium acetate, about 100 mM to about 300 mM triethylammonium acetate, about 150 mM to about 300 mM triethylammonium acetate, about 200 mM to about 300 mM triethylammonium acetate, about 25 mM to about 250 mM triethylammonium acetate, about 50 mM to about 250 mM triethylammonium acetate, about 75 mM to about 250 mM triethylammonium acetate, about 100 mM to about 250 mM triethylammonium acetate, about 150 mM to about 250 mM triethylammonium acetate, about 200 mM to about 250 mM triethylammonium acetate, about 25 mM to about 200 mM triethylammonium acetate, about 50 mM to about 200 mM triethylammonium acetate, about 75 mM to about 200 mM triethylammonium acetate, about 100 mM to about 200 mM triethylammonium acetate, about 150 mM to about 200 mM triethylammonium acetate, about 25 mM to about 150 mM triethylammonium acetate, about 50 mM to about 150 mM triethylammonium acetate, about 75 mM to about 150 mM triethylammonium acetate, or about 100 mM to about 150 mM triethylammonium acetate.

[0289] In certain embodiments, the ion pair chromatography of the present disclosure (such as ion pair reversed-phase HPLC) includes a mobile phase, characterized in that it includes dibutylammonium acetate and triethylammonium acetate. In certain exemplary embodiments, the ion pair chromatography of the present disclosure (such as ion pair reversed-phase HPLC) includes a mobile phase, characterized in that it includes about 50mM dibutylammonium acetate and about 100mM triethylammonium acetate. In various embodiments, the ion pair chromatography of the present disclosure (such as ion pair reversed-phase HPLC) includes two or more mobile phases shown here. For example, in some embodiments, the ion pair chromatography of the present disclosure (such as ion pair reversed-phase HPLC) includes: a first mobile phase, characterized in that it includes about 50mM dibutylammonium acetate and about 100mM triethylammonium acetate; and a second mobile phase, characterized in that it includes about 50mM dibutylammonium acetate and about 100mM triethylammonium acetate. In certain embodiments, the ion pair chromatography of the present disclosure (such as ion pair reversed-phase HPLC) can be carried out at any pH (i.e., 0 to 14). In various embodiments, ion-pair chromatography can be performed at a pH between about 7 and about 8.

[0290] In various embodiments, the ion pair chromatography of the present disclosure (e.g., ion pair reversed phase HPLC) can be performed at any temperature. In various embodiments, the ion pair chromatography of the present disclosure (e.g., ion pair reversed phase HPLC) can be performed at a temperature of about 20° C. to about 80° C. (e.g., about 65° C.).

[0291] In various embodiments, nucleic acids separated by ion pair chromatography are retained in the chromatography matrix and / or column at different retention times, exit the chromatography matrix and / or column at different times, and / or are detected at different times, any or all of which constitute separation as disclosed herein. In various embodiments, separation can also include physical separation of eluates containing different nucleic acid populations and / or detection of different nucleic acid populations.

[0292] In various embodiments, the ion pair chromatography of the present disclosure separates a first nucleic acid from a second nucleic acid in the resulting chromatogram by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., at a flow rate of 0.45 mL / min. In various embodiments, the ion pair chromatography of the present disclosure separates an approximately 100mer RNA from an approximately 1000mer RNA by at least or about 1, 2, or 3 (e.g., at least or about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0), e.g., at a flow rate of 0.45 mL / min. In various embodiments, the ion pair chromatography methods of the present disclosure separate about 100mer RNA from about 4500mer RNA by at least or about 1, 2, 3, 4, or 5 (e.g., at least or about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0), for example, at a flow rate of 0.45 mL / min. In various embodiments, the ion pair chromatography of the present disclosure separates an approximately 1000mer RNA from an approximately 4500mer RNA by a difference of at least or about 1 or 2 (e.g., at least or about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0), for example, at a flow rate of 0.45 mL / min. The separation of the first nucleic acid from the second nucleic acid can be calculated according to various methods known in the art, for example, based on peak or based on half-peak width.

[0293] Detection of nucleic acids and determination of nucleic acid concentration

[0294] The present disclosure includes methods for evaluating lipid nucleic acid assembly compositions as provided herein that can include determining the absolute concentration and / or relative concentration of one or more isolated nucleic acids (e.g., nucleic acids in a lipid nucleic acid assembly composition used to prepare a nucleic acid extract and perform chromatography according to the present disclosure). Chromatography methods of the present disclosure can include a variety of detectors for detecting nucleic acids (e.g., nucleic acids in a column eluate), and the detection can, in particular, provide the absolute concentration and / or relative concentration of one or more nucleic acids, and / or absolute and / or relative total nucleic acid concentration.

[0295] Those skilled in the art will appreciate that any one of the various detectors is applicable to detecting nucleic acid during chromatography or when combined with chromatography. For example, in some embodiments, nucleic acid can be detected by measuring absorbance. In some embodiments, absorbance can be measured at 260nm and / or at 260nm and 280nm. In some embodiments, absorbance can be measured using a UV detector or a spectrophotometer. In certain embodiments, the UV detector can be a fixed wavelength UV detector, a variable wavelength UV detector (VWD) or a photodiode array UV detector (DAD). In some instances, nucleic acid can be detected by using nucleic acid binding dye and fluorescence measurement, by eluent electrophoretic analysis (for example, on agarose gel) and / or by PCR (for example, quantitative PCR, reverse transcription PCR and / or quantitative reverse transcription PCR) amplification of the nucleic acid sequence of interest.

[0296] In some embodiments, nucleic acid concentration and / or purity is assessed using UV absorbance or UV spectroscopy, wherein the absorbance of a diluted RNA sample is measured at 260 nm and / or at 260 nm and 280 nm. According to the Beer-Lambert Law, the concentration of nucleic acids in a sample can be calculated based on the amount of light absorbed at 260 nm: A = εbc, where A is the absorbance (also known as optical density), ε is the extinction coefficient, b is the optical path length of the sample cuvette, and c is the concentration of the compound in solution.

[0297] It will be appreciated by those skilled in the art that a calibration curve can be designed to provide calibration for the detection of an analyte of interest within a concentration range of interest (e.g., nucleic acid, RNA, sgRNA, mRNA, total nucleic acid, and / or total RNA concentrations disclosed herein). It will be further appreciated by those skilled in the art from the present disclosure that a calibration curve is a form of reference or control and is therefore subjected to the same or comparable analytical process or conditions (e.g., the same or comparable chromatography and / or the same or comparable detection, e.g., by absorbance detection at 260 nm) as the test composition (e.g., lipid nucleic acid assembly composition or nucleic acid extract) being compared. The present disclosure includes, for example, that in various embodiments, a dilution series of a nucleic acid of known concentration (e.g., an mRNA of known concentration and / or an sgRNA of known concentration) can be used to generate one or more calibration curves, e.g., where the dilution series represents a series of linear concentration values. In certain embodiments, a dilution series of a reference mRNA of known concentration and / or a reference sgRNA of known concentration, e.g., an mRNA and / or sgRNA known or expected to be present in a lipid nucleic acid assembly composition, can be used to generate one or more calibration curves, e.g., where the dilution series represents a series of linear concentration values. In various embodiments, the concentration of a first nucleic acid (e.g., an mRNA present in a lipid-nucleic acid assembly composition or a nucleic acid extract) can be determined based on a calibration curve for a first reference nucleic acid (e.g., a reference mRNA), and the concentration of a second nucleic acid (e.g., an sgRNA present in a lipid-nucleic acid assembly composition or a nucleic acid extract) can be determined based on a calibration curve for a second reference nucleic acid (e.g., a reference sgRNA). In various embodiments, such as embodiments in which the nucleic acids present in the lipid-nucleic acid assembly composition consist of or consist essentially of the first and second nucleic acids, the total nucleic acid concentration (e.g., total RNA concentration) can be determined as the sum of the concentrations of the first nucleic acid (e.g., mRNA) and the second nucleic acid (e.g., sgRNA).

[0298] In various embodiments, a series of reference dilutions of the calibration curve includes the concentration of the first reference nucleic acid and the concentration of the second reference nucleic acid in each dilution, for example, wherein each dilution in the series has or is intended to have the same or approximately the same ratio of the first reference nucleic acid to the second reference nucleic acid (a "constant" or "maintained" ratio). In various embodiments, the constant or maintained ratio is the same or approximately the same as or intended to be the same as the actual or expected ratio of the first nucleic acid and the second nucleic acid in the lipid nucleic acid assembly composition and / or nucleic acid extract. In various embodiments, a series of reference dilutions of the calibration curve can have a range of total nucleic acid concentrations, each dilution including the concentration of the first nucleic acid and the concentration of the second nucleic acid. In various embodiments, the total nucleic acid concentration of the reference dilution represents a series of linear values, each dilution including the concentration of the first nucleic acid and the concentration of the second nucleic acid.

[0299] It will be appreciated by those skilled in the art that the concentration range of the dilution series of the calibration curve can be selected to cover a relevant range, and that the relevant range can be determined by the user through the dilution or concentration of the nucleic acid extract. In various embodiments, the nucleic acid extract and the calibration curve are prepared and / or selected so that the nucleic acid extract has a concentration (e.g., mRNA concentration, sgRNA concentration, and / or total concentration) at or near the midpoint of the calibration curve, e.g., based on measured values ​​or expected values. For exemplary embodiments only, the calibration curve for the reference mRNA molecule can represent a concentration range between and / or including a lower limit of about 12 μg / mL and an upper limit of about 60 μg / mL, or between and / or including a lower limit of about 12 μg / mL and an upper limit of about 36 μg / mL. To provide only exemplary embodiments, a calibration curve for a reference sgRNA molecule can represent a concentration range between and / or including a lower limit of about 8 μg / mL and an upper limit of about 40 μg / mL, or between and / or including a lower limit of about 8 μg / mL and an upper limit of about 24 μg / mL. In various embodiments, each dilution in the dilution series can include a reference mRNA and a reference sgRNA, wherein the mRNA and sgRNA are present in the same ratio (e.g., 2:1 or 3:2) in each dilution, and can have a total RNA concentration between and / or including a lower limit of about 20 μg / mL and an upper limit of about 100 μg / mL, or between and / or including a lower limit of about 20 μg / mL and an upper limit of about 60 μg / mL.

[0300] Using the Beer-Lambert law, a calibration curve can be obtained by preparing various diluted nucleic acid samples in nuclease-free water and plotting the absorbance of the diluted nucleic acid samples measured at 260 nm against their respective concentrations. For example, using the Beer-Lambert law, an A260 reading of 1.0 is equivalent to approximately 40 μg / ml of single-stranded RNA. In some embodiments, the A260 / A280 ratio can be used to assess DNA / RNA purity. For example, an A260 / A280 ratio of 1.8-2.1 indicates that RNA purity is high (e.g., an A260 / A280 ratio of 1.85 for nuclease-free water (pH 6-7)).

[0301] application

[0302] The compositions and methods provided herein can be used to purify and / or separate one or more nucleic acids (e.g., a first nucleic acid and a second nucleic acid, e.g., an mRNA and an sgRNA) present in a lipid nucleic acid assembly composition. The compositions and methods provided herein can be used to determine the concentration of one or more (e.g., a first nucleic acid and a second nucleic acid, e.g., an mRNA and an sgRNA) present in a lipid nucleic acid assembly composition. The compositions and methods provided herein can be used to determine the ratio of two or more (e.g., a first nucleic acid and a second nucleic acid, e.g., an mRNA and an sgRNA) present in a lipid nucleic acid assembly composition. The compositions and methods provided herein can be used to determine the absolute and / or relative total concentration of nucleic acids present in a lipid nucleic acid assembly composition.

[0303] The present disclosure includes that information such as absolute and / or relative nucleic acid concentrations and / or ratios determined according to the compositions and methods shown in the present disclosure can be used for a variety of purposes, including research and drug preparation. For example, the compositions and methods disclosed herein can be used to determine the absolute concentration and / or relative concentration of nucleic acids in a lipid nucleic acid assembly composition, the ratio of nucleic acids in a lipid nucleic acid assembly composition, and / or the total nucleic acid concentration in a lipid nucleic acid assembly composition, for example, wherein the nucleic acids present in the lipid nucleic acid assembly composition include two nucleic acids (e.g., mRNA and sgRNA), are composed of them or are essentially composed of them. In various embodiments, the absolute concentration and / or relative concentration of nucleic acids in a lipid nucleic acid assembly composition, the ratio of nucleic acids in a lipid nucleic acid assembly composition, and / or the total nucleic acid concentration in a lipid nucleic acid assembly composition can be compared with reference values ​​characterizing a therapeutic product. In various embodiments, the lipid nucleic acid assembly composition is a pharmaceutical or bulk drug, for example, a pharmaceutical or bulk drug comprising a nucleic acid, wherein the nucleic acid comprises mRNA and sgRNA, is composed of mRNA and sgRNA, or is essentially composed of mRNA and sgRNA. In some embodiments, if the absolute concentration and / or relative concentration of each of one or more (e.g., two) nucleic acids, the ratio of the two nucleic acids, and / or the total nucleic acid concentration of the drug product or drug substance or a lipid-nucleic acid assembly composition derived therefrom is determined to be the same or substantially the same (e.g., within 99%, 98%, 97%, 96%, 95%, or 90% of the reference value) as a reference value characterizing the drug product, then the drug product or drug substance is formulated for pharmaceutical use. In some embodiments, if the absolute concentration and / or relative concentration of each of one or more (e.g., two) nucleic acids, the ratio of the two nucleic acids, and / or the total nucleic acid concentration of the drug product or drug substance or a lipid-nucleic acid assembly composition derived therefrom is determined to be not the same or substantially different (e.g., not within 99%, 98%, 97%, 96%, 95%, or 90% of the reference value) as a reference value characterizing the therapeutic product, then the drug product or drug substance is not formulated for pharmaceutical use.

[0304] Reagent test kit

[0305] The present disclosure particularly includes a kit comprising a SEC matrix and instructions for using it to separate two or more nucleic acids present in a lipid nucleic acid assembly composition. The present disclosure particularly includes a kit comprising a SEC matrix and one or both of the following: (i) at least one reagent for SEC, such as a mobile phase solution; and / or (ii) a reagent for ethanol precipitation, isopropanol precipitation, butanol precipitation, or acetonitrile precipitation of nucleic acids in a lipid nucleic acid assembly composition. The present disclosure particularly includes a kit comprising a SEC matrix and one or both of the following: (i) at least one reagent for SEC, such as a mobile phase solution; and / or (ii) a reagent for ethanol precipitation, isopropanol precipitation, butanol precipitation, or acetonitrile precipitation of nucleic acids in a lipid nucleic acid assembly composition, and instructions for using it to separate two or more nucleic acids present in a lipid nucleic acid assembly composition. Various such kits may also include instructions for determining absolute and / or relative total nucleic acid concentrations, individual nucleic acid concentrations, and / or concentration ratios. In various such kits, the SEC matrix is ​​a SEC column, or is included in a SEC column. In various such kits, the SEC matrix, reagents for SEC, SEC mobile phase solutions, and / or reagents for ethanol solutions are described elsewhere herein.

[0306] In at least one aspect, the present disclosure provides a method for isolating nucleic acids present in a lipid-nucleic acid assembly composition, the method comprising: a) preparing a nucleic acid extract from the lipid-nucleic acid assembly composition, wherein the preparation of the nucleic acid extract comprises ethanol precipitation of the nucleic acids in the lipid-nucleic acid assembly composition; and b) performing SEC on the nucleic acid extract, wherein the SEC separates the nucleic acids. In certain embodiments, the method further comprises determining the total concentration of nucleic acids present in the lipid-nucleic acid assembly composition.

[0307] In at least one aspect, the present disclosure provides a kit for isolating nucleic acids present in a lipid nucleic acid assembly composition, the kit comprising a SEC matrix and at least one reagent for ethanol precipitation of nucleic acids in the lipid nucleic acid assembly composition, optionally wherein the kit is for isolating two or more nucleic acids, optionally wherein the two or more nucleic acid molecules are or include gRNA and mRNA. In certain embodiments, the SEC matrix is ​​a SEC column or is present in a SEC column. In certain embodiments, the inner diameter of the SEC column is 3 mm to 6 mm, optionally its inner diameter is about 4 mm to about 5 mm, optionally its inner diameter is about 4.6 mm inner diameter. In certain embodiments, the length of the SEC column is about 100 mm to about 400 mm, optionally its length is about 300 mm long. In certain embodiments, the SEC matrix and / or SEC column has a pore size of about 5 nm to about 50 nm, optionally wherein the pore size is about 12.5 nm to about 25 nm. In certain embodiments, the SEC matrix and / or SEC column has a particle size of about 3 μm to about 5 μm, optionally its particle size is about 4 μm. In certain embodiments, the SEC matrix and / or SEC column comprises silica, optionally wherein the SEC matrix comprises silica particles. In certain embodiments, the SEC matrix and / or SEC column is compatible with a mobile phase having a pH of about 7 to about 8, optionally a mobile phase having a pH of about 7.3 to about 7.7, optionally a mobile phase having a pH of about 7.5. In certain embodiments, the SEC matrix and / or SEC column is compatible with a mobile phase comprising about 25 mM to about 200 mM NaCl, optionally a mobile phase comprising about 150 mM NaCl.

[0308] In certain embodiments of the kits provided herein, the kits include a dilution series of one or more reference nucleic acids, optionally wherein the kit includes a dilution series of reference gRNA molecules and / or reference mRNA molecules. In certain embodiments of the kits provided herein, the kits include instructions for preparing a nucleic acid extract by a method including ethanol precipitation. In certain embodiments of the kits provided herein, the kits include instructions for separating two or more nucleic acids by SEC, optionally wherein the two or more nucleic acids include or consist of RNA molecules, optionally wherein the RNA molecules include or consist of gRNA molecules and mRNA molecules. In certain embodiments of the kits provided herein, the kits include instructions for determining the concentration and / or total nucleic acid concentration of two or more nucleic acids, optionally wherein the two or more nucleic acids include or consist of RNA molecules, optionally wherein the RNA molecules include or consist of gRNA molecules and mRNA molecules.

[0309] In at least one aspect, the present disclosure provides a kit for separating nucleic acids present in a lipid nucleic acid assembly composition, the kit comprising an ion pair chromatography matrix and at least one reagent for ethanol precipitation of nucleic acids in a lipid nucleic acid assembly composition, optionally wherein the kit is for separating two or more nucleic acids, optionally wherein the two or more nucleic acid molecules are or include gRNA and mRNA; and / or for separating three or more nucleic acids, optionally wherein the three or more nucleic acid molecules are or include gRNA, a first mRNA, and a second mRNA. In certain embodiments, the ion pair chromatography matrix is ​​an ion pair chromatography column or is present in an ion pair chromatography column. In certain embodiments, the inner diameter of the ion pair chromatography column is 1 mm to 4 mm, optionally its inner diameter is about 1.5 mm to about 2.5 mm, optionally its inner diameter is about 2.1 mm inner diameter. In certain embodiments, the length of the ion pair chromatography column is about 50 mm to about 400 mm, optionally its length is about 100 mm long. In certain embodiments, the pore size of the ion pair chromatography matrix and / or the ion pair chromatography column is about to about Optionally wherein the pore size is about to about In certain embodiments, the particle size of the ion pair chromatography matrix and / or the ion pair chromatography column is about 3 μm to about 5 μm, optionally its particle size is about 4 μm. In certain embodiments, the ion pair chromatography matrix and / or the ion pair chromatography column comprises silica, optionally wherein the ion pair chromatography matrix comprises silica particles. In certain embodiments, the ion pair chromatography matrix and / or the ion pair chromatography column is compatible with a mobile phase comprising dibutylammonium acetate and / or triethylammonium acetate. In certain embodiments, the kit comprises a dilution series of one or more reference nucleic acids, optionally wherein the kit comprises a dilution series of a reference gRNA molecule, a first reference mRNA molecule, and / or a second reference mRNA molecule. In certain embodiments, the kit comprises instructions for preparing a nucleic acid extract by a method comprising ethanol precipitation. In certain embodiments, the kit comprises instructions for separating two or more nucleic acids, or three or more nucleic acids, by ion pair chromatography, optionally wherein the nucleic acids comprise RNA molecules, optionally wherein the RNA molecules comprise gRNA molecules, first mRNA molecules, and / or second mRNA molecules. In certain embodiments, the kit includes instructions for determining the concentration of two or more nucleic acids, three or more nucleic acids, and / or the total nucleic acid concentration, optionally wherein the nucleic acids include RNA molecules, optionally wherein the RNA molecules include gRNA molecules, first mRNA molecules, and / or second mRNA molecules.

[0310] Example

[0311] The present disclosure provides exemplary schemes and reagents that can be used to separate the nucleic acid in the lipid nucleic acid assembly composition shown in the present disclosure and / or determine the absolute concentration and / or relative concentration of the nucleic acid in the lipid nucleic acid assembly composition shown in the present disclosure. The present embodiment provides the determination of RNA concentration in the lipid nucleic acid assembly composition, wherein mRNA and sgRNA are encapsulated in LNP. Without wishing to be bound by any particular scientific theory, LNP is decomposed (de-formulated) by ethanol precipitation, thereby releasing RNA. Then, SEC is performed on the extracted RNA, thereby separating them according to the relative size difference of mRNA and sgRNA. Quantitative concentration is carried out by UV detection at 260nm.

[0312] Example 1: Nucleic acid extraction

[0313] This example provides exemplary steps for extracting nucleic acid (here, RNA) from a lipid-nucleic acid assembly composition, where the nucleic acid is encapsulated in LNPs. Figure 1 Exemplary procedures for extracting nucleic acids are also provided.

[0314] 1) Add 1000 μL of ethanol (absolute ethanol; 200 Proof) to the RNase-Free tube appropriate for each lipid-nucleic acid assembly composition.

[0315] 2) Add 100 μL of LNP sample to each ethanol tube and vortex for 3 seconds.

[0316] 3) Centrifuge each tube at 12,000 RCF for 15 minutes at 4°C. After centrifugation, a white RNA precipitate was observed at the bottom of the tube.

[0317] 4) Decant and discard the supernatant ethanol solution by gently inverting the tube without disturbing the pellet at the bottom of the tube.

[0318] 5) Add an additional 1000 μL of ethanol to each tube. Vortex the tubes for 3 seconds and then centrifuge at 12,000 RCF for 15 minutes at 4°C. After centrifugation, a white RNA precipitate is observed at the bottom of the tube.

[0319] 6) Decant and discard the supernatant ethanol solution by gently inverting the tube. A small amount of ethanol may still be observed after decanting. Dry the pellet in a vacuum centrifuge at 25-30°C for 10 minutes ± 5 minutes to ensure that no residual ethanol remains in the vial. If residual ethanol is present, additional drying is required.

[0320] 7) Add 500 μL of RNase-free water to each tube and vortex until the pellet is completely dissolved.

[0321] 8) Centrifuge the tube for 10 seconds to collect the solution at the bottom of the tube.

[0322] The extraction procedure dilutes the sample nucleic acid concentration by 5 times. For example, if the nominal total RNA concentration of the sample provided is 1.5 mg / mL, the extracted sample should contain 300 μg / mL of total RNA, provided that the recovery during the extraction process is 100%. The dilution can be adjusted to produce a nucleic acid extract with a nucleic acid concentration within the range of the desired calibration curve and preferably at or near the midpoint of the calibration curve. The nucleic acid extract is further denatured and cooled before being transferred to an HPLC vial for separation. These steps include heating to 70°C, holding for 10 minutes, and then cooling to 4°C. In this example, the calibration curve range is 12-36 μg / mL mRNA and 8-24 μg / mL sgRNA (i.e., total RNA is 20-60 μg / mL).

[0323] Example 2: Size Exclusion Chromatography of Nucleic Acid Extracts

[0324] This example provides reagents and procedures for SEC separation of nucleic acids in the nucleic acid extract prepared according to Example 1 and determination of nucleic acid concentration.

[0325] One step in the SEC protocol provided herein is the preparation of a dilution series for a calibration curve. This example includes a calibration curve in which a series of five dilutions (working standards, "WS") were prepared using the stock solutions, each including a reference mRNA and a reference sgRNA, as shown in the table below.

[0326] Table 1. Exemplary dilution series for calibration curves

[0327]

[0328] One step in the SEC protocol provided herein is the preparation of the SEC mobile phase. The SEC mobile phase was prepared as a 20 mM Tris-HCl, 150 mM NaCl aqueous solution at pH 7.5.

[0329] SEC was performed using Waters BioSuite UHR SEC. The HPLC system of 4 μ m, 4.6 × 300 mm column (catalog 186002161) and equipped with UV detection (260 nm) or equivalent equipment was carried out, and the HPLC system was equipped with: automatic sample injector (capable of maintaining 5 ± 2 ° C), column heater (capable of maintaining 30 ° C) and capable of delivering 10 μ L injection volume. Column preparation was performed using standard methods, including equilibrium and adjustment, and WS3 was used as a system readiness check. Operating conditions and exemplary injection sequences are provided below, showing three replicate tests for each sample.

[0330] Table 2. Operating conditions

[0331] Mobile phase A: 20 mM Tris-HCl, 150 mM NaCl, pH 7.5 aqueous solution Flow rate: 0.30mL / min column: Waters BioSuite UHR SEC, 125A, 4um, 4.6×300mm Column temperature: 30℃ Autosampler Temperature: 5℃ Injection volume: 10 μL Detector: UV detection Detection wavelength: 260nm Sample time 20 minutes

[0332] Table 3. Exemplary injection sequence

[0333] Sample / Standard Injection times Reserve fluid column adjustment ≥5 WS 3 (40 μg / mL) SRC 2 WS 3 (40 μg / mL) system suitability 5 Blank (NFW) 3* WS1 (20 μg / mL) 1 WS2 (30 μg / mL) 1 WS 3 (40 μg / mL) 1 WS 4 (50 μg / mL) 1 WS 5 (60 μg / mL) 1 WCS 1 Blank (NFW) 1 WS 3 (40 μg / mL) bracket method (Bracket) 1 Sample 1 - Replicate 1 Sample 1 - Replicate 2 Sample 1 - Repeat 3 Repeat 1 time each time Sample 2 - Repeat 1 (6 injections in total) Sample 2 - Replicate 2 Sample 2 - Replicate 3 WS 3 (40 μg / mL) bracket method 1 Sample 3 - Repeat 1 Sample 3 - Repeat 2 Sample 3 - Replicate 3 Repeat 1 time each time Sample 4 - Repeat 1 (6 injections in total) Sample 4 - Repeat 2 Sample 4 - Repeat 3 WS 3 (40 μg / mL) bracket method 1 Blank(NFW) 1

[0334] * The last injection was used for non-interference evaluation.

[0335] Using the calibration curves, the mRNA and sgRNA concentrations in the samples were calculated (in μg / mL). The calculated concentrations were corrected by the dilution factor, which was 37.5x (5x initial dilution during extraction × 7.5x post-extraction dilution). Chromatograms of samples were accepted based on a USP resolution of 1.5 or greater between the mRNA and sgRNA peaks, where the %RSD of the total RNA concentration (in mg / mL) across replicates was equal to or less than 10%. Figure 2-4 Exemplary chromatograms are provided.

[0336] It has been demonstrated that the nucleic acid separation according to Examples 1 and 2 provides various advantages illustrated herein. This example demonstrates that the nucleic acid separation methods provided in the present disclosure provide robust separation of nucleic acids and easy sample preparation (e.g., fewer and / or easier steps required for nucleic acid extraction than various methods known in the art). High-purity nucleic acid extracts were prepared from complex samples including LNPs, and these nucleic acid extracts were highly compatible with chromatography, at least because they did not contain or were substantially free of materials that are incompatible with chromatography techniques (e.g., SEC) (e.g., that may cause turbidity and / or be detrimental to chromatographic performance), such as lipids, buffers, organic solvents, and / or detergents. This example demonstrates that the nucleic acid separation methods provided in the present disclosure also provide high accuracy, precision, and recovery, including when applied to small nucleic acids (e.g., sgRNA) and low-concentration nucleic acids (e.g., total nucleic acid concentrations below 100 μg / mL).

[0337] Example 3: Nucleic acid extraction

[0338] This example provides exemplary steps for extracting nucleic acids (here, two mRNAs and one sgRNA) from a lipid-nucleic acid assembly composition, where the nucleic acids are encapsulated in LNPs. Figure 5 Exemplary procedures for extracting nucleic acids are also provided.

[0339] 1) Add 1000 μL of ethanol (absolute ethanol; 200 Proof) to the RNase-Free tube appropriate for each lipid-nucleic acid assembly composition.

[0340] 2) Add 100 μL of LNP sample to each ethanol tube and vortex for 5 seconds.

[0341] 3) Centrifuge each tube at 12,000 RCF for 10 minutes at 4°C. After centrifugation, a white RNA precipitate was observed at the bottom of the tube.

[0342] 4) Decant and discard the supernatant ethanol solution without disturbing the sediment at the bottom of the tube.

[0343] 5) Centrifuge each tube at 12,000 RCF for 2 minutes at 4°C. After centrifugation, a white RNA precipitate is observed at the bottom of the tube.

[0344] 6) Remove the remaining ethanol with a pipette.

[0345] 7) Add 1000 μL of RNase-free water to each tube and vortex until the precipitate is completely dissolved. This solution is a 10-fold dilution of the carrier.

[0346] 8) The solution was further diluted to a final RNA concentration of approximately 50 μg / mL.

[0347] The extraction procedure dilutes the sample nucleic acid concentration by a factor of 10. For example, if the sample is provided with a nominal total RNA concentration of 1.5 mg / mL, the extracted sample should contain 150 μg / mL of total RNA, assuming 100% recovery during the extraction process. The dilution can be adjusted to produce a nucleic acid extract with a nucleic acid concentration within the desired calibration curve range, preferably at or near the midpoint of the calibration curve. The nucleic acid extract is further denatured and cooled before being transferred to an HPLC vial for separation.

[0348] Example 4: Ion-pair reversed phase (IPRP) chromatography of nucleic acid extracts

[0349] This example provides reagents and procedures for performing IP RP chromatography on nucleic acids in the nucleic acid extract prepared according to Example 3 and determining the nucleic acid concentration.

[0350] One step in the IP RP chromatography protocol provided herein is the preparation of a dilution series for a calibration curve. In this example, the calibration curve range is for standards with a total RNA concentration of 12-100 μg / mL. This example includes a calibration curve in which a series of five dilutions were prepared using the stock solutions (Table 4), each containing a reference 0.1 kb sgRNA, a reference 1.0 kb mRNA, and a reference 4.5 kb mRNA at a concentration ratio of 1:2:1, and the total RNA concentrations are as shown in Table 5.

[0351] Table 4. Exemplary Standard RNA Test Kit

[0352]

[0353] Table 5. Exemplary dilution series for calibration curves

[0354] point Total concentration 1 100 μg / mL 2 75 μg / mL 3 50 μg / mL 4 25 μg / mL 5 12 μg / mL

[0355] One step of the IP RP chromatography protocol provided herein is the preparation of the chromatography mobile phase. The prepared IP RP chromatography mobile phase is 50 mM dibutylammonium acetate, 100 mM triethylammonium acetate in water, or 50 mM dibutylammonium acetate, 100 mM triethylammonium acetate, 50% acetonitrile in water.

[0356] IP RP chromatography is performed using DNAPac TM RP column, 4 μ m, 2.1 × 100 mm column (Cat. No. DX088923) and equipped with UV detection (260 nm) or equivalent HPLC system, the HPLC system is equipped with: autosampler (capable of maintaining 4 ± 2 ° C), column heater (capable of maintaining 65 ° C) and capable of delivering 10 μ L injection volume. Column preparation, including equilibration and conditioning, is performed using standard methods. Operating conditions and exemplary injection sequences are provided below, showing three replicates of each sample.

[0357] Table 6. Exemplary operating conditions

[0358]

[0359]

[0360] Table 7. Exemplary operating conditions

[0361]

[0362] Using the calibration curves, the mRNA and sgRNA concentrations in the samples were calculated (in μg / mL). The calculated concentrations were corrected for the carrier dilution factor, which was 30-fold (10-fold initial dilution during extraction × 3-fold post-extraction dilution). Chromatograms of samples were accepted based on a USP resolution between mRNA and sgRNA peaks of equal to or greater than 1.5, with a %RSD of total RNA concentration (in mg / mL) equal to or less than 10% across replicates. Figure 6-8 Exemplary chromatograms are provided.

[0363] Table 8. Example standard weighing calculations

[0364] 100 μg / mL 75 μg / mL 50 μg / mL 25 μg / mL 12.5 μg / mL 0.1kb sgRNA 25 18.75 12.5 6.25 3.125 1.0kb mRNA 50 37.5 25 12.5 6.25 4.5kb mRNA 25 18.75 12.5 6.25 3.125

[0365] Table 9. Exemplary weighing calculations for LNP extraction ( Figure 8 )

[0366]

[0367]

[0368] Other implementation plans

[0369] It should be understood that the scope of the present disclosure is defined by what should be understood in the present disclosure and claims, rather than by the specific embodiments presented by way of example. Elements described for one aspect or embodiment of the present disclosure should be considered to be encompassed in other aspects or embodiments of the present disclosure. For example, an element of a claim that is directly or indirectly subordinate to a certain independent claim presented herein may serve as support for elements presented in additional dependent claims of one or more other independent claims. Throughout the specification, when a composition or method is described as having, including or containing a particular element, a composition that is essentially composed of, consists of or does not contain the element is also disclosed herein. All references cited herein are incorporated herein by reference.

Claims

1. A method comprising: a) preparing a nucleic acid extract from the lipid-nucleic acid assembly composition, wherein the preparation of the nucleic acid extract comprises ethanol precipitation of the nucleic acids in the lipid-nucleic acid assembly composition; and b) performing size exclusion chromatography (SEC) on the nucleic acid extract.

2. The method of claim 1, wherein the method further comprises determining the total concentration of nucleic acids present in the lipid-nucleic acid assembly composition.

3. A method for determining the total concentration of nucleic acids present in a lipid-nucleic acid assembly composition, the method comprising: a) preparing a nucleic acid extract from the lipid-nucleic acid assembly composition, wherein the preparation of the nucleic acid extract comprises ethanol precipitation of nucleic acids in the lipid-nucleic acid assembly composition; b) performing size exclusion chromatography (SEC) on the nucleic acid extract; and c) determining the total concentration of nucleic acids in the lipid-nucleic acid assembly composition.

4. The method of any one of claims 1 to 3, wherein the nucleic acids present in the lipid-nucleic acid assembly composition comprise at least a first nucleic acid and a second nucleic acid, and wherein the SEC separates the first nucleic acid from the second nucleic acid.

5. The method of claim 4, wherein the method further comprises determining the concentration of the first nucleic acid and / or the second nucleic acid in the lipid-nucleic acid assembly composition.

6. The method of any one of claims 1-3, wherein the nucleic acids present in the lipid nucleic acid assembly composition comprise at least a first nucleic acid, a second nucleic acid, and a third nucleic acid, and wherein the SEC separates the first nucleic acid from the second nucleic acid and / or the third nucleic acid.

7. The method of any one of claims 4 to 6, wherein the first nucleic acid is a ribonucleic acid (RNA) molecule.

8. The method of claim 7, wherein the first nucleic acid is a messenger RNA (mRNA) molecule.

9. The method of claim 8, wherein the mRNA molecule encodes a polypeptide, optionally wherein the polypeptide comprises an RNA-guided DNA binder, optionally wherein the RNA-guided DNA binder comprises a nuclease, optionally wherein the nuclease is a Cas protein, optionally wherein the RNA-guided DNA binder is a fusion polypeptide.

10. The method of claim 8 or 9, wherein the first nucleic acid encodes a base editor.

11. The method of claim 10, wherein the base editor is a cytosine base editor.

12. The method of claim 10, wherein the base editor is an adenine base editor.

13. The method of any one of claims 4 to 12, wherein the first nucleic acid comprises 1000 to 7000 nucleotides.

14. The method of any one of claims 4 to 13, wherein the second nucleic acid is an RNA molecule.

15. The method of claim 14, wherein the second nucleic acid is a guide RNA (gRNA) molecule, optionally wherein the gRNA molecule is an sgRNA molecule.

16. The method of claim 14 or 15, wherein the second nucleic acid comprises 75-200 nucleotides, optionally wherein the second nucleic acid comprises 80-120 nucleotides.

17. The method of any one of claims 4-16, wherein the nucleic acids present in the lipid-nucleic acid assembly composition consist of or consist essentially of the first nucleic acid and the second nucleic acid.

18. The method of any one of claims 4-17, wherein the total concentration of nucleic acids in the lipid-nucleic acid assembly composition is the sum of the concentration of the first nucleic acid and the concentration of the second nucleic acid.

19. The method of any one of claims 4 to 18, wherein the concentration of the first nucleic acid and / or the second nucleic acid is determined based on a calibration curve, wherein the calibration curve represents the concentration of nucleic acids in a dilution series of at least one reference nucleic acid.

20. The method of claim 19, wherein the concentrations of the first nucleic acid and the second nucleic acid are determined separately, wherein the concentration of the first nucleic acid is determined according to a first calibration curve and the concentration of the second nucleic acid is determined according to a second calibration curve.

21. The method of claim 20, wherein the first nucleic acid is an mRNA molecule and the first calibration curve is a dilution series of a reference mRNA molecule.

22. The method of claim 20 or 21, wherein the second nucleic acid is a gRNA molecule and the second calibration curve is a dilution series of a reference gRNA molecule.

23. The method of any one of claims 20-22, wherein the first calibration curve represents a first dilution series comprising concentrations of reference mRNA molecules covering a range comprising 12 μg / mL and 60 μg / mL, and / or a range comprising 12 μg / mL and 36 μg / mL.

24. The method of any one of claims 20-23, wherein the second calibration curve represents a second dilution series comprising concentrations of reference gRNA molecules covering a range comprising 8 μg / mL and 40 μg / mL, and / or a range comprising 8 μg / mL and 24 μg / mL.

25. The method of any one of claims 20-24, wherein the first dilution series and the second dilution series comprise concentrations such that increasing the concentration in the first dilution series pairwise with the concentration in the second dilution series results in a total RNA concentration encompassing a range between 20 μg / mL and 100 μg / mL, inclusive, or a range between 20 μg / mL and 60 μg / mL, inclusive.

26. The method of any one of claims 4-25, wherein the method comprises determining the concentration of the first nucleic acid and / or the second nucleic acid, and / or the total nucleic acid concentration in the lipid-nucleic acid assembly composition, wherein the concentration is equal to or less than 2.5 mg / mL, 2.0 mg / mL, 1.5 mg / mL, 1.0 mg / mL, 0.5 mg / mL, 0.4 mg / mL, or 0.3 mg / mL.

27. The method of any one of claims 4-26, wherein the method comprises determining the concentration of the first nucleic acid and / or the second nucleic acid, and / or the total nucleic acid concentration, wherein the total nucleic acid concentration is between about 0.5 mg / mL and about 2 mg / mL, optionally wherein the total nucleic acid concentration is between about 0.75 mg / mL and about 1.8 mg / mL.

28. The method of any one of claims 4-27, wherein the SEC produces a chromatogram wherein the USP resolution between the nucleic acid peak representing the first nucleic acid and the nucleic acid peak representing the second nucleic acid is equal to or greater than 1.

5.

29. The method of any one of claims 1-28, wherein the preparation of the nucleic acid extract comprises diluting the concentration of the nucleic acid extract to the midpoint of a calibration curve.

30. The method of any one of claims 1-29, wherein the lipid-nucleic acid assembly composition comprises lipid nanoparticles (LNPs) containing the nucleic acid.

31. The method of claim 30, wherein the lipid component of the LNP comprises (i) an amine lipid, (ii) a helper lipid, and (iii) a stealth lipid, optionally wherein the LNP comprises a neutral lipid, optionally wherein the stealth lipid is a PEG lipid.

32. The method of any one of claims 4 to 31, wherein: The first nucleic acid is a messenger RNA (mRNA) molecule encoding an RNA-guided DNA-binding agent, The second nucleic acid is a gRNA molecule, The lipid nucleic acid assembly composition comprises an LNP containing the first nucleic acid molecule and the second nucleic acid molecule, and The lipid component of the LNP comprises (i) an amine lipid, (ii) a helper lipid, (iii) a stealth lipid, and (iv) a neutral lipid, optionally wherein the stealth lipid is a PEG lipid.

33. The method of any one of claims 1-32, wherein the total concentration of nucleic acids in the lipid-nucleic acid assembly composition is the total concentration of RNA in the lipid-nucleic acid assembly composition.

34. The method of any one of claims 1 to 33, wherein the ethanol precipitation comprises: a) mixing the lipid-nucleic acid assembly composition with ethanol to produce an ethanol mixture; b) centrifuging the ethanol mixture, wherein the centrifugation produces a nucleic acid precipitate and an ethanol supernatant; and c) separating the nucleic acid precipitate from the ethanol, wherein the nucleic acid precipitate comprises precipitated RNA.

35. The method of claim 34, wherein separation of the precipitate comprises decanting the supernatant and / or drying the nucleic acid precipitate.

36. The method of claim 34 or 35, wherein the preparation of the nucleic acid extract comprises resuspending the nucleic acid precipitate in water to produce an aqueous nucleic acid solution.

37. The method of any one of claims 34-36, wherein the preparation of the nucleic acid extract comprises heating the aqueous nucleic acid solution to a temperature between about 65°C and about 90°C for at least about 2 minutes to about 15 minutes, optionally wherein the preparation of the nucleic acid extract comprises cooling the aqueous nucleic acid solution after the heating, optionally wherein the cooling is to 4°C.

38. The method of any one of claims 1-37, wherein the nucleic acid extract is free or substantially free of lipids, free or substantially free of buffers, and / or free or substantially free of detergents.

39. The method of any one of claims 1-38, wherein the SEC is high performance liquid chromatography (HPLC).

40. The method of any one of claims 1-39, wherein performing SEC on the nucleic acid extract comprises contacting the nucleic acid extract with a SEC matrix, optionally wherein the SEC matrix is ​​or is present in a SEC column.

41. The method of claim 40, wherein performing SEC on the nucleic acid extract comprises contacting the nucleic acid extract with a SEC column having an inner diameter of 3 mm to 6 mm, optionally having an inner diameter of about 4 mm to about 5 mm, optionally having an inner diameter of about 4.6 mm inner diameter.

42. The method of claim 40 or 41, wherein performing SEC on the nucleic acid extract comprises contacting the nucleic acid extract with a SEC column having a length of about 100 mm to about 400 mm, optionally about 300 mm long.

43. The method of any one of claims 1-42, wherein performing SEC on the nucleic acid extract comprises contacting the nucleic acid extract with a SEC matrix and / or a SEC column having a pore size of about 5 nm to about 50 nm, optionally wherein the pore size is about 12.5 nm to about 25 nm.

44. The method of any one of claims 1-43, wherein performing SEC on the nucleic acid extract comprises contacting the nucleic acid extract with a SEC matrix and / or a SEC column having a particle size of about 3 μm to about 5 μm, optionally having a particle size of about 4 μm.

45. The method of any one of claims 1-44, wherein performing SEC on the nucleic acid extract comprises contacting the nucleic acid extract with a SEC matrix and / or a SEC column comprising silica, optionally wherein the SEC matrix and / or SEC column comprises silica particles.

46. ​​The method of any one of claims 1 to 45, wherein performing SEC on the nucleic acid extract comprises contacting the nucleic acid extract with a SEC matrix and / or a SEC column that is compatible with a mobile phase having a pH of about 7 to about 8, optionally wherein the SEC matrix and / or SEC column is compatible with a mobile phase having a pH of about 7.3 to about 7.7, optionally wherein the SEC matrix and / or SEC column is compatible with a mobile phase having a pH of about 7.

5.

47. The method of any one of claims 1 to 46, wherein performing SEC on the nucleic acid extract comprises contacting the nucleic acid extract with a SEC matrix and / or a SEC column, wherein the SEC matrix and / or the SEC column is compatible with a mobile phase comprising Tris-HCl, optionally wherein the SEC matrix and / or the SEC column is compatible with a mobile phase comprising about 5 mM to about 50 mM Tris-HCl, optionally wherein the SEC matrix and / or the SEC column is compatible with a mobile phase comprising about 20 mM Tris-HCl.

48. The method of any one of claims 1 to 47, wherein performing SEC on the nucleic acid extract comprises contacting the nucleic acid extract with a SEC matrix and / or a SEC column compatible with a mobile phase comprising about 25 mM to about 200 mM NaCl, optionally wherein the SEC matrix and / or SEC column is compatible with a mobile phase comprising about 150 mM NaCl.

49. The method of any one of claims 1-48, wherein performing SEC on the nucleic acid extract comprises contacting the nucleic acid extract with a SEC mobile phase having a pH between about 7 and about 8, optionally wherein the pH of the mobile phase is about 7.3 to about 7.7, optionally wherein the pH of the mobile phase is about 7.

5.

50. The method of any one of claims 1-49, wherein performing SEC on the nucleic acid extract comprises contacting the nucleic acid extract with a SEC mobile phase comprising Tris-HCl, optionally wherein the SEC mobile phase comprises about 5 mM to about 50 mM Tris-HCl, optionally wherein the SEC mobile phase comprises about 20 mM Tris-HCl.

51. The method of any one of claims 1-50, wherein performing SEC on the nucleic acid extract comprises contacting the nucleic acid extract with a SEC mobile phase comprising NaCl, optionally wherein the SEC mobile phase comprises about 25 mM to about 200 mM NaCl, optionally wherein the SEC mobile phase comprises about 150 mM NaCl.

52. The method of any one of claims 1-51, wherein the method comprises measuring the absorbance of the SEC eluate at 260 nm.

53. The method of any one of claims 1-52, wherein the SEC is performed at a temperature of 20-40°C, optionally wherein the SEC is performed at a temperature of 30°C.

54. A kit for isolating nucleic acids present in a lipid nucleic acid assembly composition, the kit comprising a size exclusion chromatography (SEC) matrix and at least one reagent for ethanol precipitation of nucleic acids in the lipid nucleic acid assembly composition, optionally wherein the kit is for isolating two or more nucleic acids, optionally wherein the two or more nucleic acid molecules are or include gRNA and mRNA.

55. The kit of claim 54, wherein the SEC matrix is ​​or is present in a SEC column.

56. The kit of claim 55, wherein the SEC column has an inner diameter of 3 mm to 6 mm, optionally an inner diameter of about 4 mm to about 5 mm, optionally an inner diameter of about 4.6 mm inner diameter.

57. The kit of claim 55 or 56, wherein the SEC column is about 100 mm to about 400 mm in length, optionally about 300 mm in length.

58. The kit of any one of claims 54-57, wherein the SEC matrix and / or SEC column has a pore size of about 5 nm to about 50 nm, optionally wherein the pore size is about 12.5 nm to about 25 nm.

59. The kit of any one of claims 54-58, wherein the SEC matrix and / or SEC column has a particle size of about 3 μm to about 5 μm, optionally about 4 μm.

60. The method of any one of claims 54-59, wherein the SEC matrix and / or SEC column comprises silica, optionally wherein the SEC matrix comprises silica particles.

61. The kit of any one of claims 54-50, wherein the SEC matrix and / or SEC column is compatible with a mobile phase having a pH of about 7 to about 8, optionally with a mobile phase having a pH of about 7.3 to about 7.7, optionally with a mobile phase having a pH of about 7.

5.

62. The kit of any one of claims 54-61, wherein the SEC matrix and / or SEC column is compatible with a mobile phase comprising about 25 mM to about 200 mM NaCl, optionally with a mobile phase comprising about 150 mM NaCl.

63. The kit of any one of claims 54-62, wherein the kit comprises a dilution series of one or more reference nucleic acids, optionally wherein the kit comprises a dilution series of reference gRNA molecules and / or reference mRNA molecules.

64. The kit of any one of claims 54-63, wherein the kit comprises instructions for preparing a nucleic acid extract by a method comprising ethanol precipitation.

65. The kit of any one of claims 54-64, comprising instructions for separating the two or more nucleic acids by SEC, optionally wherein the two or more nucleic acids comprise or consist of RNA molecules, optionally wherein the RNA molecules comprise or consist of gRNA molecules and mRNA molecules.

66. The kit of any one of claims 54-65, wherein the kit comprises instructions for determining the concentration of the two or more nucleic acids and / or the total nucleic acid concentration, optionally wherein the two or more nucleic acids comprise or consist of RNA molecules, optionally wherein the RNA molecules comprise or consist of gRNA molecules and mRNA molecules.

67. A method comprising: a) preparing a nucleic acid extract from the lipid-nucleic acid assembly composition, wherein the preparation of the nucleic acid extract comprises ethanol precipitation of the nucleic acids in the lipid-nucleic acid assembly composition; and b) performing ion-pair chromatography on the nucleic acid extract.

68. The method of claim 67, wherein the method further comprises determining the total concentration of nucleic acids present in the lipid-nucleic acid assembly composition.

69. A method for determining the total concentration of nucleic acids present in a lipid-nucleic acid assembly composition, the method comprising: a) preparing a nucleic acid extract from the lipid-nucleic acid assembly composition, wherein the preparation of the nucleic acid extract comprises ethanol precipitation of nucleic acids in the lipid-nucleic acid assembly composition; b) performing ion-pair chromatography on the nucleic acid extract; and c) determining the total concentration of nucleic acids in the lipid-nucleic acid assembly composition.

70. The method of any one of claims 67-69, wherein the nucleic acids present in the lipid-nucleic acid assembly composition comprise at least a first nucleic acid and a second nucleic acid, and wherein the ion pair chromatography separates the first nucleic acid from the second nucleic acid.

71. The method of claim 70, wherein the method further comprises determining the concentration of the first nucleic acid and / or the second nucleic acid in the lipid-nucleic acid assembly composition.

72. The method of any one of claims 67-69, wherein the nucleic acids present in the lipid-nucleic acid assembly composition comprise at least a first nucleic acid, a second nucleic acid, and a third nucleic acid, and wherein SEC separates the first nucleic acid from the second nucleic acid and / or the third nucleic acid.

73. The method of any one of claims 70-72, wherein the first nucleic acid is a ribonucleic acid (RNA) molecule.

74. The method of claim 73, wherein the first nucleic acid is a messenger RNA (mRNA) molecule.

75. The method of claim 74, wherein the mRNA molecule encodes a polypeptide, optionally wherein the polypeptide comprises an RNA-guided DNA binder, optionally wherein the RNA-guided DNA binder comprises a nuclease, optionally wherein the nuclease is a Cas protein, optionally wherein the RNA-guided DNA binder is a fusion polypeptide.

76. The method of claim 74 or 75, wherein the first nucleic acid encodes a base editor.

77. The method of claim 76, wherein the base editor is a cytosine base editor.

78. The method of any one of claims 70-77, wherein the first nucleic acid comprises 1000-7000 nucleotides.

79. The method of any one of claims 70-78, wherein the second nucleic acid is an RNA molecule.

80. The method of claim 79, wherein the second nucleic acid is a guide RNA (gRNA) molecule, optionally wherein the gRNA molecule is an sgRNA molecule.

81. The method of claim 79 or 80, wherein the second nucleic acid comprises 75-200 nucleotides, optionally wherein the second nucleic acid comprises 80-120 nucleotides.

82. The method of any one of claims 70-81, wherein the third nucleic acid is a ribonucleic acid (RNA) molecule.

83. The method of claim 82, wherein the third nucleic acid is a messenger RNA (mRNA) molecule.

84. The method of claim 83, wherein the mRNA molecule encodes a polypeptide, optionally wherein the polypeptide is a DNA glycosylase inhibitor, optionally wherein the DNA glycosylase inhibitor is UGI.

85. The method of any one of claims 70-84, wherein the third nucleic acid comprises 750-3000 nucleotides.

86. The method of any one of claims 70-85, wherein the nucleic acid present in the lipid nucleic acid assembly composition consists of or consists essentially of: (i) the first nucleic acid and the second nucleic acid; or (ii) the first nucleic acid, the second nucleic acid and the third nucleic acid.

87. The method of any one of claims 70-86, wherein the total concentration of nucleic acids in the lipid-nucleic acid assembly composition is the sum of: (i) the concentration of the first nucleic acid and the concentration of the second nucleic acid; or (ii) the concentration of the first nucleic acid, the concentration of the second nucleic acid, and the concentration of the third nucleic acid.

88. The method of any one of claims 70-87, wherein (i) the concentration of the first nucleic acid and / or the second nucleic acid is determined according to a calibration curve, or (ii) the concentration of the first nucleic acid, the second nucleic acid and / or the third nucleic acid is determined according to a calibration curve, wherein the calibration curve represents the concentration of nucleic acids in a dilution series of at least one reference nucleic acid.

89. The method of claim 88, wherein (i) the concentrations of the first nucleic acid and the second nucleic acid are determined separately, and / or (ii) the concentrations of the first nucleic acid, the second nucleic acid, and the third nucleic acid are determined separately, wherein the concentration of the first nucleic acid is determined according to a first calibration curve, the concentration of the second nucleic acid is determined according to a second calibration curve, and when the third nucleic acid is present, the concentration of the third nucleic acid is determined according to a third calibration curve.

90. The method of claim 89, wherein the first nucleic acid is an mRNA molecule and the first calibration curve is a dilution series of a first reference mRNA molecule, optionally wherein the length and / or sequence of the first reference mRNA molecule is identical to the first nucleic acid or differs from the first nucleic acid by no more than 10%, no more than 5%, or no more than 1%.

91. The method of claim 89 or 90, wherein the second nucleic acid is a gRNA molecule and the second calibration curve is a dilution series of a reference gRNA molecule, optionally wherein the length and / or sequence of the reference gRNA molecule is identical to the second nucleic acid or differs from the second nucleic acid by no more than 10%, no more than 5%, or no more than 1%.

92. The method of any one of claims 89-91, wherein the third nucleic acid is an mRNA molecule and the third calibration curve is a dilution series of a second reference mRNA molecule, optionally wherein the length and / or sequence of the second reference mRNA molecule is identical to the third nucleic acid or differs from the third nucleic acid by no more than 10%, no more than 5%, or no more than 1%.

93. The method of any one of claims 89-92, wherein the first calibration curve represents a first dilution series comprising concentrations of a first reference mRNA molecule encompassing a range comprising 10 μg / mL and 100 μg / mL.

94. The method of any one of claims 89-93, wherein the second calibration curve represents a second dilution series comprising concentrations of a reference gRNA molecule encompassing a range comprising 5 μg / mL and 100 μg / mL, and / or a range comprising 5 μg / mL and 50 μg / mL.

95. The method of any one of claims 89-94, wherein the third calibration curve represents a third dilution series comprising concentrations of a second reference mRNA molecule encompassing a range comprising 10 μg / mL and 100 μg / mL.

96. The method of any one of claims 89-95, wherein the first dilution series and the second dilution series comprise concentrations such that the total concentration resulting from the first dilution series, the second dilution series, and the third dilution series results in a total RNA concentration encompassing a range between 20 μg / mL and 200 μg / mL, inclusive, or a range between 20 μg / mL and 100 μg / mL, inclusive.

97. The method of any one of claims 70-96, wherein the method comprises determining the concentration of the first nucleic acid, the second nucleic acid, and / or the third nucleic acid, and / or the total nucleic acid concentration in the lipid-nucleic acid assembly composition, wherein the concentration is equal to or less than 2.5 mg / mL, 2.0 mg / mL, 1.5 mg / mL, 1.0 mg / mL, 0.5 mg / mL, 0.4 mg / mL, or 0.3 mg / mL.

98. The method of any one of claims 70-97, wherein the method comprises determining the concentration of the first nucleic acid, the second nucleic acid, and / or the third nucleic acid, and / or the total nucleic acid concentration, wherein the total nucleic acid concentration is between about 0.5 mg / mL and about 2 mg / mL, optionally wherein the total nucleic acid concentration is between about 0.75 mg / mL and about 1.8 mg / mL.

99. The method of any one of claims 70-98, wherein the ion pair chromatography produces a chromatogram wherein the USP resolution between the nucleic acid peak representing the first nucleic acid and the nucleic acid peak representing the second nucleic acid is equal to or greater than 1, 1.5, 2, 2.5, or 3.

100. The method of any one of claims 70-99, wherein the ion pair chromatography produces a chromatogram wherein the USP resolution between the nucleic acid peak representing the first nucleic acid and the nucleic acid peak representing the third nucleic acid is equal to or greater than 1, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.

101. The method of any one of claims 70-100, wherein the ion pair chromatography produces a chromatogram wherein the USP resolution between the nucleic acid peak representing the second nucleic acid and the nucleic acid peak representing the third nucleic acid is equal to or greater than 1, 1.5, or 2.

102. The method of any one of claims 67-101, wherein the preparation of the nucleic acid extract comprises diluting the concentration of the nucleic acid extract to the midpoint of a calibration curve.

103. The method of any one of claims 67-102, wherein the lipid nucleic acid assembly composition comprises lipid nanoparticles (LNPs) containing the nucleic acid.

104. The method of claim 103, wherein the lipid component of the LNP comprises (i) an amine lipid, (ii) a helper lipid, and (iii) a stealth lipid, optionally wherein the LNP comprises a neutral lipid, optionally wherein the stealth lipid is a PEG lipid.

105. The method of any one of claims 72-104, wherein: The first nucleic acid is a messenger RNA (mRNA) molecule encoding a cytosine base editor, The second nucleic acid is a gRNA molecule, optionally wherein the gRNA molecule is an sgRNA molecule, and The third nucleic acid is an mRNA molecule encoding a DNA glycosylase inhibitor, optionally wherein the DNA glycosylase inhibitor is UGI.

106. The method of claim 105, wherein the first nucleic acid does not encode a DNA glycosylase inhibitor and / or wherein the first nucleic acid does not encode a UGI.

107. The method of claim 105, wherein the cytosine base editor comprises a DNA glycosylase inhibitor, optionally wherein the DNA glycosylase inhibitor is UGI.

108. The method of any one of claims 72-107, wherein: The first nucleic acid is a messenger RNA (mRNA) molecule encoding a cytosine base editor, The second nucleic acid is a gRNA molecule, The third nucleic acid is an mRNA molecule encoding a DNA glycosylase inhibitor, optionally wherein the DNA glycosylase inhibitor is a uracil glycosylase inhibitor (UGI), the lipid nucleic acid assembly composition comprises an LNP comprising the first nucleic acid molecule and the second nucleic acid molecule, and The lipid component of the LNP comprises (i) an amine lipid, (ii) a helper lipid, (iii) a stealth lipid, and (iv) a neutral lipid, optionally wherein the stealth lipid is a PEG lipid.

109. The method of any one of claims 67-108, wherein the total concentration of nucleic acids in the lipid-nucleic acid assembly composition is the total concentration of RNA in the lipid-nucleic acid assembly composition.

110. The method of any one of claims 67-109, wherein the ethanol precipitation comprises: a) mixing the lipid-nucleic acid assembly composition with ethanol to produce an ethanol mixture; b) centrifuging the ethanol mixture, wherein the centrifugation produces a nucleic acid precipitate and an ethanol supernatant; and c) separating the nucleic acid precipitate from the ethanol, wherein the nucleic acid precipitate comprises precipitated RNA.

111. The method of claim 110, wherein separating the precipitate comprises decanting the supernatant, pipetting the supernatant, and / or drying the nucleic acid precipitate.

112. The method of claim 110 or 111, wherein the preparation of the nucleic acid extract comprises resuspending the nucleic acid precipitate in water to produce an aqueous nucleic acid solution.

113. The method of any one of claims 110-112, wherein the preparation of the nucleic acid extract comprises heating the aqueous nucleic acid solution to a temperature between about 65°C and about 90°C for at least about 2 minutes to about 15 minutes, optionally wherein the preparation of the nucleic acid extract comprises cooling the aqueous nucleic acid solution after the heating, optionally wherein the cooling is to 4°C.

114. The method of any one of claims 67-113, wherein the nucleic acid extract is free or substantially free of lipids, free or substantially free of buffers, and / or free or substantially free of detergents.

115. The method of any one of claims 67-114, wherein the ion pair chromatography is reverse phase chromatography.

116. The method of any one of claims 67-115, wherein the ion-pair chromatography is high performance liquid chromatography (HPLC).

117. The method of any one of claims 67-116, wherein the ion-pair chromatography is reverse-phase high performance liquid chromatography (HPLC).

118. The method of any one of claims 67-117, wherein subjecting the nucleic acid extract to ion-pair chromatography comprises contacting the nucleic acid extract with an ion-pair chromatography matrix, optionally wherein the ion-pair chromatography matrix is ​​or is present in an ion-pair chromatography column.

119. The method of claim 118, wherein performing ion-pair chromatography on the nucleic acid extract comprises contacting the nucleic acid extract with an ion-pair chromatography column having an inner diameter of 1 mm to 4 mm, optionally an inner diameter of about 1.5 mm to about 2.5 mm, optionally an inner diameter of about 2.1 mm inner diameter.

120. The method of claim 118 or 119, wherein performing ion-pair chromatography on the nucleic acid extract comprises contacting the nucleic acid extract with an ion-pair chromatography column having a length of about 50 mm to about 400 mm, optionally about 100 mm long.

121. The method of any one of claims 67-120, wherein subjecting the nucleic acid extract to ion pair chromatography comprises subjecting the nucleic acid extract to a pore size of about to about contacting an ion-pair chromatography matrix and / or an ion-pair chromatography column, optionally wherein the pore size is about to about 122. The method of any one of claims 67-121, wherein subjecting the nucleic acid extract to ion-pair chromatography comprises contacting the nucleic acid extract with an ion-pair chromatography matrix and / or an ion-pair chromatography column having a particle size of about 3 μm to about 5 μm, optionally having a particle size of about 4 μm.

123. The method of any one of claims 67 to 122, wherein performing ion-pair chromatography on the nucleic acid extract comprises contacting the nucleic acid extract with an ion-pair chromatography matrix and / or an ion-pair chromatography column comprising silica, optionally wherein the ion-pair chromatography matrix and / or ion-pair chromatography column comprises silica particles.

124. The method of any one of claims 67-123, wherein subjecting the nucleic acid extract to ion-pair chromatography comprises contacting the nucleic acid extract with an ion-pair chromatography matrix and / or an ion-pair chromatography column that is compatible with at least one mobile phase comprising dibutylammonium acetate and / or triethylammonium acetate.

125. The method of any one of claims 67-124, wherein the at least one mobile phase comprises 25 mM to 300 mM dibutylammonium acetate, optionally wherein the mobile phase comprises 50 mM dibutylammonium acetate.

126. The method of any one of claims 67-125, wherein the at least one mobile phase comprises 25 mM to 300 mM triethylammonium acetate, optionally wherein the mobile phase comprises 100 mM triethylammonium acetate.

127. The method of any one of claims 67-126, wherein the at least one mobile phase comprises 25% to 75% acetonitrile in water, optionally wherein the mobile phase comprises 50% acetonitrile in water.

128. The method of any one of claims 67-127, wherein the method comprises measuring the absorbance of an ion pair chromatography eluate at 260 nm.

129. The method of any one of claims 67-128, wherein the ion-pair chromatography is performed at a temperature of 20-80°C, optionally wherein the ion-pair chromatography is performed at a temperature of 65°C.

130. A kit for separating nucleic acids present in a lipid nucleic acid assembly composition, the kit comprising an ion pair chromatography matrix and at least one reagent for ethanol precipitation of nucleic acids in the lipid nucleic acid assembly composition, optionally wherein the kit is for separating two or more nucleic acids, optionally wherein the two or more nucleic acid molecules are or include gRNA and mRNA; and / or for separating three or more nucleic acids, optionally wherein the three or more nucleic acid molecules are or include gRNA, a first mRNA, and a second mRNA.

131. The kit of claim 130, wherein the ion pair chromatography matrix is ​​or is present in an ion pair chromatography column.

132. The kit of claim 131, wherein the ion pair chromatography column has an inner diameter of 1 mm to 4 mm, optionally an inner diameter of about 1.5 mm to about 2.5 mm, optionally an inner diameter of about 2.1 mm inner diameter.

133. The kit of claim 131 or 132, wherein the ion pair chromatography column is about 50 mm to about 400 mm in length, optionally about 100 mm in length.

134. The kit of any one of claims 130-133, wherein the pore size of the ion pair chromatography matrix and / or ion pair chromatography column is about to about Optionally wherein the pore size is about to about 135. The kit of any one of claims 130-134, wherein the ion pair chromatography matrix and / or ion pair chromatography column has a particle size of about 3 μm to about 5 μm, optionally a particle size of about 4 μm.

136. The kit of any one of claims 130-135, wherein the ion pair chromatography matrix and / or ion pair chromatography column comprises silica, optionally wherein the ion pair chromatography matrix comprises silica particles.

137. The kit of any one of claims 130 to 136, wherein the ion pair chromatography matrix and / or ion pair chromatography column is compatible with a mobile phase comprising dibutylammonium acetate and / or triethylammonium acetate.

138. The kit of any one of claims 130-137, wherein the kit comprises a dilution series of one or more reference nucleic acids, optionally wherein the kit comprises a dilution series of a reference gRNA molecule, a first reference mRNA molecule, and / or a second reference mRNA molecule.

139. The kit of any one of claims 130-138, wherein the kit comprises instructions for preparing a nucleic acid extract by a method comprising ethanol precipitation.

140. The kit of any one of claims 130-139, wherein the kit comprises instructions for separating the two or more nucleic acids or the three or more nucleic acids by ion pair chromatography, optionally wherein the nucleic acids comprise or consist of RNA molecules, optionally wherein the RNA molecules comprise or consist of gRNA molecules, first mRNA molecules and / or second mRNA molecules.

141. The kit of any one of claims 130-140, wherein the kit comprises instructions for determining the concentration of the two or more nucleic acids, the three or more nucleic acids, and / or the total nucleic acid concentration, optionally wherein the nucleic acids comprise or consist of RNA molecules, optionally wherein the RNA molecules comprise or consist of gRNA molecules, first mRNA molecules, and / or second mRNA molecules.

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